Abstracts

8th EBSA European Biophysics Congress,August 23rd–27th 2011, Budapest, Hungary

Plenary talks

O-001View into the ribosomal exit tunnelAda YonathDepartment of Structural Biology, Weizmann Institute,Rehovot 76100, Israel

Ribosomes, the universal polymerases for the translation ofthe genetic code into proteins, possess an elongated tunnelthrough which the nascent protein progress until they emergeinto the cell. While traveling through the tunnel the nascentproteins can interact with specific tunnel wall componentscapable of imposing transient or a relatively long elongationarrest. Furthermore, internal architecture and flexibility allowresponse to cellular signals, thereby provides a mechanismfor gene regulation.

O-002The interplay between actin dynamicsand membrane tension determinesthe shape of moving cellsKinneret KerenPhysics Department, Technion-Israel Institute of Technology,Haifa 32000, Israel

A central challenge in cell motility research is to quantitativelyunderstand how numerous molecular building blocks self-organize to achieve coherent shape and movement on cel-lular scales. We focus on one of the classic examples of suchself-organization, namely lamellipodial motility, in which for-ward translocation is driven by a treadmilling actin network.We combine detailed measurements of lamellipodial mor-phology, spatio-temporal actin dynamics and membranetension, with mathematical modelling to explain how globalshape and speed of the lamellipodium emerge from the

underlying assembly and disassembly dynamics of the actinnetwork within an inextensible membrane bag.

O-003Structure determination of dynamic macromolecularcomplexes by single particle cryo-EMHolger StarkMax-Planck-Institute for biophysical Chemistry, Goettingen,Germany

Macromolecular complexes are at the heart of central regu-latory processes of the cell including translation, transcription,splicing, RNA processing, silencing, cell cycle regulation andrepair of genes. Detailed understanding of such processesat a molecular level requires structural insights into largemacromolecular assemblies consisting of many compo-nents such as proteins, RNA and DNA. Single-particleelectron cryomicroscopy is a powerful method for three-dimensional structure determination of macromolecularassemblies involved in these essential cellular processes. Itis very often the only available technique to determine the3D structure because of the challenges in purification ofcomplexes in the amounts and quality required for X-raycrystallographic studies.In recent years it was shown in a number of publications thatit is possible to obtain near-atomic resolution structures oflarge and rigid macromolecules such as icosahedral viruses.Due to a number of methodological advances there are nowalso great perspectives for high-resolution single particlecryo-EM studies of large and dynamic macromolecules.Successful high-resolution structure determination ofdynamic complexes requires new biochemical purificationstrategies and protocols as well as state of the art electronmicroscopes and high-performance computing. In the futurecryo-EM will thus be able to provide structures at near-atomic

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DOI 10.1007/s00249-011-0734-z

resolution and information about the dynamic behavior ofmacromolecules simultaneously.

O-004Detection and rapid manipulation of phosphoinositideswith engineered molecular toolsTamas BallaSection on Molecular Signal Transduction, Programfor Developmental Neuroscience, NICHD, NIH, Bethesda,MD 20892, USA

Polyphosphoinositides (PPIs) are ubiquitous lipid regulatorsof a variety of cellular processes serving as docking sites andconformational switches for a large number of signalingproteins. The localization and dynamic changes in PPIs inlive cells have been followed with the use of protein domainGFP chimeras. In this presentation we will show experi-mental systems that allow rapid manipulation of the levels ofPPIs in specific membrane compartments. We are alsoactively pursuing strategies that will allow us to map thedistribution and possible functional diversity of the phos-phatidylinositol (PtdIns) pools within intact cells since theyare the precursors of PPIs. We will show our most recentprogress in addressing this question: the use of a PtdInsspecific PLC enzyme isolated from Listeria monocytogenestogether with a highly sensitive diacylglycerol sensor todetermine the distribution and also to alter the level of PtdInsin living cells. These studies reveal that a significant meta-bolically highly active PtdIns pool exists associated with tinymobile structures within the cytoplasm in addition to theknown ER and PM PtdIns pools. We will show our mostrecent data on the consequences of PtdIns depletion withinthe various PtdIns pools on PPI production and on themorphology and functions of various organelles.

O-005Spectroscopic explorations of the nature of proteindynamicsF. MezeiHungarian Academy of Sciences, SzFKI, 1525 Budapest, Pf.49, Hungary; European Spallation Source ESS AB, PO BOX167, SE 22100 Lund, Sweden

The functionality of proteins is known to be intimately relatedto the motion of their constituents on the atomic/molecularlevel. The study of microscopic motion in complex matter isoften reduced to the observation of some average meansquare atomic displacement, a first, very partial character-ization of the dynamics. The marked crossover in thetemperature dependence of such quantities in hydratedproteins around 200 K, the so called ‘‘dynamic transition’’ hasbeen originally observed a quarter of century ago. The origin,nature and the key characteristics of the atomic motionsbehind this remarkable evolution of the mean square dis-placement in proteins remained controversial over the pastdecades. Recent analysis of Mossbauer, dielectric relaxationand neutron scattering spectroscopic data provide unam-biguous evidence that this phenomenon is caused by the

temperature dependence of a relaxation process spread overseveral orders of magnitude in the time domain, similarly tothe b relaxation process observed in glasses. The review andcritical analysis of the available data highlights the inherentambiguities of commonly used data fitting approaches.Emerging evidence from model independent observationstend to exclude some of the proposed mechanisms.

O-006Microbial Rhodopsins: light-gated ion channelsand pumps as optogenetic tools in neuro- and cellbiologyE. Bamberg, C. Bamann, R.E. Dempski, K. Feldbauer,S. Kleinlogel, U. Terpitz, P. WoodDepartment of Biophysical Chemistry, Max-Planck-Instituteof Biophysics, Frankfurt, Germany

Microbial Rhodopsins are widely used in these days as op-togenetic tools in neuro and cell biology. We were able toshow that rhodopsins from the unicellar alga Chlamydo-monas reinhardtii with the 7 transmembrane helix motif actas light-gated ion channels, which we named channelrho-dopsins(ChR1,2). Together with the light driven Cl- pumpHalorhodopsin ChR2 is used for the non-invasive manipu-lation of excitable cells and living animals by light with hightemporal resolution and more important with extremely highspatial resolution The functional and structural description ofthis new class of ion channels is given (electrophysiology,noise analysis,flash photolysis and 2D crystallography). Newtools with increased spatial resolution and extremelyenhanced light sensitivity in neurons are presented. A per-spective for basic neurobiology and for medical applicationsis given.

Literature

Nagel et al. (2002) Science 296 2395-2398Nagel et al. (2003) PNAS 100 13940-13945Nagel et al. (2005) Current Biology 15 2279-2284Zhang et al. (2007) Nature 446 633-639Bamann et al. (2007) J. Mol. Biol. 375 686-694Feldbauer, K. et al. (2009). Proc Natl Acad Sci USA 10612317-12322

Bamann, C. et al. (2010). Biochemistry 49, 267-278 14Kleinlogel, S. et al. (2011). Nature Neuroscience 14 513-518

O-007The spatial organization of growth factor signalingsystems in cellsPhilippe BastiaensMax Planck Institute of Molecular Physiology, Departmentof Systemic Cell Biology, Dortmund, Germany

Our main objective is to elucidate how intracellular networksof nanometer-sized protein molecules process extracellularinformation thereby determining cellular phenotype thatmanifests on the micrometer scale. The cellular response to

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extracellular signals consists of the induction of specific geneexpression patterns and the re- organization in space andtime of stereo-specific macromolecular interactions thatendow the cell with its specific morphology. We developquantitative experimental and computational approaches toderive and conceptualize physical principles that underliethese dynamics of signal processing and cellular organiza-tion. We have an experimental emphasis on functionalmicroscopic imaging approaches at multiple resolutions tostudy the localization and dynamics of protein reactions/interactions, maintaining the inherent spatial organization ofthe cell. We have a strong recursion between computation ofmolecular dynamics in realistic cell geometries as sampledby microscopy, and experiments that reveal the dynamicproperties of networks in living cells. We investigate thecellular topography of activities that transmit signals fromreceptors at the cell surface. Here we ask, how spatial par-titioning of intracellular signalling activities is achieved by thecausality structure of the signalling network, and how thispartitioning affects signal response. This entails the experi-mental elucidation of connections between reactions and thedetermination of enzyme kinetic parameters in living cells.

O-008Molecular photovoltaics mimic photosynthesisMichael GratzelLaboratory of Photonics and Interfaces, Institute of ChemicalScience and Engineering, Station 6, Ecole PolytechniqueFederale, CH-1015 Lausanne, SwitzerlandE-mail: michael.graetzel@epfl.ch

The field of photovoltaic cells has been dominated so far bysolid state p-n junction devices made e.g. of crystalline oramorphous silicon, profiting from the experience and materialavailability of the semiconductor industry. However, there isan increasing awareness of the possible advantages ofdevices referred to as ‘‘bulk’’ junctions due to their intercon-nected three-dimensional structure. Their embodimentdeparts completely from the conventional flat p-n junctionsolid-state cells, replacing them by interpenetrating networks.This lecture focuses on dye sensitized mesoscopic solarcells (DSCs), which have been developed in our laboratory.Imitating natural photosynthesis, this cell is the only photo-voltaic device that uses a molecular chromophore togenerate electric charges from sunlight and that accom-plishes the separation of the optical absorption from thecharge separation and carrier transport processes. It does soby associating the molecular dye with a film constituted oftiny particles of the white pigment titanium dioxide. The DSChas made phenomenal progress, present conversion effi-ciencies being over 12 percent for single junction and 16percent for tandem cells, rendering the DSC a crediblealternative to conventional p-n junction devices. Molecularlyengineered porphyrine have recently even reached over 13percent efficiency when used in conjunction with a new redoxelectrolyte. Commercial large scale production of flexibleDSC modules has started in 2009. These solar cells havebecome viable contenders for large-scale future solar energy

conversion systems on the bases of cost, efficiency, stabilityand availability as well as environmental compatibility.

References:

(1) B. O’Regan and M. Gratzel, Nature, London 353 (1991).(2) U.Bach, D.Lupo, P.Comte, J.E.Moser, F.Weissortel,

J.Salbeck, H.Spreitzert and M.Gratzel, Nature, 395,550 (1998).

(3) M Gratzel, Nature 414, 338-344 (2001).(4) M.Gratzel, Acc. Chem. Res. 42, 1781-1798 (2009).

O-009Organizing principle of the plasma membrane: three-tiered meso-scale domain architecture revealedby single-molecule trackingAkihiro KusumiInstitute for Integrated Cell-Material Sciences (iCeMS),Institute for Frontier Medical Sciences, Kyoto University,Kyoto, Japan

Single-molecule imaging and tracking techniques that areapplicable to living cells are revolutionizing our understand-ing of the plasma membrane dynamics, structure, and signaltransduction functions. The plasma membrane is consideredthe quasi-2D non-ideal fluid that is associated with the actin-based membrane-skeleton meshwork, and its functions arelikely made possible by the mechanisms based on such aunique dynamic structure, which I call membrane mecha-nisms. My group is largely responsible for advancing high-speed single molecule tracking, and based on the observa-tions made by this approach, I propose a hierarchicalarchitecture of three-tiered meso-scale (2-300 nm) domainsas fundamental organizing principles of the plasma mem-brane. The three tiers I propose are the following.[Tier 1] 30-200 nm compartments made by partitioning theentire plasma membrane by the membrane-associated actin-based meshwork (membrane skeleton: fences) and itsassociated transmembrane proteins (pickets). Since theentire plasma membrane is partitioned by these structures,and the membrane skeleton provides important platforms forthe molecular interactions and pools, membrane compart-ments are the most basic tier for the plasma membraneorganization.[Tier 2] Meta-stable 1-5 nm raft domains that can be turnedinto stable *10-20-nm domains (receptor-cluster rafts),based on ligand-induced homo-dimers of glycosylpho-sphatidylinositol (GPI)-anchored receptors (coupling with[Tier 3]) and facilitated by raft-lipid interactions.[Tier 3] Protein complexes of various sizes (3-10 nm) andlifetimes.I will also talk about how domains of Tiers 2 and 3 are cou-pled to the membrane partitioning (Tier 1). The concept of thethree-tiered domain architecture of the plasma membraneand the cooperative interactions of different tiers provides agood perspective for understanding the mechanisms forsignal transduction and many other functions of the plasmamembrane.

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Biomolecular interactions

P-010Effects of electromagnetic fields on interactionbetween Norfloxacin and Human Serum AlbuminO. Azimi1, H. Salari1, Z. Emami2, J. Chamani11Department of Biology, Faculty of Sciences, Islamic AzadUniversity-Mashhad Branch, Mashhad, Iran, 2Departmentof Physics, Faculty of Sciences, Islamic Azad University-Mashhad Branch, Mashhad, Iran

Keywords: Human serum albumin, Norfloxacin, fluores-cence, electromagnetic fields

Introduction: In the present study we investigate the effectsof electromagnetic fields (EMF) on the binding of Norfloxacin(NRF) to Human Serum Albumin (HSA) by fluorescence,three-dimensional fluorescence and uv-visible spectroscopicapproaches. HSA is the most abundant protein in humanblood plasma which works as a carrier that transports dif-ferent materials in the body. NRF is used to treat variety ofbacterial infections. It works by stopping the bacterial growth.Methods: HSA, NRF and potassium phosphate buffer werepurchased from sigma. Fluorescence Spectrofluorometer,UV-vis spectrophotometer, Three-dimensional fluorescenceand a home-built EMF generator apparatuses were used.Results: Results obtained from this study indicated that NRFhas a strong ability to quench HSA in 280 nm. In addition,there was a slight blue shift, which suggested that themicroenvironment of protein became more hydrophobic afteraddition of NRF. Moreover, synchronous fluorescence dem-onstrated that the microenvironment around Tyrosine (Tyr)had a trivial increase. These, and the results of HSA–NRF inthe presence of EMF with 1 KHz, illustrates the same resultsinferred from quenching and blue shift. However, there was asignificant decrease in KSV of NRF with HSA in presence ofEMF exposure. Moreover, the binding parameters includingthe number of binding sites and the binding constant werecalculated form Hill equation.Conclusion: It was shown that NRF could induce confor-mational changes in HSA both in the absence and presenceof EMF with no significant difference. Yet, the affinity isdecrease significantly in the presence of EMF. The clinicalimplications are discussed in detail.

P-012Characterization of the biochemical propertiesand biological function of the formin homology domainsof drosophila daamSzilvia Barko1*, Beata Bugyi2*, Marie-France Carlier2,Rita Gombos3, Tamas Matusek3, Jozsef Mihaly3

and Miklos Nyitrai11University of Pecs, Faculty of Medicine, Departmentof Biophysics, Pecs, Szigeti str. 12, H-7624, Hungary,2Cytoskeleton Dynamics and Motility, Laboratoired’Enzymologie et Biochemie Structurales, Centre Nationalde la Recherche Scientifique, 1 Avenue de la Terrasse,91198, Gif-sur-Yvette, France, 3Biological Research Centerof the Hungarian Academy of Sciences, Institute of Genetics,Szeged, Temesvari krt. 62, H-6726, Hungary

* These authors contributed equally to this work.

We characterised the properties of Drosophila melano-gaster DAAM-FH2 and DAAM-FH1-FH2 fragments andtheir interactions with actin and profilin by using variousbiophysical methods and in vivo experiments. The resultsshow that while the DAAM-FH2 fragment does not haveany conspicuous effect on actin assembly in vivo, in cellsexpressing the DAAM-FH1-FH2 fragment a profilindepen-dent increase in the formation of actin structures isobserved. The trachea specific expression of DAAM-FH1-FH2 also induces phenotypic effects leading to the col-lapse of the tracheal tube and lethality in the larvalstages.In vitro, both DAAM fragments catalyze actin nucleation butseverely decrease both the elongation and depolymerisa-tion rate of the filaments. Profilin acts as a molecular switchin DAAM function. DAAM-FH1-FH2, remaining bound tobarbed ends drives processive assembly of profilin-actin,while DAAM-FH2 forms an abortive complex with barbedends that does not support profilinactin assembly. BothDAAM fragments also bind to the sides of the actin fila-ments and induce actin bundling. These observations showthat the Drosophila melanogaster DAAM formin representsan extreme class of barbed end regulators gated byprofilin.

P-013Electron spin echo studies of free chain-labelled stearicacids interacting with b-lactoglobulinRita Guzzi, Luigi Sportelli, Rosa BartucciDipartimento di Fisica, Universita della Calabria, 87036Rende (CS), Italy

b-lactoglobulin (bLG) binds non-covalently fatty acids withinits central calyx, a cavity in the barrel formed by the strandsbA- bH.We present results of pulsed Electron Paramagnetic Reso-nance (EPR) spectroscopy on the interaction of bLG withstearic acids spin-labelled at selected positions, n, along theacyl chain (n-SASL, n = 5,7,10,12,16).D2O-Electron Spin Echo Envelope Modulation (ESEEM)Fourier transform spectra indicate that all segments of thebound chains in the protein binding site are accessible to thesolvent. The extent of water penetration decreases pro-gressively on moving from the first segments toward theterminal methyl end of the chain. About 50% of the nitroxidesin the upper part of the chain (n = 5,7) are H-bonded by asingle water molecule and this fraction reduces to 30% at thechain terminus (n = 12,16). A lower fraction of the nitroxidesare H-bonded by two water molecules, and it decreases fromabout 15% to a vanishingly small value on going down thechain.Echo-detected ED-EPR spectra reveal subnanosecondlibrational motion of small amplitude for both 5- and 16-SASLin the protein cavity. The temperature dependence of thelibrations is more marked for 16-SASL and it arises mainlyfrom an increase in librational amplitude with increasingtemperature.

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P-014Interaction of two putative fusion peptides from SARS-CoV glycoprotein with model membranesLuis G.M. Basso1, Antonio J. Costa-Filho1,21University of Sao Paulo, Sao Carlos, Brazil, 2Universityof Sao Paulo, Ribeirao Preto, Brazil

Fusion peptides (FP) pertaining to the spike glycoproteinfrom severe acute respiratory syndrome (SARS) coronavi-rus are essential for the fusion between viral and hostcellular membranes. Here we report a biophysical charac-terization of the interaction of two putative FPs with modelmembranes. Fluorescence and DSC experiments showedthat both peptides bind stronger to anionic than to zwit-terionic lipid membranes. ESR spectra showed that TOAC-SARSIFP rotational dynamics is modulated by lipid com-position and pH as compared to the spectrum of thispeptide in solution. However, stearic acid spin labelsreported no changes on the dynamic structure of zwitter-ionic micelles, whereas the whole chain of anionicsurfactants was perturbed by the peptides. Finally, CD datarevealed a predominant b-strand structure for SARSFP andan a-helix for SARSIFP in the presence of micelles, incontrast to their disordered structures in buffer. Overall theresults point out that electrostatic and hydrophobic inter-actions are both important to the energetic behavior ofpeptide membrane interaction. These findings might pro-vide a useful rationale for the elucidation of one of thesteps involved in the fusion process, and thus helpunderstanding the more general way of action of FPs at amolecular level.

P-015Interaction of filamentous actin and ezrin within surfacemodified cylindrical nanoporesDaniela Behn1,2 and Claudia Steinem1

1Institute for Organic and Biomolecular Chemistry, Universityof Gottingen, Tammannstraße 2, 37077 Gottingen, Germany,2GGNB doctoral program: IMPRS – Physics of Biologicaland Complex Systems

Ezrin is a member of the ezrin-radixin-moesin (ERM) proteinfamily that acts as a dynamic linker between the plasmamembrane and the actin cytoskeleton and is hence involvedin membrane organization, determination of shape and sur-face structures and other cellular processes. The protein ishighly enriched in microvilli of polarized epithelial cells, whereit binds filamentous actin (F-actin) with its C-terminal domain,while the N-terminal domain is connected to the plasmamembrane via specific binding to L-a-phosphatidylinositol-4,5-bisphosphate (PIP2).Nanoporous anodic aluminum oxide (AAO) films providesimilar dimensions as microvilli and are thus a versatiletemplate to investigate the interaction of ezrin with F-actinwithin spationally confined areas. Owing to their opticaltransparency, functionalized AAOs can be used to measurethe binding process of ezrin to a PIP2 containing solid sup-ported membrane by means of time resolved opticalwaveguide spectroscopy (OWS).Confocal laser scanning microscopy (CLSM) will elucidate,whether F-actin binding to ezrin takes place within or atop the

nanopores. Furthermore, elasticity mapping of F-actin fila-ments by means of atomic force microscopy will allowdetermining binding forces and the lateral tension of the actincytoskeleton.

P-017In vitro application of porphyrin photosensitiserson MCF7, HeLa and G361 tumour cell linesBinder S., Kolarova H., Bajgar R., Tomankova K.,Daskova A.Deparment of Medical Biophysics, Faculty of Medicineof Palacky University, Olomouc, Czech Republic

Tumour treatment presents a challenge to all scientists andclinicians. Contemporary methods like radiotherapy, chemo-therapy or surgery have many undesirable side effects.Photodynamic therapy (PDT) seems to be one of alternativeswhich can be helpful in malignant cell therapy. PDT is notonly limited to cancer treatment but is also used as analternative for cardiovascular, skin and eye disease treat-ment. PDT employs photosensitive agents which need to beactivated by light which is not harmful to a patient. Theactivated photosensitive agent provokes a formation ofreactive oxygen species leading to cell damage or death.The phototoxicity of the two porphyrin photosensitizer(TMPyP, ZnTPPS4.12H2O) on the malignant cell lines (G361,HeLa, MCF7) irradiated with the 1 Jcm-2 doses was evalu-ated by ROS production assay, MTTassay and comet assay.Our results indicate higher efficiency of TMPyP overZnTPPS4.12H2O. As for the photodynamic effectiveness ofthe used photosensitizers on chosen cell lines we found thatHeLa cell line is the most sensitive to phototoxic damageinduced by TMPyP.

AcknowledgementsThis work was supported by the grant projects NS9648-4/2008 from the Ministry of Health, MSM 6198959216 from theMinistry of Education, Youth and Sports of the CzechRepublic, 303/09/H048 and 202/09/1151 from the CzechScience Foundation, LF_2011_009 and CZ.1.05/2.1.00/01.0030

P-018nmR analysis of the Respiratory syncytial virus M2-1protein structure and of its interaction with some of itstargetsC. Sizun1, M.-L. Blondot2, V. Dubosclard2, F. Bontems1,J.-F. Eleouet21Institut de Chimie des Substances Naturelles, CNRSUPR2301, Gif-sur-Yvette, France, 2Unite de Virologie etImmunologie Moleculaires, INRA UR892, Jouy-en-Josas,France

The respiratory syncytial virus (RSV) is a major cause ofacute respiratory tract infections (bronchiolitis, pneumonia) inhuman and a leading cause of viral death in infants andimmunocompromised patients. RSV genome is formed of asingle non-segmented negative strand RNA which transcrip-tion and replication is ensured by a specific RNA-dependentRNA polymerase complex formed of the large (L) polymerase

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subunit and of several cofactors. This complex has no cellularcounterpart and represents an ideal target for antiviral drugs.Among the cofactors, M2-1 acts as an antitermination factorand increases the polymerase processivity. Its centraldomains has been shown, in vitro, to bind the phosphoproteinP and genomic RNA in a competitive manner. Here we reportthe nmR structure of this central domain and its interactionwith P and RNA fragments. M2-1 shares structural similaritywith VP30, a transcription factor of Ebola virus. The bindingsurfaces for RNA and P are distinct but overlapping. RNAbinds to a basic cluster located next to residues found to becritical for transcription both in vitro and in vivo by mutationalanalysis. We speculate that M2-1 might be recruited by P tothe transcription complex, where interaction with RNA takesplace, stabilized by additional elements.

P-019Force spectroscopy at the membrane-cytoskeletoninterface: interactions between ezrin and filamentousactinJulia A. Braunger1,2, Ingo Mey1 and Claudia Steinem1

1Institute for Organic and Biomolecular Chemistry, Georg-August-University of Gottingen, Tammannstraße 2, 37077Gottingen, Germany, 2GGNB doctoral program: IMPRS -Physics of Biological and Complex Systems

Ezrin, a member of the ERM (ezrin/radixin/moesin) proteinfamily, provides a regulated linkage between the plasmamembrane and the actin cytoskeleton. It contributes to theorganization of structurally and functionally distinct corticaldomains participating in adhesion, motility and fundamentaldevelopmental processes. Ezrin is negatively regulated by anintramolecular interaction of the terminal domains that masksthe F-actin binding site. A known pathway for activationinvolves the interaction of ezrin with phosphatidylinositol 4,5-bisphosphate (PIP2) in the membrane, followed by phos-phorylation of the threonine 567 residue in the C-terminaldomain. To date, it is unclear to what extent both regulatoryinputs contribute to the activation.We developed an in vitro system that facilitates the specificanalysis of the interaction forces between ezrin and F-actinby means of atomic force spectroscopy (AFM). Applyingezrin wild type and the pseudophosphorylated mutant proteinezrin T567D, respectively, permits to monitor the individualinfluence of phosphorylation on the F-actin-ezrin interaction.Thus, a thorough characterization of the acting forces at theezrin-actin interface will elucidate the activation mechanismof ezrin.

P-020PEG-ylated LDL particles: a potential drug deliverysystemDiana Buzova1, Peter Kasak2, Daniel Jancura1,Pavol Miskovsky11Department of Biophysics, P.J. Safarik University, Kosice,Slovakia, 2Polymer Institute SAS, Bratislava, Slovakia

Low-density lipoproteins (LDL) have proven to be usefulvehicles for lipophilic drugs to cancer cells. To make this

delivery system even more efficient, we have constructednano-carrier by coating of LDL by polyethylene glycol (PEG).The hydrophilicity of PEG should reduce the interaction ofLDL with other serum proteins and consequently decreasethe redistribution of loaded drug from LDL to the (lipo)pro-teins. Dynamic light scattering was used for determination ofhydrodynamic radius of LDL-PEG particles. CD spectros-copy measurements didn’t reveal structural changes ofapoliprotein B-100 (ligand for LDL receptors on cell surface),after conjugation of PEG with LDL. Interaction of LDL-PEGcomplexes with hypericin (Hyp) a natural photosensitizer wasstudied by fluorescence spectroscopy. We have demon-strated accumulation of higher number of Hyp in LDL-PEGthan LDL particles. However, the kinetics of Hyp redistribu-tion from Hyp/LDL-PEG complex to free LDL have similarparameters as those for the kinetics of Hyp transfer betweennon-modified LDL molecules. We suggest that Hyp mole-cules are mostly localized in the vicinity of the surface of theLDL-PEG particles and they are prone to redistribution toother serum proteins.

AcknowledgmentsGrant support: LPP-0072-07, VEGA-0164-09.

P-021Reactivity of aminophospholipid’s polar headwith glucose and arabinoseC. Caldes, B. Vilanova, M. Adrover, J. Frau, J. Donosoand F. MunozInstitute of Health Sciences Research (IUNICS), Departmentof Chemistry, University of Balearic Islands, Palma deMallorca, Spain

Modification of the head-group of aminophospholipids byglycation and subsequent lipid oxidation affect membrane’sstructure causing cell death.1 These processes are involvedin the pathogenesis of aging2 and diabetes.3 Non-enzymaticglycation forms in the first step a Schiff base (SB), whichrearranges to a more stable ketoamine, Amadori product,which leads to the formation of a heteregenous group ofcompounds (AGEs). Although several studies have beenfocused on identification of aminophospholipid glycationproducts,4 less attention has been paid to kinetic mechanismof the reaction. For that reason, in the present work, wecompare the kinetic reactivity of polar head-group of phos-phatidylethanolamine (PE) and phosphatidylserine (PS), thetwo target phospholipids components of mammalian cellmembranes. The reaction of PE and PS’s head-group withglycating compounds (glucose and arabinose) was studied inphysiological conditions by using nmR spectroscopy. Theobtained formation rate constants for SB are lower thanthose determined for the SB of the peptide Ac-Phe-Lys withthe same carbohydrates.5 It suggests that the phosphategroup may delay the glycation process. Moreover, the PS’shead-group has a carboxilic group in the structure, whichaffects the stability of the SB.

(1) Obsil, T.; Amler, E.; Obsilova, V.; Pavlıcek, Z. Biophys.Chem., 1999, 80, 165-177.

(2) Bailey, A. J.; Paul, R. G.; Knott, L. Mech. Ageing Dev.,1998, 106, 1-56.

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(3) Baynes, J. W.; Thorpe, S. R. Diabetes, 1999, 48, 1-9.(4) (a) Lertsiri, S.; Shiraishi, M.; Miyazawa, T. Biosci.

Biotechnol. Biochem., 1998, 62, 893-901. (b) Lederer,M. O. and Baumann, M. Biorg. Med. Chem., 2000, 8,115-121.

(5) Adrover, M.; Vilanova, B.; Frau, J.; Munoz, F.; Donoso,J. Amino acids, 2009, 36, 437-448.

P-022Ultrasensitive protein detection in nano-immuno assaysbased on DNA directed immobilization and atomic forcemicroscopy nanograftingL. Casalis, F. Bano, L. Fruk, B. Sanavio, P. Parisse,A. Bosco, A. De Simone, C. Micheletti, G. ScolesSincrotrone Trieste S.C.p.A., Trieste, Italy

We developed ultrasensitive, ELISA-like nanoimmunoassays suitable for proteomics/interactomics studies in lowsample volumes. We exploit the approach of DNA microarraytechnologies applied to proteomics [1], in combination withatomic force microscopy (AFM) to generate functional proteinnanoarrays: semisynthetic DNA-protein conjugates areimmobilized by bioaffinity within a nanoarray of comple-mentary ssDNA oligomers produced by AFM nanografting(NG). A nanoarray of different antibodies or syntheticmolecular binders can be generated in a single operation,once the DNA nanoarray is produced. Moreover, NG allowsadjusting the packing density of immobilized biomolecules toachieve optimum bio-recognition.AFM-based immunoassayswith these nanoarrayswere shownto achieve detection limit of hundreds of femto Molar, in fewnanoliters volumes, with very high selectivity and specificity [2].To detect the hybridization efficiency of our devices, we run acombined experimental-computational study that providesquantitative relations for recovering the surface probe densityfrom the mechanical response (AFMcompressibility mea-surements) of the sample.

[1] C.M. Niemeyer, in Nanobiotechnology (Weinheim:Wiley-VCH (2004))

[2] F. Bano et al., Nano Lett (2009) 9 (7) pp. 2614-8

P-023Distinct ubiquitin binding modes exhibited by the SH3domains of CD2APSalvador Casares1, Jose L. Ortega-Roldan1,Malene Ringkjobing-Jensen2, Martin Blackledge2,Nico A.J. van Nuland3,4, Ana I. Azuaga11Departamento de Quımica Fısica e Instituto deBiotecnologıa, Facultad de Ciencias, Universidad deGranada, Fuentenueva s/n, 18071 Granada, Spain, 2ProteinDynamics and Flexibility by nmR, Institut de BiologieStructurale Jean-Pierre Ebel, CEA, CNRS, UJF UMR 5075,41 Rue Jules Horowitz, Grenoble 38027, France, 3StructuralBiology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050Brussels, Belgium, 4Department of Molecular and CellularInteractions, VIB, Pleinlaan 2, 1050 Brussels, Belgium

SH3 domains form a highly conserved family of proteindomains. Recently, they were identified as a new class ofubiquitin binding domains. Several mechanistic differences

have been proposed for ubiquitin binding to SH3 domainsinvolved in the immune signaling pathway. A key affinity andspecificity determinant has been appointed to residue Phe409inSla1SH3-3 domain.Wepreviously showed that the thirdSH3domain (SH3-C) of CD2 associated protein (CD2AP), whichpossesses that key phenylalanine residue (Phe324), indeedbinds toubiquitinbut inadifferent orientation to that inSla1SH3-3. In this work, we have performed structural and mutationalanalysis of the three SH3 domains of CD2AP and the third SH3domain of CIN85.We have found that the first and second SH3domains of CD2AP bind ubiquitin in a similar orientation to thatin Sla1 SH3-3, as opposed to the third one, despite the highsequencehomology the threeCD2AP-SH3domains share.Wehave also shown that SH3 domains displaying this differentubiquitin binding mode interact with higher affinity to ubiquitinmolecules with extended C-terminus. We conclude that theCD2AP SH3-C interaction with ubiquitin constitutes a newubiquitin binding mode involved in a different cellular function,thus changing the previously established mechanism of EGF-dependent CD2AP/CIN85 monoubiquitination.

P-024Virtual screening for the discovery of authenticinhibitors against cytosolic 5’-nucleotidase II:therapeutic use and benefit in cancer treatmentZ. Marton1, M. Rhimi2, P. Lallemand1,L. P. Jordheim3, C. Dumontet3, N. Aghajari2,C. Lionne1, S. Peyrottes4 and L. Chaloin11CPBS-UMR 5236, CNRS - Universite Montpellier Sud deFrance, 1919 route de Mende, 34293 Montpellier cedex 5,France, 2Laboratoire de Biocristallographie, Institut debiologie et Chimie des Proteines- UMR 5086, 7 passage duVercors, 69367 Lyon cedex 07, France, 3Laboratoire deCytologie Analytique – INSERM U590 – Faculte deMedecine Rockefeller, Universite Claude Bernard Lyon I,69008 Lyon, France, 4Institut des Biomolecules MaxMousseron (IBMM), UMR 5247, CNRS - UniversiteMontpellier Sud de France, Place Eugene Bataillon, 34095Montpellier cedex 5, Franceemail : laurent.chaloin@cpbs.cnrs.fr

Nucleoside analogues used as anticancerous drugs canbe rapidly degraded within treated cells, constituting amajor obstacle of their therapeutic efficiency. Among theenzymes responsible for this degradation, the cytosolic 5’-nucleotidase II (cN-II) catalyses the hydrolysis of somenucleoside monophosphates. In order to improve the effi-cacy of anticancerous drugs and to define the precise roleof cN-II, new original inhibitors have been developedagainst cN-II. Virtual screening of chemical libraries on thecrystal structure has allowed us to identify very promisingcandidates that turned to be competitive inhibitors of cN-II.One molecule was included in the anticancerous treatmentof tumoral cell lines in order to evaluate the potentialbenefit and could induce in fine a sensitization of certainanticancerous drugs. We also explore other inhibitors tar-geting the allosteric sites of this enzyme using a strategythat takes into account the dynamics of cN-II. The chem-ical structures of the newly identified allosteric inhibitors aswell as the atomic interactions with enzyme residues willbe presented. The final goal of this study is to find

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molecules that can freeze the enzyme in a conformationfor which its dynamics is severely limited and therefore itsfunction.

O-025Native mass spectrometry to Decipher interactionsbetween biomoleculesSarah CianferaniLaboratoire de Spectrometrie de Masse Bio-Organique,Universite de Strasbourg, IPHC, 25 rue Becquerel 67087Strasbourg, France. CNRS, UMR7178, 67037 Strasbourg,France

Mass spectrometry is generally understood as ‘‘molecularmass spectrometry’’ with multiple applications in biology(protein identification using proteomic approaches, recombi-nant protein and monoclonal antibody characterization). Anoriginal and unexpected application of mass spectrometryemerged some twenty years ago: the detection and thecharacterization of intact biological noncovalent complexes.With recent instrumental improvements, this approach, callednative MS, is now fully integrated in structural biology pro-grams as a complementary technique to more classicalbiophysical approaches (NMR, crystallography, calorimetry,SPR, fluorescence, etc.).Native MS provides high content information for multiproteincomplexes characterization, including the determination ofthe binding stoichiometries or oligomerization states, site-specificities and relative affinities. Recent developmentsof ion mobility / mass spectrometry instruments (IM-MS)provide a new additional level for MS-based structural char-acterization of biomolecular assemblies allowing size andshape information to be obtained through collisional crosssection measurements.These different aspects of native MS for structural charac-terization of biomolecular assemblies will be illustratedthrough several examples, ranging from multiprotein-com-plexes to protein/nucleic acid assemblies.

P-026Atomic Force Microscopy in study of gum-arabic/chitosan complexesSılvia Maria de Castro Coelho, Sandra Rocha, ManuelCoelho, Maria do Carmo PereiraLEPAE, Chemical Engineering Department, Facultyof Engineering, University of Porto, Rua Roberto Frias,4200-465 Porto, Portugal

Complex coacervation is a process which may result byelectrostatic interaction between charged polysaccharides. Itdepends essential on pH, ionic strength and biopolymersproperties like ratio, concentration and charge density.In this case, the main work was to study the structuralproperties of a colloidal system of opposite charge – chitosanand gum-arabic by atomic force microscopy (AFM).According to some of complexes show tendency toagglomerate. This depends on the molar ration of the mac-romolecules and their relative molecular weights.

AFM micrographs show, too, that some formation of irregularaggregates by both polymers were due to presence of non-charged polar monomers in chitosan molecule.At higher gum-arabic/chitosan ratios biopolymer concentra-tions, coacervates appear like a core-shell miccelarstructure composed of hydrophobic core (charge neutral-ized segments) stabilized by the excess component(positive zeta potential) and non-charged segments of gumarabic.

P-027Interaction of human serum albumin with rutintheoretical and experimental approachesIcaro P Caruso1, Wagner Villegas2, Marinonio L Cornelio11Instituto de Biociencias, Letras e Ciencias Exatas (IBILCE),Sao Paulo State Univesity, Sao Jose do Rio Preto, SP, Brazil,2Instituto de Quımica (IQ), Sao Paulo State University,Araraquara, SP, Brazil

Human serum albumin (HSA) is the principal extracellularprotein with a high concentration in blood plasma and carrierfor many drugs to different molecular targets. Flavonoids area large class of naturally occurring polyphenols widely dis-tributed in plants. Rutin (quercetin-3-rutinoside) is theglycoside between flavonoids quercetin and disacchariderutinose. Like other flavonoids, rutin displays anti-inflamma-tory and anti-oxidant properties. The interaction betweenHSA and rutin was investigated by fluorescence spectros-copy, ab initio and molecular modeling calculations.Fluorescence titration was performed by keeping the HSAconcentration (4 lM) constant and stoichiometrically varyingthe rutin concentration (1-4 lM). The emission spectra wereobtained in the range of 305 to 500nm, with the excitationwavelength at 295nm. The obtained fluorescence data werecorrected for background fluorescence and for inner filtereffects. The Stern-Volmer quenching constant values were3.722 9 105 and 1.868 9 105 M-1 at 298 and 303 K,respectively. From the modified Stern-Volmer associationconstants 2.285 9 105 (at 298 K) and 2.081 9 105 M-1

(at 303 K) were calculated the thermodynamic parametersDH = 14.048 KJ mol-1, DG298K = -30.557 KJ mol-1 andDG303 K = -30.834 KJ mol-1, and DS = 55.4 KJ mol-1 K-1.Fluorescence quenching method was used also to study thebinding equilibria thus determining the number of bindingsites 1.085 and 1.028, and binding constant 1.094 9 106 M-1

and 0.255 9 106 M-1 at 298 and 303 K, respectively. Theefficient quenching of the Trp214 fluorescence by rutin indi-cates that the binding site for the flavonoid is situated withinsubdomain IIA of HSA. The distance r = 2.397 nm betweenthe donor (HSA) and the acceptor (Rutin) was obtainedaccording to fluorescence resonance energy transference(FRET). Wavelength shifts in synchronous fluorescencespectra showed the conformation of HSA molecules ischanged in the presence of rutin.The structure of rutin utilized in molecular modeling calcula-tion was obtained by Gaussian 98 program. The optimizationgeometry of rutin was performed in its ground states by usingab initio DFT/B3LYP functional with 6-31G(d,p) basis setused in calculations. The molecular electrostatic potential(MEP) was calculated to provide the molecular charge

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distribution of rutin. The gap energy value between theHOMO and LUMO of the rutin molecule was about 4.22 eVwhich indicates that rutin is classified as a reactive molecule.From molecular modeling calculation the interaction betweenHSA and rutin was investigated using the AutoDock programpackage. The three-dimensional coordinates of humanserum albumin were obtained from the Protein Data Bank(entry PDB code 1AO6) and of rutin were obtained fromoutput optimization geometry of DFT. The best energyranked result shows that rutin is localized in the proximity ofsingle tryptophan residue (Trp214) of HSA that is in agree-ment with the fluorescence quenching data analysis.

P-029The effect of Toxofilin on the structure of monomericactinLıvia Czimbalek, Veronika Kollar, Roland Kardos, Gabor HildUniversity of Pecs, Faculty of Medicine, Departmentof Biophysics, Pecs, Hungary

Actin is one of the main components of the intracellularcytoskeleton. It plays an essential role in the cell motility,intracellular transport processes and cytokinesis as well.Toxoplasma gondii is an intracellular parasite, which canutilise the actin cytoskeleton of the host cells for their ownpurposes. One of the expressed proteins of T. gondii is the 27kDa-sized toxofilin. The long protein is a monomeric actin-binding protein involved in the host invasion.In our work we studied the effect of the actin-binding site oftoxofilin69-196 on the G-actin. We determined the affinity oftoxofilin to the actin monomer. The flourescence of the actinbound e-ATP was quenched with acrylamide in the presenceor absence of toxofilin. In the presence of toxofilin theaccessibility of the bound e-ATP decreased, which indicatesthat the nucleotide binding cleft is shifted to a more closedconformational state.The results of the completed experiments can help us tounderstand in more details what kind of cytoskeletal changescan be caused in the host cell during the invasion of the hostcells by intracellular parasites.

P-030Porphyrin conjugates in virus inactivation—roleof porphyrin binding and singlet oxygen productionBianka Tamas1, Katalin Toth2, Gabor Mez}o3,Levente Herenyi1, Gabriella Csık11Institute of Biophysics and Radiation Biology, SemmelweisUniversity, Budapest, Hungary, 2Biophysik derMakromolekule, DKFZ, Heidelberg, Germany, 3ResearchGroup of Pepetide Chemstry, HAS, ELTE, Budapest,Hungary

Here we tested the virus inactivation capability of tetra-peptideconjugates of meso-tri(4-N-methylpyridyl)-mono-(4-carboxy-phenyl)porphyrin (TMPCP) or meso-5,10-bis(4-N-methyl-pyridyl)-15,20-di-(4-carboxyphenyl)porphyrin (BMPCP) in thedark and upon irradiation.

T7 bacteriophage, as a surrogate on non-enveloped viruseswas selected as a test system. Both TMPCP and BMPCPand their peptide conjugates proved to be efficient photo-sensitizers of virus inactivation. The binding of porphyrin tophage DNA was not a prerequisite of phage photosensiti-zation, moreover, photoinactivation was more efficientlyinduced by free than by DNA bound porphyrin.Mechanism of photoreaction (Type I. versus Type II) and thecorrelation between DNA binding, singlet oxygen productionand virus inactivation capacity was also analyzed.DNA binding reduced the virus inactivation due to thereduced absorbance and singlet oxygen production of boundphotosensitizer, and altered mechanism of photoinactivation.As optical melting studies of T7 nucleoprotein revealed,photoreactions of porphyrin derivatives affected the struc-tural integrity of DNA and also of viral proteins, even if theporphyrin did not bind to NP or was selectively bound toDNA.

P-031Antimicrobial photodynamic therapy of porphyrinson bacterial cellsAdela Daskova1, Katerina Bogdanova2, KaterinaTomankova1, Svatopluk Binder1, Robert Bajgar1,Milan Kolar2, Jirı Mosinger3, Hana Kolarova11Department of Medical Biophysics, Institute of Molecularand Translational Medicine, 2Department of Microbiology,Faculty of Medicine and Dentistry, Palacky University,Hnevotinska 3, 775 15 Olomouc, Czech Republic,3Department of Inorganic Chemistry, Faculty of Sciences,Charles University in Prague, Prague, Czech Republic

Keywords: Porphyrin, Photosensitizer, Cyclodextrin, Bacte-rial Strains

Photodynamic therapy is usually used against malignant andnon-malignant tumors. Nowadays due to resistance of bac-terial strains we look for a new antimicrobial strategy todestroy bacteria with minimal invasive consequences.Development antimicrobial technology combines nontoxicphotosensitizer, visible light of appropriate wavelength andgeneration reactive oxygen species. In this work photosen-sitizers TMPyP and ZnTPPS4 are investigated forphotodynamic antimicrobial therapy. We tested these twophotosensitizers on two bacterial strains for comparison:Gram – positive S. aureus and Gram – negative P. aerugin-osa. We applied photosensitizers alone and bound in thecomplex created with hp-b-cyclodextrin and we comparedthe effectiveness to bacteria. The light emitting diodes (LEDs414 nm) were used as a source in PDTat the doses 0, 25, 50and 100 J. Bacteria were grown in the presence of photo-sensitizers at the concentration from 0.78 to 100 lM. Up tonow our result suggest that TMPyP and ZnTPPS4 are effi-cient alone and the efficiency even more increases incomplex created with hp-b-cyclodextrin.This work was supported by the grant projects 2192/2011/G3from the Ministry of Education, Youth and Sports of theCzech Republic, 303/09/H048 from the Czech ScienceFoundation, CZ.1.05/2.1.00/01.0030 and LF_2011_009.

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P-032Characterization of tetracaine: cyclodextrin inclusioncomplexes by nuclear magnetic resonanceRoberta A.F. de Lima1, Marcelo Bispo de Jesus1, LuisFernando Cabeca3, Leonardo F. Fraceto2, Eneida de Paula11Departamento de Bioquımica, Instituto de Biologia,Universidade Estadual de Campinas, Campinas SP, Brazil,2Departamento de Engenharia Ambiental, Univ. EstadualJulio de Mesquita Filho, Sorocaba, SP, Brazil, 3EmbrapaInstrumentacao Agropecuaria CNPDIA/EMBRAPA, SaoCarlos, SP, Brazil

Anesthesia is a medical milestone (Friedman & Friedland,Medicine’s 10 Greatest Discoveries, 2000) and local anes-thetics (LA) are the most important compounds used tocontrol pain in surgical procedures. However, systemic tox-icity is still a limitation for LA agents as well as low solubility,as for tetracaine (TTC). Approaches to improve LA effectsinclude macrocyclic systems formation, such as in cyclo-dextrins (CD). We have studied complexes formed betweenTTC and b-CD or hydroxylpropyl (HP)-b-CD through nmRand other (UV-VIS, fluorescence, DSC and X-ray diffraction)techniques. At pH 7.4 a 1:1 stoichiometry of complexationwas detected for both complexes, with association constantsof 777 M-1 and 2243 M-1 for TTC:b-CD and TTC:HP-b-CD,respectively. The nuclear overhauser nmR data disclosedtrough the space proximities between hydrogens Hh and Hi -at the aromatic ring of TTC - and hydrogens from the innercavity of the cyclodextrins, allowing us to propose thetopology of TTC:CD interaction. Complex formation did notcurb TTC association with model (liposomes) and biologicalmembranes since the total analgesic effect (infraorbital nerveblockade in rats) induced by 15mM TTC increased 36% uponcomplexation. Supported by (FAPESP # 06/121-9, 03838-1)Brazil.

P-033ITC as a general thermodynamic and kinetic tool to studybiomolecule interactionsPhilippe Dumas1, Dominique Burnouf1, Eric Ennifar1,Sondes Guedich1, Guillaume Bec1, Guillaume Hoffmann1,Barbara Puffer1, Mireille Baltzinger2, Daniele Altschuh21Equipe de Biophysique et Biologie Structurale, UniteArchitecture et Reactivite de l’ARN, Universite deStrasbourg, IBMC-CNRS 15 rue Rene Descartes, F67084Strasbourg, France, 2ESBS, Bd. Sebastien Brant, F67412Illkirch, France

Isothermal Titration Calorimetry (ITC) is a powerful techniquefor thermodynamic investigations that is little used to obtainkinetic information. We have shown that, in fact, the shape ofthe titration curves obtained after each ligand injection isstrictly governed by the kinetics of interaction of the twopartners. A simple analysis allowed us to explain severalfacts (e.g. the variation of time needed to return to equilibriumduring a titration experiment). All simplifications were furtherreleased to obtain a very realistic simulation of an ITCexperiment. The method was first validated with the bindingof the Nevirapine inhibitor onto the HIV-1 Reverse trans-criptase by comparison with results obtained by BiacoreTM.

Importantly, for more complex systems, the new methodyields results that cannot be obtained in another way. Forexample, with the E. coli transcription-regulator ThiaminePyroPhosphate riboswitch, we could resolve kinetically andthermodynamically the two important successive steps: (1)the binding of the TPP ligand and (2) the subsequent RNAfolding. Our results show that initial TPP binding is controlledthermodynamically by TPP concentration, whereas theoverall transcription regulation resulting from RNA folding iskinetically controlled.

P-034Characterization of human primary amine oxidase/Sialicacid binding Ig-like lectin 10 interactionH. Elovaara1, V. Parkash2, T. A. Salminen2,S. Jalkanen11Medicity Research Laboratory, University of Turkuand National Institute of Health and Welfare, Turku, Finland,2Biochemistry, Department of Biosciences, Abo AkademiUniversity, Turku, Finland

Leukocyte trafficking into inflamed tissues from the bloodvessels is essential in maintaining health. Human membraneprimary amine oxidase (hAOC3) is involved in this trafficking.The mechanism of the role of hAOC3 in the trafficking isunknown, but its amine oxidase activity is important.Recently the first hAOC3 counter molecule was identifiedbeing sialic acid binding Ig-like lectin -10 (Siglec-10; Kiviet al., 2009, Blood 114:5385-5392). Siglec-10 is an adhesionmolecule expressed on the B-cells, monocytes and eosino-phils. The hAOC3 binding of Siglec-10 site has beensuggested to be in the second C2 domain of Sigec-10. Forthe characterization of hAOC3/Siglec-10 interaction we havecloned Siglec-10 fragments and produced the protein ininsect cells. We have been able to demonstrate specificinteraction between hAOC3 and Siglec-10 fragment. Inaddition, the fragment is processed by hAOC3. This is thefirst time that natural ligand of hAOC3 has been shown to beprocessed using purified proteins.

P-035Investigating the oligomerization state of cyanfluorescent protein, from cell cytoplasm to solutionAgathe Espagne,1 Marie Erard,1 Ronald Melki,2 HelenePasquier1 and Fabienne Merola11Laboratoire de Chimie Physique, Universite Paris-Sudand CNRS, Orsay, France, 2Laboratoire d’Enzymologie etBiochimie Structurales, CNRS, Gif-sur-Yvette, France

The sensitivity of GFP-like fluorescent proteins to theirenvironment is a potential source of artifacts in protein-pro-tein interaction studies based on FRET. Besides parametersthat vary in the course of metabolic processes, such as pH orreactive oxygen species concentration, protein expressionlevel is likely to influence the fluorescence properties of

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GFPs, due to their tendency to dimerize at high concentra-tion. We have characterized for the first time the self-association properties of CFP (Cyan Fluorescent Protein),the fluorescent protein mostly used as FRET donor. Wefound that the fluorescence quenching observed at highexpression level in the cell cytoplasm and the fluorescencedepolarization measured at high concentration in vitro areinsensitive to the A206K mutation, shown to dissociate otherGFP dimers. Both phenomena are satisfactorily accounted forby a model of non-specific homo-FRET between CFP mono-mers due to molecular proximity. Modeling the expectedcontributions to fluorescence depolarization of rotational diffu-sion, homo-FRET within a hypothetical dimer and proximityhomo-FRET shows that CFP has a homo-affinity at least 30times lower than GFP. This difference is due to an intrinsicmutation of CFP (N146I), originally introduced to increase itsbrightness and that by chance also disrupts the dimers.

P-036Coupling of the hydration shell of B-DNAto conformational substates and peptide recognitionstudied by time-resolved FTIR spectroscopyHassan Khesbak, Olesya Savchuk, Satoru Tsushima,Karim FahmyDivision of Biophysics, Institute of Radiochemistry,Helmholtz-Zentrum Dresden-Rossendorf D-01314Dresden, PF 510119, Germany,E-mail: k.fahmy@hzdr.de

Biomolecular recognition typically proceeds in an aqueousenvironment, where hydration shells are a constitutive part ofthe interacting species. The coupling of hydration shellstructure to conformation is particularly pronounced for DNAwith its large surface to volume ratio. Conformational sub-states of the phosphodiester backbone in B-DNA contributeto DNA flexibility and are strongly dependent on hydration.We have studied by rapid scan FTIR spectroscopy the iso-thermal BI-BII transition on its intrinsic time scale of seconds.Correlation analysis of IR absorption changes induced by anincremental growth of the DNA hydration shell identifieswater populations w1 (PO2

- -bound) and w2 (non-PO2- -bound)

exhibiting weaker and stronger H-bonds, respectively, thanthose in bulk water. The BII substate is stabilized by w2. Thewater H-bond imbalance of 3-4 kJ mol-1 is equalized at littleenthalpic cost upon formation of a contiguous water network(at 12-14 H2O molecules per DNA phosphate) of reduced!(OH) band width. In this state, hydration water coopera-tively stabilizes the BI conformer via the entropically favoredreplacement of w2-DNA interactions by additional w2-watercontacts, rather than binding to BI-specific hydration sites.Such water rearrangements contribute to the recognition ofDNA by indolicidin, an antimicrobial 13-mer peptide frombovine neutrophils which, despite little intrinsic structure,preferentially binds to the BI conformer in a water-mediatedinduced fit. In combination with CD-spectral titrations, thedata indicate that in the absence of a bulk aqueous phase, asin molecular crowded environments, water relocation withinthe DNA hydration shell allows for entropic contributionssimilar to those assigned to water upon DNA ligand recog-nition in solution.

P-037Segmental-labeling expression of SH3 domainsof CD2AP protein to study interaction with their ligandI.F. Herranz-Trillo1, J.L. Ortega-Roldan1, N.A.J. van Nuland2,M.A. Ceregido1, A.I. Azuaga11Departamento de Quımica Fısica e Instituto deBiotecnologıa, Facultad de Ciencias, Universidad deGranada, 18071 Granada, Spain, 2Molecular RecognitionUnit. Structural Biology Brussels (VUB/VIB) building E,4th Floor, Peinlaan 2, 1050 Brussels, Belgium

Transient and low affinity interactions within the cell can beenhanced by the combination of more than one domain. Upto now most of the effort has been put on the study of theregulation in the affinity and specificity of the binding toisolated single domains but little is known about the effectof the presence of a second or third domain. Multipleexamples of proteins containing tandem domains exist inthe genome like the CIN85/CMS family of adaptor proteins.In this family all three N-terminal SH3 domains are involvedin a wide variety of different interactions, they share highersimilarity among themselves than to any other SH3domains, suggesting an overlapping specificities in binding.CD2 associated protein (CD2AP) is an adaptor protein andbelongs to this family, its N-terminus consists of three SH3domains and the interaction of each one of them with itstarget(-s) might be ultimately modulated by the presence ofits next-door-neighbor. In this work we present theexpression and purification of the tandem CD2AP-SH3A/SH3B produced by segmental labeling techniques thatallow us to express the domains with different isotopiclabel, improving the nmR signal and facilitating to study theinteraction of the natural ligand in the presence of next-door-neighbor domain.

P-038Kinetics of the interaction of DDP with DNA and nuclearproteinsMaria Feofilova1,2, Mikhail Ogarkov2,,Alexander Polyanichko1,2, Elena Chikhirzhina11Institute of Cytology of the Russian Academy of Sciences,Saint-Petersburg, Russia, 2Faculty of Physics of Saint-Petersburg State University, Saint-Petersburg, Russia

Platinum based coordination compounds are consideredsome of the most effective anti-tumor drugs. One of the mosteffective compounds in this group is cisplatin (cis-diammin-edichloroplatinum(II) or cis-DDP). It is used in treatment ofseveral types of tumors, yet its detailed mechanism of actionremains unknown.The anti-tumor mechanism of cis-DDP is based on its abilityto form DNA crosslinks. It has been shown that sulfur con-taining amino acids are possible intermediates for DNA-cisplatin interactions. Proteins that contain such amino acidsmay be involved in transportation of cis-DDP into the cellnucleus. One of the most interesting examples of such aprotein is non-histone protein HMGB1, which demonstratesan ability to interact with the DDP molecule. It has also beenshown that HMGB- proteins preferentially bind to DNA plat-inum adducts.

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Our research is focused on kinetic aspects of interactions ofDDP with DNA and proteins in aqueous solutions. Using UV-spectroscopy, electrophoresis, kinetic and thermodynamicapproaches we investigated DNA-DDP and HMGB-DDPinteractions. Using chemometrics and principle componentanalysis we have determined kinetic constants of the majorreactions.

P-039Quantifying Molecular Partition of SupramolecularComplexes in Lipid Model Systems – Studies on DengueVirus Supramolecular ComponentsJoao Miguel Freire1, Ana Salome Veiga1, Nuno C. Santos1,Wioleta Kowalczyk2, David Andreu2, Andrea T. Da Poian3

and Miguel Castanho11Instituto de Medicina Molecular, Faculdade de Medicina daUniversidade de Lisboa, Av., Prof. Egas Moniz, 1649-028Lisbon, Portugal, 2Department of Experimental and HealthSciences, Pompeu Fabra University, Barcelona, BiomedicalResearch Park, E-08003 Barcelona, Spain, 3Instituto deBioquımica Medica, Universidade Federal do Rio de Janeiro,21941-902, Rio de Janeiro, BrazilE-mail: joaofreire@fm.ul.pt

There are plenty of molecules that exert their effects at thecell membrane. The evaluation of these interactions, fre-quently quantified by the Nernst lipid/water partition constant(Kp), helps to elucidate the molecular basis of these pro-cesses. We present here a recently derived and testedmethod to determine Kp for single solute partitions using f-potential measurements. The concept was then extended tothe interaction of supramolecular complexes with modelmembranes. A simultaneous double partition with an aque-ous equilibrium is considered in this partition model. Theresults were validated by dynamic light scattering - DLS,f-Potential, fluorescence spectroscopy and laser confocalmicroscopy experiments. We evaluated the interaction ofsupramolecular complexes (peptides derived from denguevirus proteins with oligonucleotides) with LUV to study ourbiophysical models. Dengue virus (DV) infects over 50-100million people every year and may cause viral hemorrhagicfever. No effective treatment is available and several aspectsof its cellular infection mechanism remain unclear. Theextension of the interactions of these complexes with bio-membranes helps to elucidate some steps of DV life cycle.

P-040The aggregation of amphotericin B in the lipidmembranes induced by K+ and Na+ ions: Langmuirmonolayers studyMarta Arczewska, Mariusz GagosDepartment of Biophysics, University of Life Sciencesin Lublin, Poland

The polyene antibiotic amphotericin B (AmB) is currently thedrug of choice in the treatment of fungal infections despite

its undesirable side effects. According to the general con-viction, the biological action of the drug is based on theformation of transmembrane channels which affect physio-logical ion transport, especially K+ ions. This work reportsthe results of Langmuir monolayers study of the effect of K+

and Na+ ions on the molecular organizations of AmB in themodel lipid membrane. The two-component monolayerscontaining AmB and phospholipid (DPPC) have beeninvestigated by recording surface pressure-area isothermsspread on aqueous buffers containing physiological con-centration of K+ and Na+ ions. The strength of the AmB-DPPC interactions and the stability of the mixed monolayerswere examined on the basis of surface pressure measure-ments, the compressional modulus and the excess freeenergy of mixing. The obtained results proved a high affinityof AmB towards lipids in the presence of K+ than Na+ ions.The most stable mixed monolayers were formed with the 1:1and 2:1 stoichiometry in the presence of K+ and Na+ ions,respectively.This research was financed by Ministry of Education andScience of Poland within the research project N N401015035.

P-041Microcalorimetric study of antibiotic amphotericin Bcomplexes with Na+, K+ and Cu2+ ionsArkadiusz Matwijczuk, Grzegorz Czernel,Mariusz GagosDepartment of Biophysics, University of Life Sciencesin Lublin, Poland

Amphotericin B (AmB) as a metabolite of Streptomycesnodosus is one of the main polyene antibiotics applied inthe treatment of deep-seated mycotic infections. We pre-sented microcalorimetric (DSC) study of molecularorganization of amphotericin B in lipid membranes inducedby Na+, K+ and Cu2+ ions. The analysis of DSC curvesindicates the influence of Na+and K+ ions on the mainphase transition of pure DPPC lipid. For the molar fractionsof 3, 5, 10, 15 mol% AmB in DPPC we observed thethermal shift towards higher temperatures in respect to purelipid, both in the presence of Na+ and K+ ions. This resultmay be connected with the changes in dynamic propertiesof the model membrane system. In case of AmB-Cu2+

complexes in aqueous solution at two pH values, 2.0 and10.6, the DSC measurements reported endothermic heateffect. This phase transition was related to the dissociationprocess of AmB-Cu2+ complexes. The formation of AmB-Cu2+ complexes are accompanied by changes of themolecular organization of AmB especially disaggregation.These all observed effects might be significant from amedical point of view.This research was financed by Ministry of Education andScience of Poland within the research project N N401015035.

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P-042GSH-Ferrocene conjugates as electrochemical sensorsfor gluthatione S-transferasesRamiro Tellez-Sanz1, Manuel Martos-Maldonado2,Concepcion Mesa-Valle3, Juan ManuelCasas-Solvas2, Antonio Vargas-Berenguel2,Luis Garcia-Fuentes11Dept. of Physical Chemistry, Biochemistry and InorganicChemistry, University of Almerıa, Almerıa, Spain, 2Dept.of Geometry, Topology and Organic Chemistry, Universityof Almerıa, Almerıa, Spain, 3Dept. of Applied Biology,University of Almerıa, Almerıa, Spain

Enzymes belonging to the glutathione S-transferase (GST)family catalyze the conjugation of glutathione (GSH) to dif-ferent compounds, mainly xenobiotics, so that they can beexcreted from the cell. Over-expression of GSTs was dem-onstrated in a number of different human cancer cellsassociated with the resistance to many anticancer chemo-therapeutics. Thus, a quick and specific determination ofGST levels would be very helpful in cancer diagnosis. Vol-tammetric techniques are simple, sensitive and suitable forreal time monitoring of chemical and biological reactions, butcannot be used to examine the GST-GSH interactions.However, it is known that metallocenes such as ferrocene(Fc) are neutral, chemically stable and nontoxic compoundswith excellent redox properties. They have found applicationsin different fields as biosensors. GSH-ferrocene conjugatescould be of particular interest as their reversible and tunableredox properties could be applied for the development ofsensors for the electrochemical detection of GST. To evalu-ate the potential of GSH-ferrocene conjugates as redoxprobes, we have synthesized two conjugates, GSFc andGSFcGS, and have performed thermodynamic studies withthe dimeric GST of Schistosoma japonicum (SjGST) and thetwo mentioned ferrocenyl conjugates along with oxidizedglutathione. All of them are competitive inhibitors of SjGSTand the voltammetric properties of the ferrocene-SG conju-gates were examined. Our results show that the GSFcGSconjugate exhibits an affinity for SjGST approximately twoorders of magnitude higher than the other two, and threeorders higher than GSH alone. Furthermore, it shows nega-tive cooperativity with the affinity for the second site twoorders of magnitude lower than that for the first one. Wepropose the reason for this negative cooperativity is stericsince our docking studies have shown that, when bound, partof the first bound ligand invades the second site due to itslarge size. We suggest the combination of its quasi 1:1stoichiometry, enhanced voltammetric signal and high affinitymakes GSFcGS a good redox probe for the electrochemicaldetection of GST levels.

AcknowledgmentsThis work was supported by the ‘‘Consejerıa de Economıa,Innovacion y Ciencia de la Junta de Andalucıa’’ of theregional Andalusian Administration, grants numbers CVI-6028 and FQM-3141.

O-43Crowding of membrane proteins and peptidesStephan L. Grage1, Sergii Afonin1, Parvesh Wadhwani1,Pavel Mykhailiuk2, Marina Berditsch1, Tamta Turdzeladze1,Roland P. May3, Michael Haertlein3, Philip Callow3,Anthony Watts4, Kerwyn C. Huang5, Boris Martinac6,Anne S. Ulrich11Karlsruhe Institute of Technology, Germany, 2TarasShevchenko University, Kiev, Ukraine, 3Institut LaueLangevin, Grenoble, France, 4Department of Biochemistry,University of Oxford, UK, 5Department of Bioengineering,Stanford University, California, USA, 6Victor Chang CardiacResearch Institute, Sydney, Australia

Membrane proteins and peptides are acting in an envi-ronment rich in other proteins or peptides. Aim of our studywas to understand how such molecular crowding andresulting intermolecular interactions can influence thebehavior of membrane proteins, using various antimicrobialpeptides and membrane proteins as examples. In the caseof antimicrobial peptides we have previously described achange in their alignment in the membrane at a charac-teristic threshold concentration. To understand whether thischange is due to unspecific crowding or specific peptide-peptide interactions, we tested if this re-alignment dependson the presence of additional peptides. In most cases wefound a similar re-orientation behavior irrespective of theadded peptide type, indicating unspecific crowding. Whenpairing PGLa and magainin-2, however, we observed adistinctly different sequence of PGLa re-orientation in themembrane, indicating a specific interaction between thesetwo peptides, which correlates well with their known syn-ergistic activity.A rather different effect of crowding was observed for thelarger channel protein MscL, which was found to form clus-ters of functionally active proteins in the membrane. Wepropose that this clustering is caused by lipid-mediatedprotein-protein interactions.

P-044Water, hydrophobic interaction, and protein stabilityJ. Raul Grigera and C. Gaston FerraraInstituto de Fısica de Lıquidos y Sistemas Biologicos(IFLYSIB), CONICET-UNLP, La Plata, Argentina

Although there are several forces maintaining proteinstructure, it is well know that hydrophobic interaction is thedominant force of protein folding. Then, we can infer thatany factor that alters hydrophobic interaction will affect theprotein stability. We have study by computer simulationa model system consisting in solution of Lenard-Jonesparticles in water (SPC/E model) at different pressuresand temperatures and analyzed the solubility i.e. the

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aggregation properties, of such a system. From theobtained data we are able to build up the phase surfacedetermining the critical point. The computing results wherecompared with experimental data of binary mix of non polarsubstance in water and of protein denaturation, finding highcoincidence on the critical point. Since the behavior of ourmodel system can only be due to hydrophobic effects, thecoincidences with the denaturation of proteins allow us toconclude that the dominant factor that determine tempera-ture and pressure denaturation of proteins is thehydrophobic interaction. The temperature and pressures atwhich the denaturation, as well the disaggregation of simplenon-polar particles, starts agree with what we could expectbased on the cross over line of the low to high densitystructure water transition.

P-045Ligand induced changes of a functionallyreconstituted membrane protein in a supportedlipid bilayerClaire Hatty1,3, Vanessa Lake2, Guo Jun Liu1,3,Richard Banati1,31Medical Imaging and Radiation Sciences, Faculty of HealthSciences, The University of Sydney, Sydney, NSW 2006,Australia, 2National Deuteration Facility, Bragg Institute,Australian Nuclear Science and Technology Organisation,Lucas Heights, NSW 2234, Australia, 3Life Sciences,Australian Nuclear Science and Technology Organisation,Lucas Heights, NSW 2234, Australia

The functional reconstitution of a mitochondrial membraneprotein into a lipid bilayer was studied using a quartz crystalmicrobalance. The 6xHis-tagged protein was immobilisedvia specific binding to a Cu2+ terminated sensor surface,with a change in frequency indicating approximately 75%coverage of the sensor surface by the protein. A lipidbilayer was reconstituted around the protein layer, with afinal change in frequency that is consistent with theremaining area being filled by lipid. Incubation with a spe-cific ligand for the protein resulted in a significant change infrequency compared to the interaction with the surface orlipid alone. The change is greater than expected for themass of the ligand, indicating a possible conformationalchange of the protein, such as the opening of a channeland increased water content of the layer. Electrical imped-ance measurements on the same system have providedadditional evidence of protein-lipid bilayer formation, and itis intended that this system will be studied with neutronreflectometry to characterise potential ligand inducedchannel formation. Valuable functional and structural infor-mation about this membrane protein was obtained by usingsurface sensitive techniques to study the protein in a bio-mimetic lipid bilayer.

O-046Visualizing and quantifying HIV-host interactionswith fluorescence microscopyJelle Hendrix1,*, Zeger Debyser2, Johan Hofkens3

and Yves Engelborghs11Laboratory for Biomolecular Dynamics, University ofLeuven, Belgium, 2Laboratory for Molecular Virologyand Gene Therapy, University of Leuven, Belgium,3Laboratory for Photochemistry and Spectroscopy (*presentaddress), University of Leuven, Belgium

Protein-chromatin interactions are classically studied with invitro assays that only provide a static picture of chromatinbinding. Fluorescence correlation spectroscopy (FCS) is anon-invasive technique that can be used for the same pur-pose. Being applicable inside living cells it provides dynamicreal-time information on chromatin interactions. Transcrip-tional co-activator LEDGF/p75 has well characterized proteinand chromatin interacting regions. We studied LEDGF/p75 invitro and inside living cells with FCS and other techniques(luminescent proximity assay, spot/half-nucleus fluorescencerecovery after photobleaching, continuous photobleaching).Protein-protein interactions in living cells can be monitoredwith fluorescence cross-correlation spectroscopy (FCCS)using fluorescent proteins as genetic labels. Advantagesover using Forster resonance energy transfer (FRET) are theindependence from intermolecular distance and knowledgeof absolute protein concentrations. We characterized FCCSwith fluorescent proteins in vitro and then studied the intra-cellular complex of LEDGF/p75 and the HIV-1 integrase (IN)enzyme both with FRET and FCCS.

P-047Do nucleolar and apoptotic proteins interactin actinomycin D-treated cells?Ales Holoubek, Petra Otevrelova and Barbora BrodskaInstitute of Hematology and Blood Transfusion,U Nemocnice 1, 128 20 Prague 2, Czech Republic

Nucleus and its compartment nucleolus are a seat of enor-mous biosynthetic activity in human cancer cells. Nucleolarproteins, e.g. B23 or C23, play an important role in regulationof cell division and proliferation. One of the strategies how tointermit malignant cell proliferation is affecting, e.g. by drugtreatment, a net of intracellular protein interactions to bringthe cell on a way of apoptosis. A cytostatic agent actinomycinD initiates apoptosis in human cancer cells, as well as innormal peripheral blood lymphocytes. At the same time,translocation of B23 and C23 into nucleoplasm is observed inthe treated cells. Therefore interaction between nucleolarand apoptotic proteins comes into a question. Co-immuno-precipitation, fluorescence microscopy and yeast two hybridanalysis are used to answer it. In co-immunoprecipitationexperiments, tumor suppressor p53 showed up to be apromising candidate for the interaction. Fluorescence

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microscopy showed that actinomycin D-stabilized p53 pro-tein is localized mainly in nucleoplasm and therefore there isa possibility of its interaction with the relocalized nucleolarproteins. The yeast two-hybrid system is used for screeningcDNA library with the baits of interest and also for furthertesting of possibly interacting candidates.This work was supported by the grants IGA NS 9637-4 andIGA VZ00023736 from the MHCR

P-049Anisotropy study of OK kidney cells in the presenceof aminoglycoside gentamicinC. Istrate, T. Savopol, L. Saplacan, M.-G. Moisescu,E. KovacsDepartment of Biophysics and Cell Biotechnology, CarolDavila Medical University, Bucharest, Romania

It is known that gentamicin causes nephrotoxicity by inhi-bition of protein synthesis in renal cells. This causesnecrosis of proximal tubular cells, leading to acute renalfailure. The aim of this study was to investigate the mem-brane fluidity of ‘‘Opossum kidney’’ (OK) cells treated withgentamicin at different temperatures in order to establish thecapacity of gentamicin to influence the packing of membranelipids. For determination of membrane fluidity, OK cells wereincubated with 1mM gentamicin. Dependence of membranefluidity with temperature was recorded by measuring thefluorescence depolarization of TMA-DPH incubated cells, inthe temperature range between 100 - 40�C. The anisotropy ofOK cells treated with gentamicin is higher than in control cellsat temperatures above 35�C (t- Student test showed that thedifferences become significant at temperatures higher than35�C). This may be due to loss of lipids packing above thistemperature which favors a higher exposure of negative lipidpolar heads to the gentamicin positive charges and a moreeffective electrical action of gentamicin on each negativelycharged membrane constituent. In conclusion, gentamicininduces a significant increase in membrane fluidity in OKrenal cells at temperatures above 35�C.

P-050Thermodynamics: a tool for affinity optimization of drugcandidates for transthyretin amyloidosisCatarina S. H. Jesus1,2, Carlos J. V. Simoes1,2,Elsa S. Henriques2, Adrian Velazquez-Campoy3

and Rui M. M. Brito1,21Chemistry Department, Faculty of Science and Technology,University of Coimbra, Coimbra, Portugal, 2 Centerfor Neuroscience and Cell Biology, University of Coimbra,Coimbra, Portugal, 3 Instituto de Biocomputacion y Fısica deSistemas Complejos (BIFI), Universidad de Zaragoza,Zaragoza, Spain

Among the pathological conditions associated with amyloidfibrils, familial amyloid polyneuropathies (FAP) exhibit fibril

deposits mostly constituted by variants of Transthyretin(TTR), a homotetrameric plasma protein implicated in thetransport of thyroxine and retinol [1].Nowadays, the only effective therapy for TTR amyloidosis isliver transplantation. New therapeutic strategies are beingdeveloped taking advantage of our current understanding ofthe molecular mechanisms of amyloid formation by TTR [2].A significant effort has been devoted to the search andrational design of compounds that might decrease TTR tet-ramer dissociation, for example, through ligand binding at thethyroxine binding sites of TTR [3, 4].Here, we use isothermal titration calorimetry (ITC) to char-acterize the thermodynamic binding signature of potentialTTR tetramer stabilizers, previously predicted by computer-assisted methods [3]. ITC allows the measurement ofthe magnitude of the binding affinity, but also affords thecharacterization of the thermodynamic binding profile of aprotein-ligand interaction. High affinity/specificity TTRligands, enthalpically and entropically optimized, may pro-vide effective leads for the development of new and moreeffective drug candidates against TTR amyloidosis.

(1) Brito, R. M. M., Damas, A. M., and Saraiva, M. J. (2003),Current Medicinal Chemistry - Immunology, Endocrine& Metabolic Agents 3, 349-360

(2) Quintas, A., Vaz, D. C., Cardoso, I., Saraiva, M. J.,and Brito, R. M. M. (2001), J. Biol. Chem. 27627,202-27213

(3) Simoes, C.J.V., Mukherjee, T., Brito, R.M.M., Jackson,R.M. (2010), J. Chem. Inf. Model. 50, 1806-1820

(4) Baures, P. W., Peterson, S. a, and Kelly, J. W. (1998),Bioorg. Med. Chem. 6, 1389-401

P-051Fluorescence lifetime as a tool to study protein-protein interactions of GroEL and PRD1viral proteinsJenni Karttunen1, Sari Mantynen1, Hanna M. Oksanen2,Janne Ihalainen1 and Jaana K.H. Bamford11Department of Biological and Environmental Scienceand NanoScience Center, University of Jyvaskyla, Finland,2Institute of Biotechnology and Department of Biologicaland Environmental Science, University of Helsinki, Finland

We have established a set of vectors to promote easycloning of eCFP and eYFP fusions with any protein ofinterest. We exploit these fluorescent fusion proteins to studyprotein-protein interactions by fluorescence lifetime of eCFP.The decrease of eCFP lifetime reveals FRET between eCFPand eYFP and hence the interaction between proteins inquestion.GroEL-GroES chaperonin complex is required for the properfolding of Eschericia coli proteins. Bacteriophage T4 and itsdistant relative coliphage RB49 encode co-chaperon proteins(respectively gp31 and CocO) that can replace GroES in thechaperonin complex. GP31 is also required in the folding ofthe major capsid protein of the phage. PRD1 is a largemembrane-containing bacteriophage infecting gram-negative

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bacteria such as E. coli and Salmonella enterica. It has 15 kblong linear dsDNA genome and the capsid has an icosahedralsymmetry. The GroEL-GroES chaperonin complex is neededin the assembly of PRD1. We have found evidence that PRD1protein P33 can work similar way as other viral co-chaper-ones and substitute GroES in chaperonin complex.Fluorescence lifetime studies between proteins GroEL andP33 reveals an interaction that backs up the theory.

P-052Structural modification of model membranes by fibrillarlysozyme as reaveled by fluorescence studyA.P. KastornaV.N. Karazin Kharkiv National University, 4 Svobody Sq.,Kharkiv, 61077, Ukraine

Recent experimental findings suggest that protein aggrega-tion, leading to the formation and depositions of amyloidsplay a central role in the neurodegenerative diseases, type IIdiabetes, systemic amyloidosis, etc. In the present study wefocused our efforts on investigation of the influence of fibrillarlysozyme on the structural state of model lipid membranescomposed of phosphatidylcholine and its mixtures with car-diolipin (10 mol %) and cholesterol (30 mol %). To achievethis purpose, two fluorescent probes with different bilayerlocation, 1,6-diphenyl-1,3,5-hexatriene (DPH) distributing inmembrane hydrocarbon core and 6-lauroyl-2-dimethylami-nonaphthalene (Laurdan) locating at lipid-water interface,have been employed. The changes in membrane viscosityunder the influence of amyloid lysozyme were characterizedby fluorescence anisotropy of DPH. This fluorescenceparameter was not markedly affected by fibrillar protein in alltypes of model membranes. The changes in emissionspectra of Laurdan were analysed by the generalizedpolarization value (GP). It was found that adding of amyloidlysozyme resulted in the increment of GP value. Our datasuggest that lysozyme fibrils cause reduction of bilayerpolarity and increase of lipid packing density.

P-053A new ITC assay for measuring ultratight and low-affinityprotein–ligand interactionsSandro Keller1, Georg Krainer2, Jana Broecker11Molecular Biophysics, University of Kaiserslautern, Erwin-Schrodinger-Str. 13, 67663 Kaiserslautern, Germany,2Biophysics of Membrane Proteins, Leibniz Instituteof Molecular Pharmacology (FMP), Robert-Rossle-Str. 10,13125 Berlin, Germany

Isothermal titration calorimetry (ITC) is the gold standard forthe quantitative characterisation of protein–ligand and pro-tein–protein interactions. However, reliable determination ofthe dissociation constant (KD) is typically limited to the range100 lM[KD[1 nm. Nevertheless, interactions character-ised by a higher or lower KD can be assessed indirectly,provided that a suitable competitive ligand is available whoseKD falls within the directly accessible window. Unfortunately,the established competitive ITC assay requires that the high-

affinity ligand be soluble at high concentrations in aqueousbuffer containing only minimal amounts of organic solvent.This poses serious problems when studying protein bindingof small-molecule ligands taken from compound librariesdissolved in organic solvents, as is usually the case duringscreening or drug development. Here we introduce a newITC competition assay that overcomes this limitation, thusallowing for a precise thermodynamic description of high-and low-affinity protein–ligand interactions involving poorlywater-soluble compounds. We discuss the theoretical back-ground of the approach and demonstrate some practicalapplications using examples of both high- and low-affinityprotein–ligand interactions.

P-054Interaction of myoglobin with oxidized polystyrenesurfaces studied using rotating particles probeM. Kemper1,2, D. Spridon1, L.J. van IJzendoorn1,M.W.J. Prins1,31Eindhoven University of Technology, department of AppliedPhysics, Eindhoven, The Netherlands, 2Dutch PolymerInstitute, Eindhoven, The Netherlands, 3Philips Research,Eindhoven, The Netherlands

The interaction of proteins with polymer surfaces is of pro-found importance for the sensitivity of biosensors. Polymersurfaces are often treated in order to tune their chemicaland physical properties, for example by oxidation pro-cesses. To get a better understanding of the association ofproteins to treated polymer surfaces, we use the rotatingparticles probe (X.J.A. Janssen et al., Colloids and SurfacesA, vol. 373, p. 88, 2011). In this novel technique, proteincoated magnetic particles are in contact with a substrateand the binding is recorded for all individual particles usinga rotating magnetic field. We investigate the interaction ofmyoglobin coated magnetic particles to spincoated poly-styrene surfaces that have been oxidized with a UV/ozonetreatment. The surfaces have been characterized by XPS,AFM and water contact angle measurements. We willdemonstrate a clear influence of polystyrene oxidation onthe binding fractions of the myoglobin coated particles. Weinterpret the results in terms of DLVO-theory: electrostaticas well as electrodynamic properties of the surfaces will beinfluenced by the oxidation.

P-055The effects of mouse twinfilin-1 on the structureand dynamics properties of actinVeronika Kollar1, Pekka Lappalainen2, Roland Kardos1,Gabor Hild11University of Pecs, Faculty of Medicine, Departmentof Biophysics, 7624 Pecs, Szigeti street 12, Hungary,2University of Helsinki, Institute of Biotechnology, Programin Cell and Molecular Biology, Helsinki, Finland

The actin cytoskeleton of eukaryotic cells plays a key role inmany processes. The structure and dynamics of the cyto-skeleton are regulated by a large number of proteins that

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interact with monomeric and/or filamentous actins. Twinfilin isa 37-40 kDa protein composed of two ADF-homologuedomains connected by a short linker.In our work we studied the effects of the mouse twinfilin-1(TWF1) on the monomeric actin. We determined the affinityof TWF1 to the ATP-actin monomer with fluorescenceanisotropy measurement (KD = 0.015 lM). The fluores-cence of the actin bound e-ATP was quenched withacrylamide in the presence or absence of TWF1. In thepresence of twinfilin the accessibility of the bound e-ATPdecreased, which indicates that the nucleotide binding cleftis shifted to a more closed conformational state. It wasconfirmed with stopped-flow experiments that the kinetics ofnucleotide-exchange of actin decreased in the presence ofTWF1. We determined the thermodynamic properties ofTWF1 and investigated the effect of twinfilin on the stabilityof actin monomer with differential scanning calorimetry. TheTWF1 stabilized the stucture of the G-actin. Our results canhelp us to understand the regulation of actin cytoskeleton inmore details.

P-056Nephrotoxicity of i.v. administered oleic acid coatedFe3O4 nanoparticles (NP) in ratsKollarova R.1, Klenovicsova K.1, Vlkova B.2,Celec P.2, Kebis A.1, Kovacikova Z1, Volkovova K.1,Sebekova K.21Slovak Medical University, Bratislava, Slovakia, 2ComeniusUniversity Medical Faculty, Bratislava, Slovakia

Magnetic NP have attracted attention due to their potential ofcontrast enhancement of magnetic resonance imaging andtargeted drug delivery, e.g. tumor magnetic hyperthermiatherapy. Potential nephrotoxicity of single i.v. administrationof Fe3O4 NP was studied in female Wistar rats i.v. adminis-tered either placebo (10% v/v rat serum in 0.9% NaCl),suspension of TiO2 NP (positive control, bimodal 84/213 nmdistribution), or Fe3O4 NP (bimodal 31/122 nm distribution) indoses of 0.1, 1.0 or 10.0 mg/kg. Rats were sacrificed 24h, 7-,14- and 28-days after NP injection (n=9-10/each group).Administration of NP did not alter kidney size significantly;renal function of NP administered rats as monitored byplasma creatinine and urea concentrations, creatinineclearance and protein excretion rate did not differ signifi-cantly in either interval from rats administered placebo. Oneweek after administration significant rise in plasma Ca, itsurinary and fractional excretion was observed in ratsadministered 10 mg Fe3O4/kg. Plasma Mg levels rose in thisgroup 1 and 2 weeks after administration. No significantchanges in the expression of TNF-a, TGF-b, and collagen IVgenes in renal cortex were revealed. No obvious nephrotoxiceffects were observed in rats after a single i.v. dose of Fe3O4

NP.Study was supported by 7FP EC EU: NanoTEST (Develop-ment of methodology for alternative testing strategies for theassessment of the toxicological profile of nanoparticles usedin medical diagnostics.), Grant No.: 201335.

P-057Biomimetic supramolecular assemblies for studyingmembrane interactions in vitro and in vivoS. Kolusheva, R. JelinekBen-Gurion University, Beer-Sheva, Israel

We designed a novel biomimetic sensor, composed of con-jugated polydiacetylene (PDA) matrix embedded within lipidvesicles. The system is capable of detecting various com-pounds occurring within lipid membranes through rapidcolorimetric as well as fluorescent transitions. The colori-metric response of the sensor is correlated to the extent ofcompound-membrane binding and permeation and quanti-fied binding sensitivity to lipid composition. We describe anew disease diagnostic approach, denoted ‘‘reactomics’’,based upon reactions between blood sera and an array ofvesicles comprising different lipids and polydiacetylene(PDA), a chromatic polymer. We show that reactionsbetween sera and such a lipid/PDA vesicle array producechromatic patterns which depend both upon the sera com-position as well as the specific lipid constituents within thevesicles. Through attachment of chromatic polydiacetylene(PDA) nanopatches onto the plasma membrane, real-timevisualization of surface processes in living cells is possible.

P-058Mechanistic studies of the Ras-superfamily by time-resolved FTIR spectroscopyCarsten Kotting and Klaus GerwertRuhr-Universitat Bochum, Lehrstuhl fur Biophysik, ND04/352, D-44780 Bochum, GermanyE-mail: carsten.koetting@rub.de

The Ras protein is mutated in 30% of human tumors. Rasacts as a switch, transmitting a growth signal in an activeGTP-bound form and turning the signal off in an inactiveGDP-bound form. The switch off is accomplished by GTPhydrolysis, which is catalyzed by Ras and can be furtheraccelerated by GTPase activating proteins (GAPs). Muta-tions which prevent hydrolysis cause severe diseasesincluding cancer.We investigate the reaction of the Ras GAP protein-proteincomplex by time-resolved FTIR spectroscopy.1 Detailedinformation on the mechanism and the thermodynamics ofthe reaction was revealed:2 First, the catalytic arginine-fin-ger of GAP has to move into the GTP binding pocket, thencleavage of GTP is fast and H2PO4 hydrogen-bonded in aneclipsed conformation to the b-phosphate of GDP isformed.Further, we performed for the first time ATR-FTIR spectros-copy of Ras in its native environment, a lipid membrane.3 Inthis setup we are able to do difference spectroscopy of theimmobilized protein. Interactions with other proteins can bedetermined in a similar way as in SPR experiments but withthe additional information from the infrared spectra.

1. Kotting, Gerwert, ChemPhysChem 6, 881 (2005).2. Kotting et al. PNAS 105, 6260 (2008).3. Guldenhaupt et al., FEBS J. 275 5910 (2008).

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P-059ATR-FTIR Spectroscopy of immobilized proteinsCarsten Kotting, Jorn Guldenhaupt, Philipp Pinkerneiland Klaus GerwertPD Dr. Carsten Kotting, Ruhr-Universitat Bochum, Lehrstuhlfur Biophysik, ND04/352, D-44780 Bochum, GermanyE-mail: carsten.koetting@rub.de

We report on trFTIR1 investigations on the Ras protein, whichis mutated in 30% of the human tumors. Ras proteinsundergo distinct post-translational lipid modifications. Theseare required for appropriate membrane targeting. While bio-logical function of Ras in vivo is always located at amembrane, most in vitro studies have been conductedwithout the lipid anchor in solution.We performed for the first time ATR-FTIR spectroscopy ofmembrane anchored Ras:2 A monolayer of a model mem-brane was tethered to a germanium ATR-crystal.Subsequently Ras is immobilized at the membrane. This canbe done with bioorganically synthesized lipidated Ras (H.Waldmann)5 or by protein tags. We compare the differentlymembrane bound Ras proteins among themselves and withRas is solution.Since our setup includes a flow-through cell, we are able todo difference spectroscopy of the immobilized protein, e.g.by exchanging the nucleotide. By this method we canobserve the absorption of individual functional groups. Fur-ther, the interactions with other proteins can be determined ina similar way as in SPR experiments but with the additionalinformation from the infrared spectra. Since immobilization ispossible e.g. with a His-tag our system can be useduniversal.

1. Kotting, Gerwert, ChemPhysChem 6, 881 (2005).2. Guldenhaupt et al., FEBS J. 275 5910 (2008).

P-060Carbohydrate recognition domains play different rolesin galectin functionP.S. Kumagai1, F.H. Dyszy1, M.C. Nonato2,M. Dias-Baruffi2, A.J. Costa-Filho1,31Instituto de Fısica de Sao Carlos, Universidade de SaoPaulo, Sao Carlos, SP, Brazil, 2Faculdade de CienciasFarmaceuticas de Ribeirao Preto, Universidade de SaoPaulo, Ribeirao Preto, SP, Brazil, 3Faculdade de Filosofia,Ciencias e Letras de Ribeirao Preto, Universidade de SaoPaulo, Ribeirao Preto, SP, BrazilE-mail: patriciasuemy@ursa.ifsc.usp.br

Galectins are a family of animal lectins that specifically bindb-galactosides and have gained much attention due to theirinvolvement in several biologic processes such as inflam-mation, cell adhesion and metastasis. In such processes,several issues are still not clear including the mechanisms ofinteraction with different carbohydrates. Galectin-4 (Gal-4) isa tandem-repeat type galectin that contains two carbohy-drate recognition domains (CRD-I and CRD-II) connected bya linking peptide. In this study, we performed spectroscopicstudies of the carbohydrate-recognition domains from humanGal-4. Our goals are two-fold: (1) to monitor conformationalchanges in each domain upon its binding to specific ligands

and then to correlate the observed changes with structuraldifferences between the CRDs and (2) to investigate theinteraction between the CRDs and lipid model membranes.To achieve such objectives we used a combined approach ofspectroscopic techniques involving Circular Dichroism andElectron Spin Resonance. Overall the results obtained so farshow that CRD-I and CRD-II have distinct behaviors in termsof carbohydrate recognition and membrane binding. Thismay be due to specific differences in their structures andcertainly suggests a non-equivalent role in protein function.

P-061Hemoglobin influence on lipid bilayer structureas revealed by fluorescence probe studyO.K. Kutsenko, G.P. Gorbenko, V.M. TrusovaV.N. Karazin Kharkov National University, Kharkov, Ukraine

Hemoglobin (Hb) is a red blood cell protein responsible forthe oxygen transport. Its affinity for lipid bilayers representsinterest for gaining insight into protein biological function aswell as for some applied aspects such as development ofblood substitutes or biosensors. Hb influence on lipid bilayerstructure was investigated using fluorescent probes pyreneand Prodan. Model membranes were prepared of phospha-tidylcholine (PC) and its mixtures with phosphatidylglycerol(PG) and cholesterol (Chol). Hb penetration into membraneinterior is followed by the increase of relative intensity ofpyrene vibronic bands and decrease of Prodan generalpolarization value suggesting an enhancement of bilayerpolarity. This implies that Hb incorporation into membraneinterior decreases packing density of lipid molecules, pro-moting water penetration into membrane core. Cholcondensing effect on lipid bilayer prevents protein embed-ment into bilayer, thus decreasing membrane hydrationchanges as compared to PC bilayers. In the presence ofanionic lipid PG Hb-induced increase of bilayer polarity wasfound to be most pronounced, pointing to the modulatoryrole of membrane composition in Hb bilayer-modifyingpropensity.

P-063Thermodynamic evaluation of high affinity CD22antagonistsKatrin Lemme1, Stefanie Mesch1, Oliver Schwardt1,Hendrik Koliwer-Brandl2, Soerge Kelm2, Beat Ernst11Institute of Molecular Pharmacy, University of Basel,Switzerland, 2Department of Physiological Biochemistry,University of Bremen, Germany

CD22, expressed on the surface of B cells, is a member ofthe sialic-acid binding immunoglobulin-like lectin (Siglec)family. It is a negative regulator of B cell signaling and plays acrucial role in the regulation of activity, homeostasis andsurvival of B cells [1]. Furthermore, the presence of cyto-plasmic CD22 was proven to be a marker in B cellmalignancies [2]. The physiological ligands of CD22 are a2,6-linked sialic acids on glycoproteins and gangliosides. Theminimal binding motif is Siaa2,6-Gal (or GalNAc, GlcNAc) [3].

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We present optimized sialic acid-based mimics binding inthe low nanomolar range. Molecular interactions weredetermined with surface plasmon resonance (SPR), char-acterizing the affinity and the kinetics of binding.Furthermore, isothermal titration calorimetry (ITC) wasapplied to dissect the standard free energy of binding (DG�)into the standard enthalpy of binding (DH�) and the standardentropy of binding (DS�).

[1] P.R. Crocker, and A. Varki, Siglecs in the immunesystem. Immunology 2001, 103, 137-45.

[2] D.Y. Mason, H. Stein, J. Gerdes, K.A. Pulford, E.Ralfkiaer, B. Falini, W.N. Erber, K. Micklem, and K.C.Gatter, Value of monoclonal anti-CD22 (p135) antibod-ies for the detection of normal and neoplastic Blymphoid cells. Blood 1987, 69, 836-40.

[3] L.D. Powell, D. Sgroi, E.R. Sjoberg, I. Stamenkovic, andA. Varki, Natural ligands of the B cell adhesion moleculeCD22 beta carry N-linked oligosaccharides with alpha-2,6-linked sialic acids that are required for recognition. JBiol Chem 1993, 268, 7019-27.

P-064Importance of the low enantioselectivity of PGKfor the phosphorylation of antiviral L-nucleosidesPerrine Lallemand1, Beatrice Roy2, Laurent Chaloin1,Christian Perigaud2, Tom Barman1, Corinne Lionne11UMR5236, Centre d’etudes d’agents Pathogenes etBiotechnologies pour la Sante (CPBS), Montpellier, France,2UMR5247, Institut des Biomolecules Max Mousseron(IBMM), CNRS – University Montpellier 1 & 2, Montpellier,France

D- and L-nucleoside analogues are compounds of clinicalinterest in antiviral therapies. In order to pass the cell mem-branes, most of these medicines has to be administrated topatients as nucleoside pro-drugs and not directly as trip-hosphorylated forms. Because of the poor phosphorylation ofthe nucleoside analogues used in therapy, it is important tounderstand and to optimize their metabolism. Our aim is tounderstand how compounds of chirality L turn away3-phosphoglycerate kinase (PGK) from its normal glycolyticfunction to be converted into the triphosphate forms. In orderto elucidate PGK mechanism and substrate specificity, wehave measured the kinetics of the different steps of theenzymatic pathways by rapid mixing techniques and studiedthe influence of the nature of the nucleotide substratethereon. We first performed an extensive study with D- andL-ADP (see poster by P. Lallemand). We are now extendingthe studies to other nucleotide diphosphates (some of themused in therapies). Changes in the nature of the nucleobaseor deletion of hydroxyl group of the sugar affect the efficiencyof phosphorylation by PGK, either by decreasing dramaticallytheir affinity or by altering the phospho-transfer step itself.Structural explanations are given based on docking data.

P-065New insights into the translocation route of enrofloxacinand its metalloantibioticsS. C. Lopes, C. Ribeiro, P. GameiroRequimte, Faculdade de Ciencias, Universidade do Porto,Rua do Campo Alegre, 4169-007 Porto, PortugalE-mail: silvia.lopes@fc.up.pt

Probing drug/lipid interactions at the molecular level repre-sents an important challenge in pharmaceutical research,drug discovery and membrane biophysics. Previous studiesshowed that enrofloxacin metalloantibiotic has potential asan antimicrobial agent candidate, since it exhibits antimicro-bial effect comparable to that of free enrofloxacin but adifferent translocation route. These differences in uptakemechanism can be paramount in counteracting bacterialresistance. In view of lipids role in bacterial drug uptake, theinteraction of these compounds with different E. coli modelmembranes were studied by fluorescence spectroscopy.Partition coefficients determined showed that lipid/antibioticinteractions were sensitive to liposomes composition andthat the metalloantibiotic had a higher partition than freeenrofloxacin. These results corroborate the different mech-anism of entry proposed and can be rationalized on the basisthat an electrostatic interaction between the metalloantibioticpositively charged species, present at physiological pH, andthe lipids negatively charged head groups clearly promotesthe lipid/antimicrobial association.

P-066Oligomerization and fibril assembly of amyloid b peptide(1-40) in the presence of amphiphilesJoana Loureiro, Sandra Rocha, Maria do Carmo PereiraLEPAE, Departamento de Engenharia Quımica, Faculdadede Engenharia, Universidade do Porto, Rua Dr. RobertoFrias, 4200-465 Porto, Portugal

Amyloid b peptide (Ab) forms a large amount of extracellulardeposits in the brain of Alzheimer’s disease (AD) patientsand it is believed that this peptide is related to the patho-genesis of that disease. The most abundant monomeric formof physiological Ab (*90%) is constituted by 40 amino acidsand is benign, but by an unknown mechanism this endoge-nous material becomes aggregated and neurotoxic.Increasing evidence suggests that membrane interactionplays an important role in Ab neurotoxicity.In this work it will be studied the interactions of Ab(1-40) withCTAC (cationic), SDS (anionic), PFOA (anionic with fluorineatoms) and OG (nonionic) amphiphiles in monomeric andmicellar forms.The results demonstrated that Ab(1-40) forms fibrils withdifferent morphologies in the presence of micelles. In addi-tion, the presence of micelles accelerates the formation offibrils and decreases the lifetime of oligomers.

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P-067On chip multiplex label free bio-affinity analysisand MALDI-MS characterisation of bound analyteBellon Sophie1, Boireau Wilfrid2, Ducoroy Patrick3,Elodie Ly-Morin1, Chiraz Frydman11Horiba Scientfic, ZA de la Vigne aux Loups – 5 avenueArago, 91380 Chilly-Mazarin, France, 2Institut FEMTO-ST,32 avenue de l’Observatoire – 25044 Besancon Cedex,France, 3CLIPP, 1 rue du Professeur Marion – 21079 DijonCedex, FranceE-mail: elodie.ly-morin@horiba.com

We present here the exploitation of the powerful approach ofSurface Plasmon Resonance imaging and Mass Spectrom-etry coupling for protein fishing in biological fluids such ashuman plasma at the same sensitivity. On one hand, multiplexformat SPRi analysis allows direct visualization and thermo-dynamic analysis of molecular avidity, and is advantageouslyused for ligand-fishing of captured bio-molecules on multipleimmobilized receptors on a SPRi-Biochip surface. On theother hand, MALDI mass spectrometry is a powerful tool foridentification and characterization of molecules captured onspecific surface. Therefore, the combination of SPRi and MSinto one concerted procedure, using a unique dedicatedsurface, is of a great interest for functional and structuralanalysis at low femtomole level of bound molecules.To reach these goals, particular surface engineering hasbeen engaged to maintain a high level of antibody graftingand reduce non-specific adsorption. Thus, various chemis-tries have been tested and validated towards biological fluidssuch plasma, keeping in mind the capacity of the in situinvestigation by MS.Finally, signal to noise ratio was magnified leading to thecharacterization of protein LAG3, a potential marker of breastcancer, in human plasma.

P-069Atenolol incorporation into PNIPA nanoparticlesinvestigated by Isothermal Titration CalorimetryMihaela Mic, Ioan Turcu, Izabell Craciunescu,Rodica Turcu,National Institute for R&D of Isotopic and MolecularTechnologies, 400293 Cluj-Napoca, RomaniaE-mail: Mihaela.Mic@itim-cj.ro

Poly(N-isopropylacrylamide) (PNIPA) is a thermo-sensitivehydrogel undergoing a volume phase transition at about of32�C close to the body temperature. This volume phasetransition is envisaged as a key property for drug binding andrelease.The purpose of our research is the thermodynamic charac-terization of the binding of atenolol by PNIPA polymericnanoparticles. The thermodynamic parameters which char-acterize the binding process are obtained using theIsothermal Titration Calorimetry (ITC) as the main investi-gation technique.When polymeric nanoparticles bind drug molecules, heat iseither generated or absorbed depending on the amount ofbond molecules and also on the exothermic or endothermiccharacter of the binding process. The heat measurementallows the determination of binding constants, reaction stoi-chiometry and the thermodynamic profile of the interaction.

ITC technique has been used to investigate the bindingproperties of nanoparticles which shrink from the swollen tothe collapsed state. The capacity of such nanogels to bindatenolol molecules is directly related to relevant differencesbetween the binding properties in the swollen and in thecollapsed state respectively.

P-070Aggregation study of x-(alkyldimethylamonium)-alkylaldonamide bromidesP. Misiak1, B. Ro _zycka-Roszak1, E. Wozniak1, R. Skrzela1,K.A. Wilk21Department Physics and Biophysics, Wrocław Universityof Environmental and Life Sciences, Wrocław, Poland,2Department of Chemistry, Wrocław Universityof Technology, Wrocław, Poland

Sugar-based surfactants are of considerable research inter-est because they have improved surface and performanceproperties, reduced environmental impact, and have poten-tial pharmaceutical and biomedical applications. x-(alkyldimethylamonium)alkylaldonamide bromides (CnGAB)with different chain lenghs (n = 10, 12, 14, 16) belonging tocationic sugar-based surfactants were newly synthesised.The aggregation processes of CnGABs were studied bymeans of isothermal titration calorimetry (ITC), electric con-ductance method and molecular modelling methods. Thecritical micelle concentrations (cmc), the degree of micelleionization (b), the enthalpies (DHm) and the entropies (DSm)of micellization as well as the contributions of the headgroupsto the Gibbs free energies (DGm

0 (hy)) were calculated. Theobtained values were compared with those for dodecyldi-methylethylammonium bromide and literature data foranalogical glucocationic surfactants. The latest compoundsdiffer from CnGAB surfactants by substitution of sugar chainby gluco ring. Molecular modelling methods were used torelate the molecular properties of the compounds with theirexperimentally studied properties in solution. This work wassupported by grant N N305 361739.

P-071Characterization of membrane active regions of denguevirus (DENV) proteins C and EHenrique de Castro Nemesio, M. Francisca PalomaresJerez, Lidia Nuno y Jose Villalaın.Instituto de Biologıa Molecular y Celular, Universidad MiguelHernandez, E-03202 Elche (Alicante), Spain

Every year over 50 million people are infected with denguevirus (DENV), transmitted by a mosquito (Aedes aegypti).This enveloped virus, member of the Flaviviridae family, hasfour distinct serotypes. It has a single stranded positive RNAmolecule with a single open reading frame that encodes asingle poliprotein, which, after appropriate processing by viraland host proteases, gives rise to three structural proteins (C,prM and E) and seven non-structural proteins (NS1, NS2A,NS2B, NS3, NS4A, NS4B and NS5) [1]. The surface of theimmature virion is composed of E and prM heterodimers thatare arranged as trimers protruding from the membrane [2].The virus is thought to enter the host cell via a receptor-

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mediated endocytosis, although, if any, the specific denguereceptors have not been described. Once inside the cell, theacidified environment inside the endocytic vesicle triggers anirreversible trimerization of the envelope (E) protein, inducingthe release of the nucleocapsid (composed of viral RNA andmultiple copies of C protein) to the cytoplasm, thus startingthe infection process, where the poliprotein is translated andprocessed, originating all viral proteins. Considering thestructural proteins C and E, these are essential for the viralinfection, specifically, protein C is thought to be involved inthe viral assembly and specific encapsidation of the genomeand protein E (a class II fusion protein) plays a major role inthe fusion process. As recently described by some studies[3], protein C is composed of four a helices connected by fourshort loops and has a highly hydrophobic region forming aconcave groove that could interact with lipid membranes anda region with an increased concentration of positive charges,possibly interacting with the viral RNA. As for protein E, it iscomposed of three b stranded domains. It is proposed thatthe fusion loop is located in domain II of this protein and theputative receptor binding sites, considered essential for theviral entry, are supposedly located in domain III. In this work,we describe the identification of the membrane active regionsof both these proteins, considering both theoretical studies,hydrophobic moments, hydrophobicity and interfaciality val-ues as well as experimental ones, namely fluorescencespectroscopy, where a fluorescent probe is encapsulated inmodel membrane systems, and differential scanning calo-rimetry [4]. We have found one region in protein C and fourregions in protein E with membranotropic activity. This is thefirst work describing experimentally the putative membraneinteracting zones of both these proteins.

[1] Mukhopaday, S., Kuhn, R.J., and Rossmann, M.G.(2005) Nat. Rev.Microbiol. 3, 13-22.

[2] Qi, R.F., Zhang, L., and Chi, C.W. (2008) Acta Biochim.Biophys. 40(2), 91-101.

[3] Ma, L., Jones, C.T., Groesch, T.D., et al. (2004) Proc.Natl. Acad. Sci. USA 101(10), 3414-3419.

[4] Guillen, J., Perez-Berna, A.J., Moreno, M.R., Villalaın, J.(2005) J Virol. 79(3), 1743-1752.

This work was funded by grant BFU2008-02617-BM fromMinisterio de Ciencia y Tecnologia, Spain, granted to JoseVillalaın.

P-072Investigation of membrane-membrane interactionmediated by coiled coil lipopeptidesGesa Pahler, Andreas JanshoffGeorg-August-University, Tammannstrasse 6, 37077Gottingen, GermanyE-mail: gpaehle@gwdg.de

Specific cellular membrane interaction and fusion are crucialpoints in vivo which are in eukaryotic cells mediated bySNARE proteins. The definite mechanism behind theseprocesses is still poorly understood, but the coiled coil for-mation of a SNARE core complex consisting of four a-helicesseems to generate a fusogenic driving force. This offers thepossibility to design a straightforward experimental setup tomimic the complex protein-mediated membrane-membraneinteraction by using mere protein fragments or peptides

attached to artificial lipid bilayers which self-assemble to acoiled coil structure.In our approach, two artificial three heptad repeat coiled coilforming peptides were synthesized and attached to malei-mide functionalized membranes via an in situ-couplingreaction. Thus, secondary structure changes, kinetic char-acteristics and binding energetics were monitored duringcoiled coil formation with real time ellipsometry, IR and CDspectroscopy. The lipopeptide mediated membrane-mem-brane interaction itself is investigated by colloidal probespectroscopy and TIRFM.These techniques and the setup of our model system allowscreening the energetic and structural properties of variablecoiled coil forming peptides, i.e. linker-modified or biologicallyinspired sequences.

O-073Enzymatic reactions in nanostructured surfaces:unzipping and cutting the double helixPietro Parisse1, Matteo Castronovo2, Bojana Lucic3,Alessandro Vindigni3, Giacinto Scoles2 andLoredana Casalis11Sincrotrone Trieste S.C.p.A., Trieste, Italy, 2TempleUniversity, Philadelphia, USA, 3ICGEB, Trieste, Italy

Protein-DNA interactions are vital for living organisms. Fromviruses to plants and humans, the interactions of these twodifferent classes of biopolymers control processes asimportant and diverse as the expression of genes and thereplication, rearrangement, and repair of DNA itself.To understand these processes at the molecular level, and tofollow changes in cellular pathways due to different kinds ofperturbations and/or diseases it is necessary the identifica-tion and quantification of proteins and their complex networkof interactions.We have exploited the high spatial resolution given byAtomic Force Microscopy to generate DNA arrays of variabledensity by means of Nanografting. On such nanostructures,we investigate the mechanism of different enzymatic reac-tions (from restriction enzymes to helicases).Registering with high precision the height variation due to theaction of the enzyme onto the engineered DNA sequences(in the case of restriction enzymes) or taking advantage ofthe different mechanical properties of single and doublestranded DNA (in the case of helicases, where for the firsttime kinetic data were obtained on RecQ1 human helicase),we were able to monitor either the activity and/or the actionmechanisms of these two important classes of enzymes.

P-074Electric hyperfine interactions ab initio studyin biological systems: Cd in DNA basesP. A. D. Petersen1, M. B. Goncalves1, A. S. Silva2,A. L. Lapolli2, A. W. Carbonari2, H. M. Petrilli11Universidade de Sao Paulo, Instituto de Fısica,Departamento de Fısica de Materiais e Mecanica DFMT,Brazil, 2Instituto de Pesquisas Energeticas e Nucleares,IPEN, Sao Paulo, BrazilE-mail:hmpetril@if.usp.br

Electric hyperfine interactions can be a powerful tool toidentify local environments in many different systems.

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Recently it has been drawn attention to the possible appli-cation of the Time Differential Perturbed Angular Correlation(TDPAC) technique to study differences in the NuclearQuadrupole Coupling constants (tQ) at Cd probes in mouseDNA infected with the Trypanosoma Cruzi (Chagas diseasevector). The electric contribution to the hyperfine interactionis usually expressed as the nuclear quadrupole couplingfrequency tQ which is given by the product of the nuclearquadrupole moment Q and the Electric Field Gradient (EFG)at the nucleus. The EFG can be theoretically obtained froman ab initio electronic structure calculation. The 111In ?111Cd c-c decay can be used in a TDPAC measurement toinvestigate the Cd metal binding to DNA. The interaction ofthe metal with the DNA bases can change many aspects ofthe base pairing [1]. Here we study electric hyperfine prop-erties of Cd bound to some DNA bases. The methodologyused for the electronic structure calculations is based on theKohn Sham [2] scheme of the Density Functional Theory(DFT) and the Car-Parrinello [3] method. We use the Pro-jector Augmented Wave [4] method as embodied in the CP-PAW computational code. The results of EFG and energiesare discussed as function of water molecules present in theCd2+ environment and compared with TDPAC measure-ments at Cd probes in mouse DNA infected with theTrypanosoma Cruzi. The study of Cd2+ interactions withDNA has also a very broad interest due to Cadmium-inducedcarcinogenesis.

[1] J. V. Burda, J. Sponer, J. Leszczynski, P. Hobza, J.Phys. Chem. B., 101, 9670 (1997).

[2] W. Kohn, L. J. Sham, Phys. Rev. B., 140, 1133 (1965).[3] P. E. Blochl, Phys. Rev. B., 50, 17953 (1994).[4] R. Car e M. Parrinello, Phys. Rev. Lett. 55, 2493 (1985).

P-075Structure and stability of cyclic peptide basednanotubes: a computational chemistry investigationR. Vijayaraj1,2, S. Sundar Raman1, R. Mahesh Kumar1,Patrick Bultinck2, V. Subramanian11Chemical Laboratory, Central Leather Research Institute,Council of Scientific and Industrial Research, Adyar, Chennai600 020, India, 2Department of Inorganic and PhysicalChemistry, Ghent University, Krijgslaan 281 S-3, B-9000Gent, Belgium

In this study an attempt has been made to investigate thestructure, dynamics and stability of cyclic peptide nanotubes(CPNTs) formed by the self-assembly of cyclic peptides(CPs) using classical molecular dynamics (MD) simulationand semiempirical quantum chemistry calculation employingPM6. The structure and energetics of monomer and variousoligomeric CPNTs have been investigated by considering the(cyclo-[(D-Ala-L-Ala)4]) peptide as the model for CP. Variousgeometrical parameters extracted from the MD simulationreveal that the terminal residues are loosely hydrogen bon-ded to the inner subunits regardless of degree ofoligomerization. The hydrogen bonds present in the innercore regions are stronger than the terminal residues. As the

degree of oligomerization increases, the stability of the tubeincreases due to the hydrogen bonding and stacking inter-actions between the subunits. The results show that thebinding free energy increases with the extent of oligomeri-zation and reaches saturation beyond CPNT5. In addition,hydrophobic and electrostatic interactions play crucial rolesin the formation of CPNTs. Analysis of both structural andenergetics of formation of CPNTs unveils that the self-assembly of dimer, trimer and tetramer CPNTs are theessential steps in the growth of CNPTs.

Reference

Vijayaraj, R.; Sundar Raman, S.; Mahesh Kumar, R.; Subr-amanian, V. Studies on the structure and stability of cyclicpeptide based nanotubes using oligomeric approach: Acomputational chemistry investigation. J. Phys. Chem.B 2010, 114(49), 16574–16583.

P-076Studies at the liquid-air interface: a powerful toolboxto study interactions between biomoleculesBeaufils S.1, Vie V.1, Pezennec S.2,3, Le Floch-Fouere C.2,3,Bouhallab S.2,3, Pezolet M.4, Ameziane S.1, Renault A.11Institut de Physique de Rennes, UMR CNRS 6251,Universite de Rennes, Bat 11A Campus de Beaulieu, 35042Rennes Cedex, France, 2INRA, UMR1253 Science etTechnologie du Lait et de l’OEuf, F-35042 Rennes, France,3AGROCAMPUS OUEST, UMR1253 Science et Technologiedu Lait et de l’OEuf, F-35042 Rennes, France, 4Centre deRecherche sur les Materiaux Avances, Departement deChimie, Universite Laval, Quebec, G1V 0A6 Canada

Monolayers on a Langmuir trough constitute a great biomi-metic model to characterize protein-protein or protein-lipidinteraction, where the physical state of the interfacial layer iscompletely controlled. We present here three studies per-formed on monolayers, with a wide panel of experimental(optical, spectroscopical, rheological) techniques.i) Surface properties and conformation of Nephila clavipesSpider recombinant silk proteins (MaSpI and MaSp2) wasstudied at the air-water interface: we show that the mecha-nism of assembly of both proteins is different, although bothproteins share the same sequence pattern and a closehydrophobicity. They both exhibit a certain propensity to formb-sheets that may be important for the efficiency of the nat-ural spinning process.ii) The dystrophin molecular organization and its anchoring ina lipidic environment depend on the rod fragment used andon the lipid nature. Moreover the interaction is guided by thelateral surface pressure. This lipid packing variation isessential to understand the role of the dystrophin duringcompression-extension cycle of the muscle membrane.iii) We evidence that non additive behavior of mixtures offood globular proteins leads to enhanced foaming propertiesor to self assembled objects.

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P-078Membrane interactions and translocation assaysfor nucleolar-targeting peptidesMargarida Rodrigues1, Andrea Santos1, Beatriz G. de laTorre2, Gandhi Radis-Baptista3, Miguel Castanho1,David Andreu2, Nuno C. Santos11Instituto de Medicina Molecular, Faculdade de Medicina daUniversidade de Lisboa, Lisbon, Portugal, 2Departament deCiencies Experimentals i de la Salut, Universitat PompeuFabra, Barcelona, Spain, 3Laboratorio de Bioquımica eBiotecnologia – Instituto de Ciencias do Mar, UniversidadeFederal do Ceara, Fortaleza, Brazil

Nucleolar-targeting peptides (NrTPs) were designed bystructural dissection of crotamine, a toxin from the venom ofa South-American rattlesnake. At lM concentration, NrTPspenetrate different cell types and exhibit exquisite nucleolarlocalization. The aim of this work was to pursue with thestudy of NrTPs molecular mechanism for translocation, aswell as to determine the ability of NrTP to delivery largemolecules into cells. For the translocation experiments, rho-damine B-labeled NrTPs were used and tested with giantmultilamellar vesicles. Confocal microscopy results showthat there is an efficient translocation across model mem-branes. High levels of intracellular peptide were also seen indifferent cell lines and PBMC, soon after incubation withNrTP. Furthermore, a conjugate of NrTP (NrTP6C) bound tob-galactosidase was prepared by chemical synthesis andtested in HeLa cells. This conjugate maintains enzymaticactivity and is stable at 4�C for several days. The work doneso far with this new family of cell-penetrating peptidesrevealed strong interaction and translocation with lipid modelsystems. Moreover, successfully cellular delivery of b-galactosidase was observed and quantified.

P-079Interaction of zinc phthalocyanine with ionic and nonionic surfactants: UV-vis absorption and fluorescencespectroscopy for application in Photodynamic TherapyM. P. Romero, S. R. W. LouroPhysic Department, Pontifıcia Universidade Catolica de Riode Janeiro PUC-Rio, Brazil

Among the second-generation photosensitizer (PS) devel-oped for the treatment of neoplastic diseases byphotodynamic therapy (PDT), metallo-phthalocyanines(MPc) have been proposed as an alternative to the currentlyused PS in clinical application. Unsubstituted MPc are notsoluble in physiological solvents and their in vivo adminis-tration relies upon their incorporation into carriers or theirchemical conversion into water-soluble dyes by the attach-ment of selected substituents. In this work, uv-vis absorptionand fluorescence spectroscopy were used to study the abilityof different micelles for dispersing zinc phthalocyanine(ZnPc). The following surfactants were tested: SDS, CTAB,HPS, Tween 80, Tween 20, and Pluronic F127. ZnPc has lowsolubility in virtually all solvents, but DMF and DMSO areobserved to dissolve ZnPc in concentrations of the order of0.9 and 0.2 mmol/L, respectively. Stock solutions of ZnPc inDMF and DMSO were prepared.Micelles of the different surfactants containing ZnPc wereprepared by dissolving in aqueous medium (milli-Q water or

phosphate buffer pH 7.4) small amounts of the stock solu-tions previously mixed with each surfactant. The amounts ofeach surfactant were calculated to give an average ratio ofone ZnPc molecule per micelle in the final solution. Theabsorption and fluorescence spectra of ZnPc in the micellarsystems were obtained, and were observed to change intime. Immediately after dissolution the spectra are charac-teristic of monomeric ZnPc, suggesting formation ZnPc-containing nanoemulsions with the mixture of ZnPc-organicsolvent in the hydrophobic region of the micelle. SinceDMSO and DMF are miscible with water, the solvent diffusesout of the micelle and ZnPc stays inside the micelle in amonomeric or aggregated form. The different surfactantslead to different time evolution of ZnPc aggregation. Aggre-gation lifetimes vary from one hour, in the case of PluronicF127, to more than twelve hours, in the case of CTAB andHPS. It was observed that the ionic surfactants were moreefficient than non ionic ones for monomeric delivery of ZnPc .Work partially supported by CNPq, INAMI and FAPERJ.

P-080Nucleobase-containing peptides: a spectroscopic studyof their properties and nucleic acids recognition abilityGiovanni N. Roviello1, Domenica Musumeci1,Enrico M. Bucci1 and Carlo Pedone21Istituto di Biostrutture e Bioimmagini – CNR, ViaMezzocannone 16, 80134 Napoli, Italy, 2Dipartimento delleScienze Biologiche, Universita di Napoli ‘‘Federico II’’, 80134Napoli, ItalyE-mail: giroviel@unina.it

Nucleobase-containing peptides are an important class of mol-ecules comprising both artificial (synthetic nucleopeptides) andnatural (peptidyl nucleosides and willardiine-containing pep-tides) compounds characterized in many cases by interestingbiological properties.1,2 In this work, we report a spectroscopicstudy on the properties of a chiral nucleobase-bearing peptideobtained by chemical synthesis starting from commercialsources. The findings of this research strongly encourage fur-ther efforts in the field of the use of nucleopeptides assupramolecular assembling systemsand open theway to noveldrug delivery approaches based on nucleobase recognition.

1. N. P. Dolman, et al. J. Med. Chem. (2006), 49, 2579.2. H.C. Lee, et al. Arch. Pharm. Res. (2003), 26, 446.

P-081Controlled immobilization and nanoscalesupramolecular assembly of intrinsically disorderedproteinsBarbara Sanavio1, Stefania Corvaglia1,2, Barbara Sorce3,Stefania Sabella3, Pier Paolo Pompa3, Giacinto Scoles4,Loredana Casalis11ELETTRA Synchrotron Light Source, Trieste, Italy,2University of Trieste, Trieste, Italy, 3Italian Instituteof Technology, Center for Bio-Molecular Nanotechnology,Arnesano, Lecce, Italy, 4University of Udine, Udine, Italy

Intrinsically Disordered Proteins are characterized by thelack of a well-defined 3-D structure and show high

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conformational plasticity. Their structure depends tremen-dously on their local environment and confinment, and mayaccommodate several unrelated conformations, that are astrong challenge for the traditional characterizations ofstructure, supramolecular assembly and biorecognitionphenomena. Atomic Force Microscopy (AFM) has beensuccessfully exploited for both highly controllable nanoli-thography of biomolecules and for biorecognition studies,such as oriented prion protein - antibody interaction(Sanavio et al., ACS Nano (2010) 4(11): 6607, Bano et al.Nano Lett (2009) 9(7): 2614-8). Here, we report differentstrategies for selective, oriented confinement of alpha-synuclein at the nanoscale for sensitive and accurate directdetection, via precise topographic measurements on ultra-flat surfaces, of biomolecular interactions in confinedassemblies.

P-082Screening and characterization of membrane-peptideinteractions in new crotamine-derived CPPsAndrea Santos1, Margarida Rodrigues1, Beatriz G. de laTorre2, Gandhi Radis-Baptista3, Miguel Castanho1,David Andreu2, Nuno C. Santos11Instituto de Medicina Molecular, Faculdadede Medicina da Universidade de Lisboa, Lisbon, Portugal,2Departament de Ciencies Experimentals i de la Salut,Universitat Pompeu Fabra, Barcelona, Spain,3Laboratorio de Bioquımica e Biotecnologia,Instituto de Ciencias do Mar, Universidade Federaldo Ceara, Fortaleza, Brazil

A new class of cell penetrating peptides (CPPs) was gen-erated by splicing the (1-9) and (38-42) segments ofcrotamine, a toxin from Crotalus durissus terrificus venom[1]. As they localize preferably on the nucleolus, these novelCPPs were named nucleolar-targeting peptides (NrTPs).The extent of NrTP partition to zwitterionic (POPC;POPC:cholesterol 67:33) and anionic (POPG; POPC:POPG70:30) lipid vesicles was studied following the intrinsic Tyr orTrp fluorescence of the peptides. The partition curves intoPOPC zwitterionic vesicles were characterized by downwardslopes and higher partition coefficients (Kp*104-105). Forpure POPG, an upward curve and smaller partition coeffi-cient point out for a different type of membrane-peptideinteraction. POPC:POPG membranes present characteris-tics of both types of interaction. From red edge excitation shiftand quenching experiments similar conclusions wereattained. Leakage assays ruled out lipid vesicle disruption bycrotamine or NrTPs. Further studies on NrTP cellular trans-location mechanism and large molecule delivery arecurrently in progress.

[1] Radis-Baptista et al., J. Med. Chem. 2008, 51, 7041-4.

P-083Modulation of the interaction and organizationof Human Dystrophin repeats 11-15 subdomainwith MembraneJoe Sarkis1,2, Jean-Francois Hubert1, Baptiste Legrand1,Estelle Robert2, Angelique Cheron1, Julien Jardin3,Eric Hitti4, Elisabeth Le Rumeur1, Veronique Vie2

Universite Europeenne de Bretagne (UEB), 1UMR-CNRS6026 - IFR 140, Equipe RMN-Interactions lipides proteines,Faculte de Medecine, CS 34317, Universite de Rennes 1,2UMR-CNRS 6251, Institut de Physique de Rennes (IPR),Universite de Rennes 1, 3INRA, AGROCAMPUS-OUEST,UMR 1253, Science et Technologie du Lait et de l’œufand 4Laboratoire Traitement du Signal et de l’Image (LTSI),INSERM 642, Rennes, France

Dystrophin is essential to skeletal muscle function and con-fers resistance to the sarcolemma by interacting withcytoskeleton and membrane.We characterized the behaviour of DYS R11-15, a fivespectrin-like repeats from the central domain of humandystrophin, in the presence of liposomes and monolayersas membrane models. Interaction of DYS R11-15 dependson the lipid nature, anionic or zwitterionic, with SUVs, andon the lipid packing when comparing LUVs to SUVs. Lat-eral pressure of lipid monolayers modifies the proteinorganization and leads DYS R11-15 to form a regular net-work as revealed by AFM. Trypsin proteolysis assays showthat the protein conformation is modified following itsbinding to monolayer and SUVs. Label free quantificationby Nano-LC/MS-MS allowed identifying the helical aminoacid sequences in repeats 12 and 13 that are involved inthe interaction with anionic SUVs. Results indicate thatDYS R11-15 constitutes a part of dystrophin that interactswith anionic as well as zwitterionic lipids and adapts itsinteraction and organization depending on lipid-packing andlipid nature.We provide here strong experimental evidence for a physi-ological role of the central domain of dystrophin onsarcolemma scaffolding through modulation of lipid-proteininteractions.

P-084Structural and functional investigation of astaxanthinbinding to the catalytic domain of matrix metalloproteaseMMP-13Ahmed Sayed1, Mervat M. Elsayed2, Paul Tucker3,Karim Fahmy11Div. Biophysics, Institute of Radiochemistry, Helmholtz-Zentrum Dresden-Rossendorf, PF 510119,01314 Dresden,Germany, 2Chemistry Dep., Cairo University, El Gamaastreet, 12613 Cairo, Egypt, 3EMBL-Hamburg c/o DESY,Building 25A, Notkestrasse 85, 22603 Hamburg, GermanyE-mail: a.sayed@hzdr.de

Matrix metalloproteases (MMPs) are zinc-containing prote-ases involved in the remodeling and breakdown of

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extracellular matrix proteins. Overexpression of the MMPshas been associated with a variety of diseases rangingfrom periodontal disease and arthritis to tumor invasionand metastasis. The majority of the more powerful syn-thetic inhibitors of MMPs incorporate a hydroxamate group,but exhibit low selectivity and are toxic. In a recent mod-eling study, Astaxanthin (AST), a carotenoid with potentantioxidant property, has been shown to be a potentialinhibitor of MMP-13 function by occupying a binding sitenear the active center of the enzyme (Bika0di et al. 2006).In our ongoing project, we investigate the binding of ASTto the catalytic domain of MMPs using biochemical andultimately crystallization to validate the proposed action ofAST. Along these lines, the catalytic domain of MMP-13(cdMMP-13) was expressed in E.coli BL21(DE3) Codon-Plus and refolded using a novel effective refolding method.Our results reveal that AST has a potent inhibitory effecton cdMMP-13 activity, however, determination of IC50% orKi is difficult due to fast oxidation and structural instabilityof AST. Ongoing work aims at optimizing the inhibitionconditions and improving the refolding yield to allow ana-lyzing structure and function of the AST-bound MMP-13 inmore detail.

Bikadi et al. (2006) Bioorg Med Chem 14:5451-8.

P-085Macromolecular transport across hyaluronic acidbarriersMichaela Schatz and Claudia SteinemInstitute for Organic and Biomolecular Chemistry, Universityof Gottingen, Tammannstraße 2, 37077 Gottingen, Germany

Hyaluronic acid (Hyaluronan, HA) is a linear polysaccharidewith a molar mass in the range of 105 to 107 Da and is builtfrom alternating units of glucuronic acid and N-acetylgluco-samine. Synthesized in the cellular plasma membrane, it is anetwork-forming and space-filling component in the extra-cellular matrix of animal tissues.Here, we create hyaluronic acid films atop a porous aluminasubstrate, where they act as a barrier for macromoleculartransport depending on their length and geometry. Thegeometry of the hyaluronic acid switches between a fullystretched and a mushroomlike state and is dependent on theconcentration of hyaluronic acid.To bind hyaluronic acid selectively atop the nanoporousanodic aluminum oxide (AAO), the AAO is orthogonallyfunctionalized by silane chemistry. By means of timeresolved optical waveguide spectroscopy (OWS) the trans-port of macromolecules, e.g. avidin, across the hyaluronicacid barriers can be recorded as a function of the porediameter and hyaluronic acid concentration in a timeresolved and label free manner. Confocal laser scanningmicroscopy (CLSM) provides an alternative method toinvestigate the orthogonal functionalization of the pores andto elucidate whether a molecule can cross the barrier at thepore entrance.

P-086Immobilization of ABCC3 with Defined Orientationin an Environment Suitable for Transport StudiesPatrick Seelheim, Isabelle Halstrick, Hans-Joachim GallaDepartment of Biochemistry/Graduate School of Chemistry,University of Muenster Wilhelm-Klemm-Str. 2, 48149Munster, Muenster, GermanyE-mail: patrick.seelheim@uni-muenster.de; gallah@uni-muenster.de

We functionalized gold surfaces with a hydroxy-terminatedself-assembled thiol monolayer exposing an adjustablefraction of biotin moieties.[1] By in-situ acetylation or fluori-nation, surface properties could be fine-tuned to differentprotein immobilization scenarios. Using streptavidin as alinker protein, immobilization of human ABCC3[2] in liposomaland planar bilayer systems was possible.ABCC3-containing proteoliposomes doped with a biotinyla-ted anchor lipid were successfully tethered to ourstreptavidin-coated surfaces.Biotinylation of the extracellular glycosylation of ABCC3allowed direct immobilization with inside-up orientation andsubsequent assembly of a lipid bilayer. Outside-up orienta-tion was achieved by exploiting the C-terminal histidin tag ofrecombinant ABCC3 for immobilization via Ni2+ and biotin-nitrilotriacetate.All systems were thoroughly characterized by quartz crystalmicrobalance, atomic force microscopy and surface plasmonresonance techniques with respect to monitoring ABCC3-mediated substrate transport in real time.

[1] M. Seifert, M. T. Rinke, H.-J. Galla, Langmuir 2010, 26,6386.

[2] B. Zehnpfennig, I. L. Urbatsch, H.-J. Galla, Biochemistry2009, 48, 4423.

P-089Controversial sixth heme-ligand in non native humancytochrome c conformationsMatthieu J Simon, Valerie Metzinger, Olivier Delalande,Arnaud BondonCNRS UMR 6026, RMN-ILP, Universite de Rennes 1,CS 34317 35043 Rennes, Cedex, FranceE-mail: matthieu.simon@univ-rennes1.fr

Because of its role in the apoptotic pathway, conformationaltransitions of cytochrome c (cyt c) have gain on interest.In native cyt c, Met 80 and His 18 residues serve as hemeaxial ligands. Cyt c interaction with the membrane causes thedisruption of the iron–Met80 bond. This allows the binding ofothers endogenous ligands forming alternative low spinspecies1,2 or induces peroxidase activity through the forma-tion of a five coordinated high spin iron specie. Acquisition ofthis peroxydase activity by cyt c has been shown to be a keystage before its release from the mitochondria3.In order to study these non native low spin species bychecking the possible amino acids able to bind the human cytc heme, different mutants have been designed and pro-duced: H26Q, H33N, and the double one H26Q/H33N. SDS

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micelles and pH titration permit us to characterize all thepossible ligands of the cyt c in the alternative low spin form.An original model of the conformational changes of micellarcyt c will be proposed.

1. Oellerich S.; Wackerbarth H.; Hildebrandt P.; Eur.Biophys. J. EBSA 2003, 32: 599-613

2. Chevance S.; Le Rumeur, E.; De Certaines, J. D.;Simonneaux, G.; and Bondon, A.; Biochemistry, 2003,42, 15342-15351

3. Kagan et al; Nat. Chem. Biol. 1, 2005, 1, 223-232.

O-090Unfolding of the cod parvalbumin Gad m 1 allergenby high pressureJudit Somkuti1, Merima Bublin2, Heimo Breiteneder2,Laszlo Smeller11Dept. Biophysics and Radiation Biology, SemmelweisUniversity, Budapest, Hungary, 2Dept. Pathophysiologyand Allergy Research, Medical University Vienna, Vienna,Austria

In countries where seafood is an integrate part of the diet,fish are among the most common food allergen sources. Themajor fish allergen parvalbumins are abundant in the whitemuscle of many fish species. Parvalbumin belongs to thefamily of EF-hand proteins and has a globular shape con-taining six helical parts.High pressure is known to unfold proteins. We performedhigh pressure FTIR experiments, to explore the p-T phasediagram of cod parvalbumin, Gad m 1, to test the possibilityof its inactivation by high pressure treatment.The infrared spectrum of parvalbumin is characteristic for thehelical conformation, in agreement with the crystal structure.A marked transition in the structure of the parvalbumin wasobserved with the central point of 0.5 GPa (at room tem-perature). The amide I position shifts to a wavenumber whichis between the helical and the unfolded position. We assignthis change to a native-molten globule transition. It wasreversible as seen from the infrared spectra.Temperature experiments show a two-step unfolding of theprotein. Above 45 �C we have molten globule structure.Complete unfolding occurs above 70 �C. The unfolding isaccompanied with aggregation of the protein. The aggrega-tion can however be prevented by relatively low pressure of0.2 GPa.

P-091Development of a biomembrane sensor basedon reflectometryMilena Stephan, Andreas JanshoffInstitute of Physical Chemistry, Georg-August Universityof Gottingen, Germany

It has been proven in the past that reflectometric interferencespectroscopy (RIfS) is a powerful measurement system for

the detection of protein-protein interactions1. We presenthere the development of a reflectometric sensor whichallows for the detection of membrane-protein interactions inthe micromolar regime. In this study we employ two differ-ent instrumental assemblies. The first installation enablesdirect detection and quantification of the interaction ofmembrane proteins with solid supported lipid bilayers. Inthe second assembly the original instrument is combinedwith an upright fluorescence microscope. The advantagesof this installation are the direct optical control of theexperiment as well as a smaller sensing area. The set-upallows for the detection of interactions on lipid-patches ofjust several micrometers in diameter. The aim of this work isan experimental system that enables the measurement oftransport processes through lipid membranes. We attemptto achieve this by covering a closed porous substrate with alipid membrane. The first steps to reach this goal were doneby spanning membranes over anodized aluminum oxidesubstrates.

1. Schwartz, M.P.; Alvarez, S. D.; Sailor, M.J.: AnalyticalChemistry 2007, 79, 327-334.

P-092The incorporation of Hypericin and Emodin into artificiallipid membranesA. Strejckova1, J. Stanicova2, G. Bano1,K. Stroffekova1, G. Fabriciova1, P. Miskovsky11Department of Biophysics, P. J. Safarik University in Kosice,Slovakia, 2Institute of Biophysics and Biomathematics,UVMF, Kosice, Slovakia

Hypericin (Hyp) has been studied because of its possibleapplication in photodynamic therapy of cancer. Emodin(Emd) is used as a precursor for Hyp synthesis. The trans-port of Hyp and Emd through the cell membrane is of majorimportance for their delivery into target tumor cells. Hyp andEmd form non-fluorescence aggregates in aqueous solu-tions, but are dissolved in the lipid membrane in theirmonomer form. Black lipid membranes (BLMs) were formedby painting DPhPC onto a hole connecting two chambers ofa Teflon cell. After forming the BLM Hyp or Emd was addedinto one of the chambers. Hyp and Emd monomers incor-porated into the BLM were detected by measuring their auto-fluorescence signal using 532 nm and 405 nm lasers exci-tation, respectively. Characteristic time of the fluorescencesignal increase during the incorporation of Hyp and Emd intothe BLM was found to be approx. 50 and 10s, respectively.This time period has two components. First, there is a diffu-sion time of the studied drug molecules across the boundarylayer near the BLM surface. Second, there is the time needfor the incorporation of the adsorbed molecules into the BLM.This work was supported by the grants of Slovak Ministry ofEducation VEGA No. 1/1154/11, LPP-0290-09, and APVV-0449-07.

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P-093FRET, FCCS and MD-modeling reveal unexpectedhomodimerization of the Fos transcription factor in livecellsN. Szaloki,1 I. Komaromi,3 S. Damjanovich,1

K. Toth,2 G. Vamosi11Cell Biology and Signaling Research Group of the Hung.Acad. Sci. and Dept. of Biophysics and Cell Biology, Medicaland Health Science Center, Univ. of Debrecen, Hungary,2Div. Biophysics of Macromolecules, DKFZ, Heidelberg,Germany, 3Haemostasis, Thrombosis and Vascular BiologyResearch Group of the Hung. Acad. Sci, Clinical ResearchCenter, Medical and Health Science Center, Univ.of Debrecen, Hungary

Fos and Jun, members of the activator protein 1 family oftranscription factors, play a role in the regulation of a widerange of physiological processes and are also known asoncogenes. They belong to the basic-region leucine-zipperproteins binding to DNA as heterodimers. Whereas purifiedJun can also form homodimers (less stable than the het-erodimer), earlier in vitro studies showed that Fos was mainlymonomeric. The instability of Fos homodimers was attributedto the electrostatic and hydrophilic properties of the leucin-zipper.We have previously shown by fluorescence crosscorrelationspectroscopy that Fos and Jun formed stable complexes inHeLa cells and described their C terminal conformation usingFRET and molecular dynamic modeling. Here we giveevidence that Fos can also form homodimers in live cells,and estimate their Kd. FRETefficiency between Fos-CFP andFos-YFP increased with increasing expression level andincreasing acceptor-to-donor ratio up to E*4%. Truncationof the C termini by 165 AA brought the dyes closer togetherresulting in E*10%. FCCS corroborated stable association.MD modeling also indicated that stable homodimers could beformed. Our results introduce Fos homodimers as a newform of AP-1, possibly a new oncogenic form in Fos-over-expressing tumors.

P-094Expression and biophysical characterization of cardiacleiomodin 2David Szatmari, Reka Dudas and Miklos NyitraiUniversity of Pecs, Medical Faculty, Departmentof Biophysics, Pecs, Hungary

Initiation of actin polimerization in cells requires nucleationfactors. A pointed-end-binding protein of F-actin -the lei-omodin2- acts as a strong filament nucleator in muscle cells.The dynamical, structural and kinetic properties of a proteincan provide important information to understand the intra-molecular events underlying its function. We are interested inhow does the leiomodin2 regulate the actin polimerization.Our aim is to determine the dissociation constant of the actin-leiomodin2 complex, and study a possible side-binding effectof the leiomodin2. The cardiac leiomodin2 of Rattus nor-vegicus is a 50 kDa molecular weight protein, which containsa 17 kDa N-terminal, a 18 kDa leucin reach repeat (LRR)and a 15 kDa C-terminal region. The N-term and the LRRregions are together tropomodulin homologues. We

expressed the wild type the N-term+LRR the LRR+C-termand the C-term protein fragments by using a pTyB1 vectorthat contains an amplicillin resistance gene. The expressionof the proteins was carried out with the Twin-CN (NE Bio-Labs) kit, which is a chitin-intein self-cleavage andpurification system. The nucleation activity of leiomodin andthe polimerization speed of actin in the presence of tropo-myosin and leiomodin were studied by using pyrene-actinpolimerization assay. We measured the stoichiometric, con-formational and kinetic properties of the leiomodin-actincomplexes with co-sedimentation assay, fluorescencespectroscopic and rapid-kinetic methods. The resultsshowed that the rate of actin polimerization depended on theleiomodin2 concentration. The nucleator activity of leiomo-din2 was ionic strength dependent. The data also confirmedthat leiomodin2 is a side-binding and pointed-end bindingprotein of F-actin. The binding of leiomodin2 to the sides ofthe actin filaments was slower than to the pointed-end of theF-actin. The structure of F-actin was changed by the side-bound leiomodin2. These observations will contribute to thebetter understanding of the development and function of thinfilaments in cardiac and other muscle tissues.

P-095The influence of doxorubicin transferrin conjugateon the human leukemic lymphoblast cellsMarzena Szwed, Katarzyna Wisniewska, Aneta Rogalska,Agnieszka Marczak, Zofia JozwiakDepartment of Thermobiology, Institute of Biophysics,Faculty of Biology and Environmental Protection, Universityof Lodz, Pomorska 141/143 Street, 90-237 Lodz, Poland

Leukemias are one of the most common malignancy world-wide. There is a substantial need for new chemotherapeuticdrugs effective against these diseases. Doxorubicin (DOX),used for treatment of leukemias and solid tumors is poorlyefficacious when administered systemically at conventionaldoses. Therefore, in our study, to overcome these limitations,we used transferrin (TRF) as a drug carrier. We comparedthe effect of DOX and doxorubicin-transferrin conjugate(DOX-TRF) on human leukemic lymphoblasts (CCRF-CEM).The in vitro growth-inhibition test, XTT assay, indicated thatDOX-TRF was more cytotoxic for leukemia cells than DOXalone.In our researches we also evaluated the alternations ofmitochondrial transmembrane potential (DWm), and pro-duction of reactive oxygen species (ROS). We monitoredthe DWm using dye JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tet-raethylbenzimidazolcarbocyanine). The level of ROS wasstudied using the fluorescent probe DCFH2-DA (2’, 7’-dichlorodihydrofluorescein diacetate). The results demon-strate that DOX-TRF induced, decrease of mitochondrialmembrane potential and significantly higher production ofROS compared with DOX treated cells. Moreover, all theseresults seem to be correlated with DNA fragmentationanalyzed by DNA ladder. The tested processes were par-tially inhibited by the antioxidant, N – acetylocysteine(NAC).The changes induced by DOX-TRF conjugate and free drugwere suggest the different mechanism of action of DOXalone and conjugated with transferrin.

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O-096Time-resolved detection of protein-protein interactionMasahide TerazimaKyoto University, Kyoto, 606-8502, Japan

Revealing molecular mechanism of a protein reaction hasbeen a central issue in biophysics. For that purpose, avariety of time-resolved spectroscopic methods have beendeveloped. However, most of them can monitor onlydynamics associated with an optical transition and it hasbeen very difficult to trace processes without optical tran-sition. We used the pulsed laser induced transient grating(TG) method to study spectrally silent reactions of variousproteins in time-domain. Here we will show studies onPixD.PixD is a 17 kDa short protein which consists of the BLUFdomain and additional short helices, and is involved inphototactic movement. The photochemical reaction studiedby absorption spectroscopy revealed that this proteinexhibits typical photochemistry of the BLUF proteins. Thered-shifted intermediate is generated within a 100 ps. Thespectrum does not change after this initial reaction,and returns back to the dark state with a time constant of12 s at room temperature. We studied the reaction of thisprotein by our method and found that the protein-protein interaction is drastically changed during the reac-tion. The details and the biological meaning will bepresented.

P-097Molecular motions and exchange processes in humanileal bile acid-binding proteinGerg}o Horvath, Gabor Tarkanyi and Orsolya T}okeChemical Research Center of the Hungarian Academyof Sciences, Pusztaszeri ut 59-67, H-1025 Budapest,Hungary

Human ileal bile acid-binding protein (I-BABP) plays a keyrole in the enterohepatic circulation of bile salts. Previouslywe have shown that the protein has two binding sites and tri-and dihydroxy bile salts bind with strong and moderatepositive cooperativity, respectively. Positive cooperativity isthought to be related to a slow conformational change in theprotein.Our current study is directed at the structural and dynamicaspects of molecular recognition in human I-BABP usingnmR spectroscopy and other biophysical techniques. As afirst step in the investigation, 15N relaxation nmR experi-ments have been employed to characterize the backbonemotion in the apo and holo protein on a wide range oftimescales. Our results show a moderately decreased ps-nsflexibility in the ligated protein, with most significant orderingnear the portal region. In addition, the measurements indi-cate a slow ls-ms fluctuation at four distinct segments in theapo protein, a motion not observed in the doubly-ligated format room temperature. Our studies support the hypothesis ofan allosteric mechanism of binding cooperativity in humanI-BABP. To shed more light on the molecular details of thebinding mechanism, a site-directed mutagenesis study is inprogress.

AcknowledgementsThis work was supported by Hungarian GVOP-3.2.1.-2004-04-0210/3.0 and OTKA F68326.

P-098DNA binding of newly synthesized porphyrin-tetrapeptide conjugatesKatalin Toth1, Adam Orosz2, Gabor Mezo3, LeventeHerenyi2, Zsuzsa Majer4, Gabriella Csık21Biophysik der Makromolekule, DKFZ, Heidelberg, Germany,2Institute of Biophysics and Radiation Biology, SemmelweisUniversity, Budapest, Hungary, 3Research Group of PepetideChemstry, HAS, ELTE, Budapest, Hungary, 4Departmentof Organic Chemistry, Eotvos Lorand University, Budapest,Hungary

Cationic porphyrin-peptide conjugates were recently shownto enhance the delivery of peptide moiety to the close vicinityof nucleic acids but their interaction with DNA is not yetstudied. We synthesized two cationic porphyrin-peptideconjugates: Tetra-peptides were linked to the tri-cationicmeso-tri(4-N-methylpyridyl)-mono-(4-carboxyphenyl)porphy-rin and bi-cationic meso-5,10-bis(4-N-methylpyridyl)-15,20-di-(4-carboxyphenyl)porphyrin. DNA binding of porphyrins,and their peptide conjugates was investigated with compre-hensive spectroscopic methods. Evidences provided by thedecomposition of absorption spectra, fluorescence decaycomponents, fluorescence energy transfer and CD signalsreveal that peptide conjugates of di- and tricationic porphy-rins bind to DNA by two distinct binding modes which can beidentified as intercalation and external binding. The peptidemoiety does not oppose the interaction between the DNAand cationic porphyrins.We compared the effect of complexation on structuralstability of DNA and nucleoprotein complex : HeLa nucleo-somes and T7 phage. UV and CD melting studies revealedthat the porphyrin binding increases the melting temperatureof DNA and destabilizes the DNA protein interaction in thenucleosomes but not in the T7 phage.

P-099Nanocalorimetric investigation of nanogels binding sitesIoan Turcu, Izabell Craciunescu, Mihaela Mic,Crina Socaci, Rodica TurcuNational Institute for R&D of Isotopic and MolecularTechnologies, 400293 Cluj-Napoca, Romania

Nanohydrogels are porous nanosized, water-swellable,cross linked macromolecular networks, made from hydro-philic polymers. They possess high water content, and offermany advantages for polymer-based drug delivery systems:i) a tunable size from tens of nanometers to micrometers, ii) alarge surface area appropriate for multivalent bioconjugation,and iii) a functionalized internal network for the incorporationof different drugs in particular, or many other biomolecules ingeneral.Polymeric nanoparticles based on poly(N-isopropylacrylam-ide) (PNIPA) and poly(acrylic acid) (PAA) have beencharacterized by Isothermal Titration Calorimetry (ITC)

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technique. Additionally core shell structures made by com-bining a superparamagnetic core with a polymeric or co-polymeric shell have been also investigated. These materialshave been increasingly exploited as efficient delivery vectors.The types and the number of binding sites N per nanoparticleas well as the corresponding binding constant K, and thereaction enthalpy DH have been obtained for several bio-molecules. If the small molecules are attached bynoncovalent intermolecular interactions the large biomole-cules like proteins bind mainly by electrostatic forces.

P-100Conformational and dynamical changes of AMP.PNP-bound actinKatalin Turmer, Miklos Nyitrai, Denes L}orinczy,Jozsef BelagyiUniversity of Pecs, Faculty of Medicine, Departmentof Biophysics, 12. Szigeti str., Pecs H-7624, Hungary,E-mail: katalin.turmer@aok.pte.hu

Actin monomers generally bind ATP or ADP. Duringthe polymerization of actin monomers into filaments thebound ATP is hydrolysed into ADP and inorganic phosphate(Pi). The biological function of the ATP hydrolysis in actin isunclear yet. Our aim was to shed light on the molecularmechanisms attributed to the hydrolysis process. To achievethese aims we substituted the actin bound ATP with a non-hydrolyzable nucleotide analogue, AMP.PNP, and monitoredthe local and global conformational changes in globular andfilamentous actin during the nucleotide exchange. We usedtwo biophysical methods, electron paramagnetic resonancespectroscopy (EPR) and differential scanning calorimetry(DSC).The results show that the rotational correlation time of theactin bound spin label was increased in case of theAMP.PNP-actin monomers, indicating conformation andflexibility changes in the actin filaments. Parallel to thesespectral changes the thermal transition temperatureobserved in the calorimetric experiments was shifted tohigher temperatures in AMP.PNP-actin. We concluded thatthe nucleotide replacement induced both local and globalconformational and flexibility changes the actin.

P-101Detailed characterisation of Raf-1: Rock2 pathwaycross-talkAndrea Varga1, Oliviero Carugo2, Kristina Djinovic-Carugo2

and Manuela Baccarini11Center for Molecular Biology, University of Vienna, Max F.Perutz Laboratories, Dept. of Microbiology, Immunobiologyand Genetics, Vienna, Austria, 2Max F. Perutz Laboratories,University Departments at the Vienna Biocenter, Departmentfor Biomolecular Structural Chemistry, University of Vienna,Vienna, Austria

Recent conditional mutagenesis studies identified kinaseindependent functions of Raf-1. Among these, its

interaction with the cytoskeletal-based Rho-dependentkinase Rock2 was proven to have a direct role in Ras-driven epidermal tumorigenesis. The mode of this interac-tion is entirely novel, since instead of phosphorylation, adirect protein-protein interaction causes the inhibition ofRock2: the negative regulatory domain of Raf can restrainthe activity of Rock2 kinase domain. In cells, both kinasescan exist as homodimers, but whether Rock homodimer-isation and/or homo/heterodimerisation of Raf arenecessary for the Raf-1:Rock2 interaction is still unknown.We found that Rock dimerisation is dispensable, whereasRaf dimerization is detrimentral for the interaction. Basedon molecular modelling studies, the contribution of possiblesalt-bridges to the interaction was also tested. Produc-tion of soluble proteins for further co-crystallisation studiesis underway in the laboratory, with the long-term aim ofdesigning inhibitors of the interaction. Since Raf-1 isdispensable for epidermal homeostasis, targeting theRaf-1:Rock2 interaction could be a viable strategy forthe molecular (co)therapy of Ras-driven epidermaltumours.

P-102Nucleotide promiscuity of 3-phosphoglycerate kinase:implications for design of anti-HIV compoundsAndrea Varga1, Laurent Chaloin2, Gyula Sagi3, RobertSendula3, Eva Graczer1, Karoly Liliom1, Peter Zavodszky1,Corinne Lionne2 and Maria Vas11Institute of Enzymology, Biological Research Center,Hungarian Academy of Sciences, Budapest, Hungary,2Centre d’etudes d’agents Pathogenes et Biotechnologiespour la Sante (CPBS), UMR 5236, CNRS - UniversiteMontpellier Sud de France, Montpellier, France,3Institute of Biomolecular Chemistry, Chemical ResearchCenter, Hungarian Academy of Sciences, Budapest,Hungary

The wide nucleotide specificity of 3-phosphoglycerate kinase(PGK) allows its contribution to the effective phosphorylation(activation) of nucleotide-based pro-drugs against HIV. Herethe structural basis of the nucleotide-PGK interaction ischaracterised in comparison to other kinases, namely pyru-vate kinase (PK) and creatine kinase (CK) by enzyme kineticand structural modelling studies. The results evidencedfavouring the purine vs. pyrimidine base containing nucleo-tides for PGK rather than for PK or CK. This is due to theexceptional ability of PGK in forming the hydrophobic con-tacts of the nucleotide rings that assures the appropriatepositioning of the connected phosphate chain for catalysis.The unnatural L-configurations of the nucleotides (both pur-ine and pyrimidine) are better accepted by PGK than eitherby PK or CK. Further, for the L-forms the absence of theribose OH-groups with PGK is better tolerated for thenucleotides with purine rather than pyrimidine base. Onthe other hand, positioning the phosphate chain of bothpurines and pyrimidines with L-configuration is even moreimportant for PGK, as deduced from the kinetic studies withvarious nucleotide-site mutants. These characteristics of thekinase-nucleotide interactions can provide a guideline indrug-design.

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P-103Comparison of binding ability and location of twomesoporphyrin derivatives in liposomesDaniel Veres, Barnabas B}ocskei-Antal, Istvan Voszka,Karoly Modos, Gabriella Csık, Andras D. Kaposi, Judit Fidyand Levente HerenyiDepartment of Biophysics and Radiation Biology,Semmelweis University, POB. 263, H-1444 Budapest,Hungary

Application of porphyrins as photosensitizers (PS) is basedon their light-triggered generation of reactive oxygen species(ROS) that may cause oxidative damages and ultimately killcells. Cellular membranes are the action grounds of manysensitizers due to their amphiphilic character as well as thelocation of many of the targets attacked by ROS. Hence, thebinding ability and location of porphyrins in liposomes assimple models of cellular membranes are of outstandinginterest. Here we compare two similar mesoporphyrin (MP)derivatives, namely, MP IX dimethyl ester (MPE) and MP IXdihydrochloride (MPCl). Monocomponent small unilamellarvesicles formed of different phosphatidylcholines with incor-porated MPs were investigated. We determined the bindingparameters by conventional spectroscopy and the inhomo-geneous distribution functions (IDFs) by fluorescence linenarrowing (FLN) spectroscopy. We found that the bindingability of MPE is considerably higher than that of MPCl. IDFsshow three distinct binding sites. Based on a consistentinterpretation at the molecular level the ‘‘site I’’ is between thetwo lipid layers (for MPE), ‘‘site II’’ is deeply between thehydrocarbon chains (for both) and ‘‘site III’’ is betweenthe head groups (for MPCl).

P-104The role of the enzyme types ATP-ases in the musclecontractionG. Vincze-Tiszay1, J. Vincze1, E. Balogh21HHEIF, Budapest, Hungary, 2Nove Zamky, Slovakia

The myofibrilla assuring muscle contraction gains energy tothe slipping in mechanisms and the degree of efficiency ofthis process will decisively be determined by the velocity ofrecombination of the ATP molecule. In this there play aparticular part the Na+–K+–ATP-ase and Mg++–ATP-aseenzymes. Chemical reactions taking place in the livingorganism are catalyzed by enzymes, so the recombinationfrom ADP to ATP, too. This transport process can be mod-elled from the energetic point of view on the basis of thegeneral transport theorem through the following formula:

W ¼ K

Zt2

t1

Zx2

x1

grad ax dx dt:

From the point of view of muscle contraction it is of interestthat, dependent from the type of the motions whether thelength of time is very short, some seconds, or we can speakabout a long lasting process. In the first case one cancompare the decomposition of the ATP with the avalancheeffect while in the spot. Its degree of efficiency is determinedby the migration and linkage velocity of the ions. Conclusion:

The degree of efficiency of the muscle contraction isdetermined by the quantities of the two enzymes (Na+–K+–ATP-ase and Mg++–ATP-ase) as related to each other.

P-105Development of new fluorescent probes for amyloiddetectionK. O. Vus1, V. M. Trusova1, E. Kirilova2, G. Kirilov2,I. Kalnina21V.N. Karazin Kharkiv National University, 4 Svobody Sq.,Kharkiv, 61077, Ukraine, 2Department of Chemistryand Geography, Faculty of Natural Sciencesand Mathematics, Daugavpils University, 13 Vienibas,Daugavpils LV5401, Latvia

Due to a direct correlation between protein misfolding andneurodegenerative disorders, the development of smallorganic molecules for the detection and inhibition of fibrilformation is of great importance. A set of prospective dyes,recently synthesized by a nucleophilic substitution of thebromine atom in 3-bromobenzanthrone, have been tested fortheir specificity to amyloid, pre-fibrillar and native amyloido-genic hen egg white lysozyme by analyzing their bindingisotherms (i. e. lysozyme-induced changes of the dyes’fluorescence) in terms of Langmuir adsorption model. Basedon the obtained data we concluded that the majority of newprobes could be suitable alternatives for preliminary fluo-rescent detection of both mature and immature fibrils,showing stronger affinity for aggregated protein compared tothe commercially available classical amyloid marker Thio-flavin T. Moreover, these fluorophores showed very weakbinding to the native protein, as judged from the recoveredlow association constants. Although all dyes share resem-bling spectral properties and structure, they are supposed tohave different fibril binding sites because of the different rateof water relaxation in the immediate vicinity of eachfluorophore.

P-106Fetuin-A mediated formation and ripening of colloidalcalciprotein particlesJohanna Wald1, Susanne Wiese1, Thomas Eckert1,Willi Jahnen-Dechent2, Alexander Heiss3,Walter Richtering11Institute of Physical Chemistry,2Helmholtz Institutefor Biomedical Engineering, Biointerface Group,3CentralFacility for Electron Microscopy,RWTH Aachen University, Aachen, Germany

Extracellular fluids of vertebrates are supersaturated withregard to calcium and phosphate. Hence, any tissue isprone to extensive mineral deposition unless inhibitors arepresent. On protein level, fetuin-A is supposed to be one ofthe most important factors [1]. Experiments with deletionmutants have shown that the aminoterminal domain con-tains a beta sheet with an ordered array of acidic residues,which mediates the attachment to basic calcium phosphates[2, 3].

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The inhibition is based on the formation of nanometer-sized,spherical mineral-fetuin-A colloids, denoted as CalciproteinParticles (CPPs) [2, 4]. The initially formed CPPs showhydrodynamic radii in the range of 50 nm and are onlytransiently stable. After a distinct lag time, they are subject toa morphological change towards larger prolate ellipsoids withhydrodynamic radii of 100-120 nm [5].In this context, we studied the role of fetuin-A in the formationand ripening of CPPs. On the one hand, dynamic lightscattering (DLS) was used to study the influence of tem-perature, fetuin-A concentration and mineral ionsupersaturation on the kinetics of CPP ripening [6]. On theother hand, the protein fetuin-A was investigated by means ofsmall angle X-ray scattering (SAXS) and fluorescence cor-relation spectroscopy (FCS).

[1] Ketteler, M., Bongartz, P. et al. (2003). Lancet 361:827-833

[2] Heiss, A., A. DuChesne, et al. (2003). Journal ofBiological Chemistry 278(15): 13333-13341.

[3] Jahnen-Dechent, W., C. Schafer, et al. (2001). Zeits-chrift fur Kardiologie 90(3): III/47-III/56.

[4] Price, P.A., Lim, J.E. (2003). Journal of BiologicalChemistry 278: 22144-22152

[5] Heiss, A., T. Eckert, et al. (2008). Journal of BiologicalChemistry 283(21): 14815-14825.

[6] Wald, J., S. Wiese, et al. (2011). Soft Matter 7:2869-2874.

O-107Experimental physics’ resources for studying biologicalmacromolecules; deep insight into DcpS enzymatichydrolysis of short capped mRNAsAnna Wypijewska, Elzbieta Bojarska, Maciej Lukaszewicz,Janusz Stepinski, Jacek Jemielity andEdward DarzynkiewiczDivision of Biophysics, Institute of Experimental Physics,Faculty of Physics, University of Warsaw, Warsaw, Poland

Degradation of the mRNA cap (mRNA 5’ end) by DcpS(Decapping Scavenger) enzyme is an important process ofthe gene expression regulation, but little is known about itsmechanism. The biological role of DcpS and its potentialtherapeutic applications, e.g. as a novel therapeutic target forspinal muscular atrophy, make it an interesting object forbiophysical investigations. The ability of DcpS to act onvarious short capped mRNAs will be presented. We haveexamined the substrate specificity and binding affinity of theenzyme in a quantitative manner, employing experimentalphysics’ resources, such as atomic force microscopy (AFM)and fluorescence spectroscopy for enzyme kinetics and time-synchronized-titration method (TST). Applied approachresulted in several conclusions about DcpS substrate rec-ognition. For the first time, stereoselectivity of the hydrolysisprocess catalysed by DcpS will be demonstrated. Presenteddata clearly point out the constitutive features for designingthe selective inhibitors of processes in which DcpS enzymeis involved. Detailed characteristics of the techniques intro-duced in biophysical characteristic of DcpS may beindispensable for other nucleotide binding proteins

investigation, as well as for studies of many other kinds ofmacromolecules.

P-108Antifungal activity of peach defensin involves plasmamembrane permeabilizationM. Zanetti1, V Nanni2, E. Baraldi2, C. Moser3

and M. Dalla Serra11Istituto di Biofisica, Consiglio Nazionale delle Ricerche &Fondazione Bruno Kessler, Povo (TN), Italy, 2Laboratorio diBiotecnologie - Diproval-Universita di Bologna, Bologna,Italy, Fondazione Edmund Mach, 3IASMA Researchand Innovation Centre, S. Michele a/A (TN), Italy

Plant defensins are small cysteine-rich antimicrobial peptidesoccurring in various plant species. They share a commonthree-dimensional structure, stabilized by eight disulphide-linked cysteines and composed of three antiparallel b–strands and one a–helix. Most plant defensins possessantifungal or antibacterial activity but are non-toxic to mam-malian and plant cells. Plant defensins induce membranepermeabilization, resulting in Ca2+ uptake, K+ efflux, alka-linization of the medium, and membrane potential changes.The gene encoding for a peach (P. persica) defensin PpDfn1was expressed and purified in E. coli. Defensin was tested forantimicrobial activity against fungi by Sytox Green assays.Data revealed that PpDfn1 has a inhibitory effect on sporegermination and acts through membrane permeabilization.Biophysical analysis demonstrated that this protein was ableto interact with model and natural membranes. Binding ofdefensin to lipid membranes was dependent on lipid compo-sition and surface charge. Interaction with fungal membranelipid components leads to the insertion of peach defensin intothe membrane resulting in membrane destabilization.Sponsored by Bando per progetti di ricerca scientifica ap-plicata Fondazione Cassa di Risparmio di Trento e Rovereto

P-109Quantum Dot Multispectral Imaging Detects CellSignaling for Prostate Cancer ProgressionHaiyen E. Zhau1, Peizhen Hu1, Guodong Zhu2, RuoxiangWang1, Dror Berel3, Yuzhuo Wang4, Ladan Fazli5, DanielLuthringer6, Andre Rogatko3, and Leland W. K. Chung11Uro-Oncology Research Program, 2Department of Urology,School of Medicine, Xi’an Jiaotong University, Xi’an, China,3Biostatistics & Bioinformatics, Department of Medicine;6Department of Pathology, Samuel Oschin ComprehensiveCancer Institute, Cedars-Sinai Medical Center; Los Angeles,CA, USA, 4BC Cancer Agency, University of BritishColumbia, 5Molecular Pathology Lab of the VancouverProstate Center at Vancouver General Hospital, Vancouver,B.C., Canada

Background: Immunohistochemistry (IHC) color imagingand pathological evaluation of clinical specimens have beenthe standard practice in pathology for cancer diagnosis anddetection. IHC true color imaging suffers from the inheritedcomplications of photobleaching and autofluorescence. Itcannot detect multiple biomarkers at a time on the samespecimen due to the inability to un-mix the signals. A more

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dynamic detection technology is needed. We developed amultiplexed quantum dot-IHC (mQD-IHC) protocol that candetect cell signaling in cells for their epithelial to mesenchy-mal transition (EMT) (Xing, et al. Nat Protoc. 2 (5):1152-65,2007). In this study we extended the application of mQD-IHCto investigate potential biomarkers associated with prostatecancer (PCa) invasiveness and lethal progression.Objectives: To establish a mQD-IHC protocol using QDlight-emitting nanoparticles 1) to detect the expression/acti-vation of critical cell signaling proteins at the single cell level;2) to image the plasticity and lethal progression of humanPCa with specific emphasis on EMT and c-MET signaling;and 3) to examine the utility of mQD-IHC in clinical PCaspecimens to determine its invasion ability and predict itsmetastatic capability.Results: We analyzed the co-expression and activation ofosteomimicry associated biomarkers: b2-Microlgobulin (b2-M), phosphorylated cyclic AMP responsive element bindingprotein (pCREB) and androgen receptor (AR) in 2,100 cellsfrom 14 localized PCa tissue areas (Gleason 3 and 4) of 10patients with known metastatic status. The overall median %triple positive for b2-M+/pCREB+/AR+ cells was 51.5%. Themedian triple positive for the samples with metastatic potentialwas 61% compared with those without metastatic potential(median = 0%); p = 0.01 by a Wilcoxon rank sum test. Theresults were confirmed in 11 PCa bone metastatic specimens.We also investigated the c-MET signaling in castration-resis-tant human PCa model or CRPC xenografts and the clinicalPCa specimens and found that the downstream signal com-ponents including pAkt and Mcl-1 were activated.Conclusion: To validate our findings, additional clinicalspecimens with confirmed survival data will be analyzed andthe cell-signaling-network-based mQD-IHC will be auto-mated by Vectra Image Analysis System in a high throughputmanner with the hope to predict the lethal progression of PCaprior to clinical manifestation of distant metastases.

P-110Towards intrinsic binding thermodynamicsAsta Zubrien _e, Egidijus Kazlauskas,Vilma Michailovien _e, Daumantas MatulisLaboratory of Biothermodynamics and Drug Design, VilniusUniversity Institute of Biotechnology, LT-02241, Vilnius,Lithuania

Protein ligand binding is an important field of biopharma-ceutical research. There are many techniques for quantitativedetermination of the ligand binding. The combination of iso-thermal titration calorimetry (ITC) and thermal shift assayprovides a robust estimate of the binding constant.Many binding reactions are coupled to the absorption orrelease of protons by the protein or the ligand, conforma-tional changes of the protein and other processes. Tocorrelate the structural features of binding with the energeticsof binding one needs to carry out a detailed thermodynamicstudy of the binding reaction and to determine dependenciessuch as pH, buffer and temperature.Here we present a detailed thermodynamic description ofradicicol binding to human heat shock protein Hsp90 anddetermined proton linkage contributions to observed bindingthermodynamics. We calculated the pKa of the groupresponsible for proton linkage, the protonation enthalpy of

this group and intrinsic thermodynamic parameters for rad-icicol binding. The intrinsic enthalpy of radicicol binding toHsp90 is one of the largest enthalpies observed for anyprotein – ligand binding. The structural features responsiblefor such large binding enthalpy and very favorable intrinsicbinding Gibbs free energy are discussed.

Neuronal systems and modelling

O-111Optogenetic electrophysiologyWalther Akemann, Amelie Perron, Hiroki Mutoh,and Thomas KnopfelLaboratory for Neuronal Circuit Dynamics, RIKEN BrainScience Institute, Japan

The combination of optical imaging methods with targetedexpression of protein-based fluorescent probes enables thefunctional analysis of selected cell populations within intactneuronal circuitries. We previously demonstrated optoge-netic monitoring of electrical activity in isolated cells, brainslices and living animals using voltage-sensitive fluorescentproteins (VSFPs) generated by fusing fluorescent proteinswith a membrane-integrated voltage sensor domain. How-ever, several properties of these voltage reporters remainedsuboptimal, limiting the spatiotemporal resolution of VSFP-based voltage imaging. A major limitation of VSFPs hadbeen a reduced signal-to-noise ratio arising from intracellularaggregation and poor membrane targeting upon long-termexpression in vivo. To address this limitation, we generated aseries of enhanced genetically-encoded sensors for mem-brane voltage (named VSFP-Butterflies) based on a novelmolecular design that combines the advantageous featuresof VSFP2s and VSFP3s with molecular trafficking strategies.The new sensors exhibit faster response kinetics at sub-threshold membrane potentials and enhanced localization toneuronal plasma membranes after long-term expression invivo, enabling the optical recording of action potentials fromindividual neurons in single sweeps. VSFP-Butterflies pro-vide optical readouts of population activity such as sensory-evoked responses and neocortical slow-wave oscillationswith signal amplitudes exceeding 1% DR/R0 in anesthetizedmice. VSFP-Butterflies will empower optogenetic electro-physiology by enabling new type of experiments bridgingcellular and systems neuroscience and illuminating thefunction of neural circuits across multiple scales.

References:

1. Dimitrov, D., He, Y., Mutoh, H., Baker, B.J., Cohen, L.B.,Akemann, W., Knopfel, T. (2007) Engineering andcharacterization of an enhanced fluorescent proteinvoltage sensor. PLoS ONE 5: e440, 1-5. [*** first reportof VSFPs based on the Ciona Intestinalis voltage sensordomain ***]

2. Akemann, W., Mutoh, H., Perron, A., Rossier, J.,Knopfel, T. (2010) Imaging brain electric signals withgenetically-targeted voltage-sensitive fluorescent pro-teins. Nature Methods 7(8): 643-649. [*** first report ofVSFPs based voltage imaging in living mice ***]

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P-113Biophysical visual virtual reality in retinotopic visualareasBokkon I.1,2, Tuszynski J. A.3,4, Salari V.5,61Doctoral School of Pharmaceutical and PharmacologicalSciences, Semmelweis University, Budapest, Hungary,2Vision Research Institute, 25 Rita Street, Lowell, MA 01854USA and 428 Great Road, Suite 11, Acton, MA 01720 USA,3Department of Experimental Oncology, Cross CancerInstitute, 11560 University Avenue, Edmonton, AB T6G 1Z2,Canada, 4Department of Physics, University of Alberta,Edmonton, AB Canada, 5Kerman Neuroscience ResearchCenter (KNRC), Kerman, Iran, 6Afzal ResearchInstitute, Kerman, IranE-mail: bokkoni@yahoo.com

Previously, we have pointed out that biophoton productioncan be a controlled process that originates from regulatedredox/radical reactions. Our biophoton experiments supportthe notion that various visual related phenomena such asdiscrete retinal noise, retinal phosphenes as well as negativeafterimages are due to biophotons. We have also suggesteda new model, stating that the brain is able to create bio-physical pictures in retinotopic visual areas via redoxregulated biophotons of synchronized neurons. According toour interpretation, visualization (imagery) is a special kind ofrepresentation i.e., visual imagery requires peculiar inherentbiophysical processes. Our idea of biophysical visual virtualreality in retinotopic areas may be a possible biophysicalbasis of Kosslyn’s reality simulation principle in the case ofvisual imagery. Long-term visual memories are not stored asbiophysical pictures but as epigenetic codes. During visualimagery, top-down processes control the epigenetic encodedlong-term visual information. Then, according to retrievedepigenetic information, synchronized retinotopic neuronsgenerate dynamic patterns of biophotons via redox reactionsthat can produce biophysical pictures. We have also pre-sented an iterative model involving a biophysical picture-representation without homunculus during visual imagery.

O-114Opsin stability under varying stimulation conditionsEmily A. Ferenczi1,2, Joanna Mattis1,2, CharuRamakrishnan1, Kay M. Tye1, Daniel J. O’Shea1,2,Ofer Yizhar1,2, Karl Deisseroth1,2,31Dept Bioengineering, 2Neurosciences Program, StanfordUniversity, Stanford, CA, USA, 3Howard Hughes MedicalInstitute, USA

Opsin molecules are a burgeoning new tool for temporallyprecise neuronal stimulation or inhibition. Opsin propertiesare commonly characterized in cell culture or acute brainslice preparations using whole cell patch clamp techniques,where neuronal membrane voltage is fixed at the restingpotential. However, in vivo, where neurons are firing actionpotentials, opsins are exposed to large fluctuations inmembrane voltage and transmembrane ionic concentrationswhich can influence opsin function. In the case of implantedlight delivery devices, stimulation light power varies as afunction of brain tissue volume. We therefore investigated thestability of opsin properties across a variety of in vivo-likestimulation conditions.

We find that off-kinetics of excitatory opsins vary significantlywith holding membrane potential; channelrhodopsin (ChR2)slowing with depolarisation and ChIEF (ChR1/ChR2 hybrid)in contrast, accelerating. New ChR2 point mutation variantsdemonstrate stability across all membrane potentials. Weadditionally explore responses to initial and subsequent lightpulses and find that ChIEF has the unique property ofaccelerating kinetics after the first light stimulation. Inhibitoryopsins vary in sensitivity to light in a manner which correlateswith their off-kinetics. Slower opsins, such as Mac (Lep-tosphaeria maculans), have higher sensitivity at low lightpower densities, saturating early relative to fast inhibitoryopsins such as Arch (archaeorhodopsin) and NpHR (halo-rhodopsin). We discuss the relative merits of stability versusversatility of opsins under variable stimulation conditions.

P-115Overexpression of NDM29 ncRNA induces neuron-likeproperties in neuroblastoma cellsPaola Gavazzo1, Serena Vella2,3, Carla Marchetti1,Mario Nizzari2, Ranieri Cancedda2,3, Aldo Pagano2,31Institute of Biophysics, CNR, Genoa, Italy, 2Oncology,Biology and Genetics Department, University of Genoa,Genoa, Italy, 3National Institute for Cancer Research, Genoa,Italy

It has been previously shown that overexpression of NDM29ncRNA in a SKNBE2-derived neuroblastoma (NB) cell lineleads to cell differentiation, with a decrease of malignantpotential. Here we use the patch-recording technique tocharacterize the ionic channel apparatus of NB cellsexpressing NDM29 at its basal level (Mock cells) or at 5.4fold higher levels (S1 cells). The two cell lines shared verysimilar pools of functional K channels, but S1 cells displayedlarger TTX-sensitive Na currents and were able to generateaction potentials, while Mock cells were not. In addition, whileMock cells barely express functional GABA receptors, in themajority of S1 rapid application of GABA elicited a currentwith a EC50=11.4 lM; this current was antagonized bybicuculline (10 lM) and potentiated by zaleplon (EC50= 35nm). In Mock cells, real time PCR evidenced a high level ofGABAA a3 subunit, while in S1 cells a significant expressionof a1 and a4 was detected, whereas a3 mRNA was down-regulated by 70%, confirming the development of functionalGABAA receptors. In the same cell lines, the presence ofspecific markers and the secretion of specific cytokinesconfirmed that NDM29 expression leads to a differentiationprocess toward a neuron-like, rather than glial-like,phenotype.

P-116Novel human glioma model in rat brainsV.M. Kavsan1, V.P. Baklaushev2, O.V. Balynska1,G.M. Yusubalieva2, N.Ph. Grinenko2, I.V. Victorov2,V.P. Chekhonin21Institute of Molecular Biology and Genetics, Kyiv, Ukraine,2V.P. Serbsky National Research Centre for Socialand Forensic Psychiatry, Moscow, Russia

Rodent models of human cancers have been instructionalin understanding the basic principles of cancer biology. It

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was planned therefore to reconstitute a model of braintumors in rats by orthotopic implantation of xenogenictransformed human cells. Among the genes with the mostpronounced increasing expression in tumors we revealedCHI3L1, encoding the secreted CHI3L1 protein. Overex-pression of CHI3L1 resulted in increased cell proliferation,anchor-independent growth, and formation of tumors. Twomain cellular signaling passways, MAPK and PI3K, wereidentified to be upregulated in the CHI3L1 expressing 293cells. To obtain a novel glioma model, 293 cells, whichstably expressed CHI3L1 were stereotactically implanted inthe striatum of female Wistar rats. Huge intracerebraltumors were observed in each of the rats under investi-gation. All tumors were surrounded by numerous GFAP-positive reactive astrocytes what is observed in tumors,initiated by high grade glioma cultured cells. Tumor for-mation by 293 cells stably expressing CHI3L1 in ratbrains strongly suggests that CHI3L1 is involved intumorigenesis. This model is going to be very useful fordeveloping systems of oncoprotein synthesis targetedinhibition in glial tumor cells with minimal side effects onthe normal cells.

O-117Hematite particles in human brainM. Kopani1,2, M. Caplovicova3, M. Melnik2,V. Sisovsky1, J. Jakubovsky11Institute of Pathology, Faculty of Medicine, ComeniusUniversity, Bratislava, Slovakia, 2 St. Elizabeth Universityof Health and Social Sciences, Bratislava, Slovakia,3Department of Geology of Mineral Deposits, Facultyof Natural Sciences, Comenius University, Bratislava,Slovakia

Iron is important element used for chemical reaction catalysisand physiological cell functions. The reason of iron deposi-tion is still unknown. Under conditions prevailing in humanbrain it is expected the formation of an amorphous or minutecrystalline phase. We used light, scanning (SEM) andtransmission electron microscopy (TEM), energy-dispersivemicroanalysis, electron diffraction and electron paramagneticresonance (EPR) for investigation of iron deposits in globuspallidus of human brain. SEM revealed iron rich particles withNa, Si, P, S, Cl, Ca and Cu around glial cells. TEM revealedbumpy, solid particles of platy and sometimes rounded shapewith the size of 2 lm to 6 lm. These ones were identified ashematite. EPR measurements showed the presence ofFe(III) and Cu(II), but little amount of Fe(II) can not beexcluded. We consider low-temperature process of hematiteformation in human globus pallidus in aqueous environmentinfluenced by organic and inorganic factors. Chemical pro-cesses leading to nanoparticles formation can be associatedwith neurodegenerations such as Alzheimer or Parkinsondisease.

O-118Neuronal biophysics: optimised for informationprocessing?Mate Lengyel, Peter Dayan, Balazs Ujfalussy,Jean-Pascal PfisterUniversity Of Cambridge, Department of Engineering,Cambridge, UK

Over the past 50 years our understanding of the basic biophysicalmechanismsgoverning the spatio-temporal dynamics of neuronalmembrane potentials and synaptic efficacies has significantlyexpanded and improved. Much research has focussed on howionic currents contribute to the generation and propagation ofaction potentials, how subthreshold signals propagate alongdendritic trees, how the active properties of dendrites shape theintegration of incoming signals in a neuron, and how pre- andpostsynaptic activities a and potential heterosynaptic effects adetermine the way synaptic efficacies change on the short- andlong-term. Yet, despite these advances, there have been no sys-tematicefforts to relate thebasicdynamical repertoireofneurons tothe computational challenges neural circuits face, and in particulartoexplainsystematicallyhowthebiophysicalpropertiesofneuronsareadapted toprocess informationefficientlyunder theconstraintsof noise anduncertainty in the nervous system.Asan initial step inthis direction, I will show how various biophysical properties ofneurons, in particular short-term synaptic plasticity and dendriticnon-linearities, canbeseenasadaptations to resolvean importantbottleneck in neuronal information processing: the loss of infor-mation entailed by the conversion of analogue membranepotentials to digital spike trains.

O-119An analysis of new and existing opsins for scientificapplicationJoanna Mattis1, Kay M. Tye1, Charu Ramakrishnan2,Daniel J. O’Shea1, Lisa A. Gunaydin1, Emily A. Ferenczi1,Minsuk Hyun1, Lief Fenno1, Viviana Gradinaru1,Ofer Yizhar2, Karl Deisseroth1,21Neuroscience Program, 2Dept. of Bioengineering, Stanford,Palo Alto, USA

The optogenetic toolbox has greatly expanded since thefirst demonstration of genetically-targeted optical manipula-tion of neural activity. In addition to the cation channelchannelrhodopsin-2 (ChR2), the panel of excitatory opsinsnow includes an array of ChR2 variants with mutations incritical residues, in addition to other, related cation channels,and channel hybrids. The inhibitory opsin panel has similarlyexpanded beyond the first-described halorhodopsin (NpHR),a chloride pump, to include trafficking-enhanced versions ofNpHR as well as the proton pumps Mac and Arch.While the expansion of available opsins offers researchers anincreasingly powerful and diverse selection of tools, it has alsomade it increasingly difficult to select the optimal tool for agiven experiment. One cannot extract a comparison of opsinsfrom the current literature, since studies differ across multiplevariables known to contribute to opsin performance (e.g.expression method, light power density, stimulation protocols,etc.). Here, we provide the first empirical comparison of both

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excitatory and inhibitory opsins under standardized condi-tions. Furthermore, we identify the set of parameters thatdescribe the properties of an opsin in a way that is maximallyrelevant for biological application.

O-120Subcellular compartment-specific distributionof voltage-gated ion channelsZoltan NusserInstitute of Experimental Medicine, Hungarian Academyof Sciences, Budapest, Hungary

Voltage-gatedNa+ (Nav) channels are essential for generatingthe output signal of nerve cells, the action potential (AP). Inmost nerve cells, APs are initiated in the axon initial segment(AIS). In vitro electrophysiological and imaging studies havedemonstrated that dendritic Nav channels support activebackpropagation of APs into the dendrites, but the subunitcomposition of these channels remained elusive. Here, I willpresent evidence for the somato-dendritic location of Navchannels in hippocampal pyramidal cells (PCs). Using a highlysensitive electron microscopic immunogold localization tech-nique, we revealed the presence of the Nav1.6 subunit in PCproximal and distal dendrites, where their density is 40-foldlower than that found in AISs. A gradual decrease in Nav1.6density along the proximo-distal axis of the dendritic tree wasalso detected. We have also investigated the subcellular dis-tribution of Kv4.2 voltage-gated K+ channel subunit and founda somato-dendritic localization. In contrast to that of Nav1.6channels, the density of Kv4.2 first increases then decreasesas a function of distance from the somata of PCs. Such sub-cellular compartment-specific distribution of voltage-gated ionchannels increases the computational power of nerve cells.

P-121Semantic versus syntactic: the role of the extracellularmatrix on memoryNathalie Olivi-Tran1,2, Sandra Kanani3, Ian Robbins41Universite Montpellier 2, Laboratoire Charles Coulomb UMR5221, F-34095,Montpellier, France, 2CNRS, LaboratoireCharles Coulomb UMR 5221, F-34095, Montpellier, France,3L’Oreal, Paris, France, 4IGMM, UMR-CNRS 5535, 1919route de Menden 34293 Montpellier cedex 5, France

Keywords: Memory, extra cellular matrix, random walkWe expose first a biological model of memory based on onehand of the mechanical oscillations of axons during actionpotential and on the other hand on the changes in the extracellular matrix composition when a mechanical strain isapplied on it. Due to these changes, the stiffness of the extracellular matrix along the most excited neurons will increaseclose to these neurons due to the growth of astrocytesaround them and to the elastoplastic behavior of collagen.This will create preferential paths linked to a memory effect.In a second part, we expose a physical model based onrandom walk of the action potential on the array composed ofdendrites and axons. This last model shows that repetition ofthe same event leads to long time memory of this event andthat paradoxical sleep leads to the linking of different eventsput into memory.

Calcium fluxes, sparks, & waves

P-122Fluorescence properties of quercetin in human leukemiaJurkat T-cellsI. Baran1, C. Ganea1, I. Ursu2, V. Baran2, 3,O. Calinescu1, A. Iftime1, R. Ungureanu1, I.T. Tofolean11‘‘Carol Davila’’ University of Medicine and Pharmacy, Dept.of Biophysics, 8 Eroii Sanitari, 050474 Bucharest, Romania,2IFIN-HH, 407 Atomistilor Str., 077125 Magurele, Bucharest,Romania, 3 Physics Faculty, University of Bucharest,Magurele, Bucharest, Romania

We describe the fluorescent properties of quercetin (QC) inhuman leukemia Jurkat T-cells. The intracellular QC spectradisplayed a dominant emission maximum at 540 nm and 2excitation maxima (380 nm, 440 nm). Our data suggest thatQC elicits a specific fluorescence upon binding to intracel-lular proteins and that there are two dominant molecularconfigurations of these QC-bound proteins which are sensi-tive to the intracellular Ca2+ concentration. QC fluorescenceincreased after permeabilization of the cells with digitonin,suggesting that intracellular binding of QC may be down-regulated by a cytosolic factor which is lost bypermeabilization. We performed also spectrofluorimetricmeasurements of the intracellular Ca2+ concentration whichrevealed a strong biphasic calcium release signal after cellsstimulation with 50 lM QC. QC also decreased considerablythe intracellular NAD(P)H level in intact cells. Taken together,these data add more evidence for the involvement of Ca2+

and NAD(P)H in the apoptotic pathway triggered by QC inJurkat cells.

AcknowledgementsThis work is partially supported by the Sectoral OperationalProgramme Human Resources Development (SOP HRD),financed from theEuropeanSocial Fund and by theRomanianGovernment, contract number POSDRU/89/1.5/S/64109.

P-123Changes in myelinated axons after membranemodificationBibineysvili E.Z., Brazhe A.R.,Yusipovich A.I. Maksimov G.V.Biophysics Department, Biological Faculty, Moscow StateUniversity, Moscow, Russia

Myelinated nerve fibres were studied with fluorescentmicroscopy and laser interference microscopy. Ca2+

redistribution during prolonged stimulation, changes in mor-phology and rearrangement of cytoplasmic structures werecompared in normal conditions and after membrane modifi-cation by lysolecithin and methyl-b-cyclodextrin.Lysolecithin is a detergent known to provoke demyelination,and methyl-b-cyclodextrin extracts cholesterol from mem-branes. Cholesterol extraction could lead to disruption ofmembrane caveolae-like microdomains or ‘‘rafts’’ and solu-bilisation of different proteins connected to them. Our datasuggest that methyl-b-cyclodextrin and lysolecithin lead todifferent changes in morphology and distribution of cytoplas-mic structures. The effect was different for different regions ofthe nerve (node of Ranvier, paranodal and internodal regions).The agents also altered the kinetics of Ca2+ response tostimulation in myelinated fibres.

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P-124Extracellular carbonic anhydrase contributesto the regulation of Ca2+ homeostasis and salivationin submandibular salivary glandNataliya Fedirko, Olga Kopach, Nana, VoitenkoLviv National University, Human and Animal Physiology, Lviv,Ukraine

The maintenance of pH in the oral cavity is important for theoral heath since even a minor drop in pH can result indental caries and damage to the teeth. Submandibularsalivary gland (SMG) is main source of fluid and electrolytesenriched saliva therefore its core for oral pH homeostasis.SMG secretion is activated by acetylcholine (Ach) in [Ca2+]i-dependend manner and accompanied with oral pH acidicshifts. pH shifts could be due to changes in bufferingcapacity that is regulated by carbonic anhydrase (CA).Despite the expression of different subtypes of CA in SMGthe role of CA in the regulation of SMG function is unclearyet. We found that CA inhibition by benzolamide (BZ)decreased of fluid secretion in vivo extracellular Na2+ con-centration in situ. The latter confirm the ability of CA tomodify both primarily and final saliva secretion. We alsofound correlation between the secretion and Ca2+-homeo-stasis since BZ-induced decrease of: i) total resting calciumcontent and stimulating effect of ACh; ii) activity of endo-plasmic reticulum Ca2+-ATPase; iii) mitochondrial Ca2+-uptake. Thus CA contributes to saliva pH via cytosolic Ca2+

handling in SMG acinar cells and that this effect is mediatedvia cholinoreceptors.

O-125A simple model to describe single and multichannelcalcium regulation of ryanodine receptorsMichael J. Yonkunas, Michael Fill, Dirk GillespieRush University Medical Center, Chicago, USA

In cardiac muscle, type 2 ryanodine receptor (RyR2) func-tioning relies on cellular Ca2+ levels from both backgroundcytosolic Ca2+ and from neighboring open RyRs. Activationof RyR2 is brought on by calcium-induced-calcium release(CICR), but the CICR termination mechanism remainsunknown. A simple physical model is developed that is ableto describe the Ca2+ dependence of a single RyR2 and amultichannel array of RyR2. The open probability of RyR2 isdefined through a two-site Hill function comprised of oneactivation site and one inactivation site per subunit. Thediffusion equation for a steady state point source definesthe Ca2+ concentration in the region of the RyR2 activationand inactivation sites, for which experimental Ca2+ disso-ciation constant values were used. Metropolis Monte Carlosimulations show open probabilities for one and two RyR2sconsistent with electrophysiology measurements. Themodel shows that large RyR2 clusters exhibit a sharptransition in open probability as a function of RyR Ca2+

current at physiological cytosolic Ca2+ levels. Such acharacteristic may suggest not only an activation mecha-nism for multiple RyR2, but also a method of CICRtermination in clustered RyR2 channels.

O-126High sensitivity of Ca2+ wave propagation to ryanodinereceptor inhibition in cardiac myocytesNiall MacquaideKU Leuven, Division of Experimental Cardiology, Leuven,Belgium

In ventricular cardiac myocytes, global Ca2+ release from thesarcoplasmic reticulum (SR) is accomplished through the trig-gering of ryanodine receptors (RyR) via Ca2+ influx fromvoltage-gated Ca2+ channels of the cell surface. Spontaneous,propagating Ca2+ release events (Ca2+ waves) are associatedwith the generation of arrhythmogenic currents. These alsoconstitute thedominantmodeof globalCa2+ release incellswithsparse t-tubular networks, such as atrial myocytes, represent-ing an alternate, non-triggered form of global SR Ca2+ release.Themechanismofpropagation is however still under debate 1,2.In this study it will be shown that partial, irreversible inhibition ofRyR clusters using ruthenium red (an effectively irreversibleblocker) had differential affects on Ca2+ sparks and waves.Modest inhibition of RyR clusters abolished Ca2+ wave prop-agation, whilst Ca2+ sparks were relatively unchanged.Computer modeling of this system showed a dependence ofcluster size on the affect of inhibition on Ca2+ wave propa-gation; with smaller clusters being more sensitive toirreversible inhibition; larger cluster fluxes were relativelyunchanged by modest inhibition and so propagation wasuninterrupted. This highlights the importance of smallerclusters in Ca2+ wave propagation.

[1] Keller M et al., Cardiovasc Res. 74 2007[2] Kaiser J et al., Biophys J. 75 1998

O-127Alterations of ryanodine receptor (RyR) functionand arrhythmogenic Ca2+ waves in cardiomyocytesErnst Niggli, M. Keller, N.D. UllrichDepartment of Physiology, University of Bern, Bern,Switzerland

In cardiomyocytes, spontaneous Ca2+ release from the SRcan occur in diastole, as Ca2+ sparks. Sparks arise fromaccidental openings of a few ryanodine receptors (RyRs).However, under pathophysiological conditions, chain reac-tions of sparks can occur as Ca2+ waves. SR Ca2+ overloadand (hyper)-phosphorylation are known to favor wave occu-rence. We reported that changes of the luminal SR Ca2+

concentrations mediated by SERCA contribute to wavepropagation, by sensitizing the RyRs for cytosolic Ca2+. b-Adrenergic stimulation also speeds up Ca2+ waves. Thiscould be mediated via SERCA stimulation and thus SR Ca2+

loading. Alternatively, Ca2+ waves may travel faster becauseof RyR phosphorylation. Using a transgenic mouse lackingthe RyR phosphorylation site at serine 2808 (S2808A), weobtained further insight into spreading of Ca2+ waves. Weconsistently observed acceleration of Ca2+ waves upon b-adrenergic stimulation in WT myocytes by 15%. However,Ca2+ waves in S2808A cells did not increase their velocity,even though successful SERCA stimulation was confirmed

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as faster wave decay. Taken together these results indicatethat SR luminal Ca2+ sensing and RyR phosphorylation maybe important for Ca2+ wave propagation and the Ca2+ sen-sitivity of the RyRs. Support: SNF & Transcure.

P-128Complementary methods in determining the chemicalcomposition of renoureteral calculiI.G. Oswald, S. Cavalu, C. Ratiu, N. Maghiar, D. Osvat,C. Borza, R. GaborFaculty of Medicine and Pharmaceutics, Universityof Oradea, 10, 1 Decembrie Square, 410081 Oradea,Romania

Keywords: Urinary stones, chemical composition, FTIRspectroscopy, SEM-EDAX.Renoureteral lithiasis is a disease with a relatively high inci-dence among the population. Getting the chemical structureof the stones is very important in medical practice, bothin terms of surgical approaches for applying a mini-mal invasive treatment and also for the medical drugtherapy administrated to the patients. Stones have beencollected by endourological procedures and open surgeryoperations in urologic department. Preliminary estimation onthe structure of the studied calculi was established by theX-ray exam (KUB and IVP) and urine lab tests. Structuralinvestigation and calculi composition was performed throughthe FTIR spectrophotometer method and complementarySEM-EDAX electron microscopy. The combination of FTIRspectroscopy and SEM-EDAX allowed quantitative andqualitative evaluation of the components, the spatial distri-bution and the percent of major and trace elements presentin a sample. The surface morphology of the samples andelemental analysis performed by SEM-EDAX confirms thepresence of oxalate, carbonate and cystine. The final resulthas showed some non homogeneous structures and dif-ferent concentrations of the chemical components of thestones.

References

Fazil Marickar, Y.M., P.R. Lekshmi, L. Varma, P. Koshy, EDAXversus FTIR in mixed stones, Urol. Res., 2009, 37, 271–276

P-129Effects of phosphatidyl-inositol-phosphates (PtdInsP)on calcium release events in mammalian skeletal musclefibresPeter Szentesi1, Romain Lefebvre2, Dora Bodnar1,Janos Vincze1, Beatrix Dienes1, Vincent Jacquemond2,Laszlo Csernoch11Department of Physiology, University of Debrecen,Debrecen, Hungary, 2Centre de Genetique et de PhysiologieMoleculaire et Cellulaire, Claude Bernard University Lyon 1,France

In striated muscle Ca2+ release from the sarcoplasmicreticulum (SR) occurs when ryanodine receptors (Ryr-s)

open either spontaneously or upon the stimulation fromdihydropyridine receptors that are located in the adjacenttransverse-tubular membrane and change their conformationwhen the cell is depolarized. Recent observations demon-strated that muscles from animal models of PtdInsPphosphatase deficiency suffer from altered Ca2+ homeosta-sis and excitation-contraction coupling, raising the possibilitythat PtdInsP-s could modulate voltage-activated SR Ca2+

release in mammalian muscle. The openings of a single or acluster of Ryr-s can be detected as Ca2+ release events onimages recorded from fibres loaded with fluorescent Ca2+

indicators. To elucidate the effects of PtdInsP-s on Ca2+

release events, images were recorded from skeletal musclefibers enzymatically isolated from the m. flexor digitorumbreavis of mice utilizing a super-fast scanning technique. Awavelet-based detection method was used to automaticallyidentify the events on the images. Three different PtdInsP-s(PtdIns3P, PtdIns5P, and PtdIns(3,5)P) were tested. All thesePtdInsP compounds decreased the frequency of spontane-ous Ca2+ release events. Supported by the HungarianNational Science Fund (OTKA 75604), TeT.

O-130Caffeine and depolarization alters the morphologyof calcium spark in amphibian skeletal musclePeter Szentesi1, Janos Vincze1, Dora Bodnar1,Laszlo Szabo2, Beatrix Dienes1, Henrietta CserneSzappanos3, Martin F. Schneider3, Laszlo Csernoch11Department of Physiology, University of Debrecen, Hungary,2Department of Electrical Engineering, Sapientia HungarianUniversity of Transylvania, Targu Mures, Romania, 3Centerfor Biomedical Engineering and Technology, Universityof Maryland, Baltimore, Maryland, USA

Calcium sparks elicited by 1 mmol/L caffeine and by adepolarization to -60 mV were recorded at high time resolu-tion on both x-y (30 frames/s) and line-scan images (65 lines/ms) on intact skeletal muscle fibers of the frog. While atypical spark appeared in one frame only, 17.3 and 26.0% ofspark positions overlapped on consecutive frames followingcaffeine treatment or depolarization, respectively. While bothcaffeine and depolarization increased the frequency ofsparks, as estimated from x-y images, the morphology ofsparks was different under the two conditions. Both theamplitude (in DF/F0; 0.49 ± 0.025 vs. 0.29 ± 0.001;n = 22426 vs. 23714; mean ± SEM, p\0.05) and the fullwidth at half maximum (in lm; parallel with fiber axis:2.33 ± 0.002 vs. 2.21 ± 0.005; perpendicular to fiber axis:2.07 ± 0.003 vs. 1.88 ± 0.004) of sparks was significantlygreater after caffeine treatment than on depolarized cells.These observations were confirmed on sparks identified inline-scan images. In addition, x-t images were used to ana-lyze the time course of these events. Calcium sparks hadsignificantly slower rising phase under both conditions ascompared to the control. On the other hand, while the rate ofrise of signal mass was decreased after depolarization, itincreased in the presence of caffeine.

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O-131Calcium influx analysis by TIRF microscopyon myotubes from patients with RYR1 mutations linkedto MH and CCDFrancesco ZorzatoDepartment of Experimental and Diagnostic Medicine,University of Ferrara, Italy

Prolonged depolarisation of skeletal muscle cells inducesentry of extracellular calcium into muscle cells, an eventreferred to as excitation-coupled calcium entry. Skeletalmuscle excitation-coupled calcium entry relies on the inter-action between the 1,4 dihydropyridine receptor on thesarcolemma and the ryanodine receptor on the sarcoplasmicreticulum membrane. In this study we exploited TIRFmicroscopy to monitor with high spatial resolution excitation-coupled calcium entry (ECCE) in primary coltures of humanskeletal muscle cells harbouring mutation in the RYR1 genelinked to malignant hyperthermia and central core disease.We found that excitation-coupled calcium entry is stronglyenhanced in cells from patients with central core diseasecompared to individuals with malignant hyperthermia andcontrols. In addition, excitation-coupled calcium entry indu-ces generation of reactive nitrogen species and causes thenuclear translocation of NFATc1. The activation of NFATc1dependent genes is consistent with an increase of the IL6secretion from primary coltures human myotubes from CCDpatients and with fibre type 1 predominance of skeletalmuscle of CCD patients.

Membrane lipids, microdomains & signalling

P-132FTIR and calorimetric investigation of the effectsof trehalose and multivalent cations on lipid structureSawsan Abu Sharkh, Jana Oertel, and Karim FahmyDivision of Biophysics, Institute of Radiochemistry,Helmholtz-Zentrum Dresden-Rossendorf, GermanyE-mail: s.sharkh@hzdr.de

The structure of membrane lipids is of fundamental impor-tance for the integrity of cell and organelle membranes inliving organisms. Membrane lipids are typically hydrated andtheir headgroup charges counter-balanced by solvated ions.Consequently, water loss can induce severe structuralchanges in lipid packing (lyotropic transitions) and can leadto the damage of lipid membranes even after rehydration.This can be one out of several factors that affect the viabilityof organisms undergoing desiccation. Many organisms,however, are resistant to even extreme water loss. Some ofthem synthesize trehalose which has been shown to beassociated with survival of desiccation in phylogeneticallydiverse organisms (yeast, nematodes, brine shrimp, insectlarvae, resurrection plants, and others). Here we have stud-ied hydration sensitive transitions in model lipids todetermine the effect of trehalose and electrostatics on lipidorder. Hydration pulse-induced time-resolved Fourier-trans-form infrared (FTIR) difference spectroscopy was used toaddress hydration-dependent lipid structure as a function oftrehalose. In combination with differential scanning calorim-etry and studies of Langmuir-Blodget films we arrive at astructural and energetically consistent picture of how

trehalose can affects lipidic phase behaviour and support anative lipid structure under water loss. Experiments wereperformed on model lipids with different headgroups andnative lipids from desiccation-tolerant organisms.

P-133Controlled self-assembly and membrane organizationof lipophilic nucleosides and nucleic acids: perspectivesfor applicationsMartin Loew1, Paula Pescador3, Matthias Schade1,Julian Appelfeller1, Jurgen Liebscher2, Oliver Seitz2,Daniel Huster3, Andreas Herrmann1, andAnna Arbuzova11Humboldt Universitat zu Berlin, Institute of Biology/Biophysics, Berlin, Germany, 2 Humboldt Universitat zuBerlin, Institute of Chemistry, Berlin, Germany, 3 UniversitatLeipzig, Department of Medical Physics and Biophysics,Leipzig, Germany

Lipophilic conjugates of nucleosides and nucleic acids such asDNA, RNA, and peptide nucleic acid (PNA) - combiningassembly properties of amphiphiles and specific molecularrecognition properties of nucleic acids - allow numerousapplications in medicine and biotechnology. We recentlyobserved self-assembly of microtubes, stable cylindricalstructures with outer diameters of 300 nm and 2-3 lm and alength of 20-40 lm, from a cholesterol-modified nucleosideand phospholipids. Morphology and properties of thesemicro-tubes and functionalization with lipophilic DNA will be char-acterized. We also observed that lipophilic nucleic acids, PNAand DNA differing in their lipophilic moieties, partition into dif-ferent lipid domains in model and biological membranes asvisualized by hybridization with respective complementaryfluorescently-labeled DNA strands. Upon heating, domainsvanished and both lipophilic nucleic acid structures intermixedwith each other. Reformation of the lipid domains by coolingled again to separation of membrane-anchored nucleic acids.By linking specific functions to complementary strands, thisapproach offers a reversible tool for triggering interactionsamong the structures and for the arrangement of reactions andsignaling cascades on biomimetic surfaces.

P-134Conformational dependent traffickingof P-glycoprotein with raftsZsuzsanna Gutayne Toth, Orsolya Barsony, Katalin Goda,Gabor Szabo and Zsolt BacsoUniversity of Debrecen, MHSC, Department of Biophysicsand Cell Biology, Debrecen, Hungary

P-glycoprotein (Pgp), an ABC-transporter playing a promi-nent role in multidrug resistance, demonstrate conformation-dependent endocytosis on the surface of 3T3-MDR1 cells.These cell surface transporters have a UIC2 conformation-sensitive-antibody-recognizable subpopulation, which isabout one-third of the rest persisting long on the cell surfaceand perform fast internalization via rafts. We have identifiedthat the rapid internalization is followed by quick exocytosis, inwhich the other subpopulation is not or only slightly involved.The exocytosis presents a cholesterol depletion dependent

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intensification, in contrast to the internalization, which isinhibited by cyclodextrin treatment. This continuous recyclingexamined by total internal reflection (TIRF) microscopyincreases the amount of the raft associated subpopulation ofPgps in the plasma membrane, and it might have a role inrestoring the cholesterol content of the membrane after cho-lesterol depletion. Our presentation will summarize relatedendocytotic, exocytotic and recycling processes and that howdoes our data fit into our current notions regarding to thecholesterol and sphingomyelin trafficking.

P-135Multivalent chelator lipids for targeting proteinsinto membrane nanodomainsOliver Beutel1, Joerg Nikolaus2, Alf Honigmann3,Christian Eggeling3, Andreas Herrmann2, Jacob Piehler11University of Osnabruck, Department Biology, Divisionof Biophysics, Osnabruck, Germany, 2 Humboldt UniversityBerlin, Department Biology/ Molecular Biophysics, Berlin,Germany, 3Max-Planck-Institut fur biophysikalische ChemieGottingen, Abteilung NanoBiophotonik, Gottingen, GermanyE-mail: oliver.beutel@biologie.uni-osnabrueck.de

Membrane nanodomains based on phase-segregating lipidmixtures have been emerged as a key organizing principle ofthe plasma membrane. They have been shown to play impor-tant roles in signal transduction and membrane trafficking. Wehave developed lipid-like probes carrying multivalent nitrilotri-acetic acid (tris-NTA) headgroups for selective targeting ofHis-tagged proteins into liquid ordered or liquid disordered phases.In giant unilamellar vesicles strongpartitioningof tris-NTA lipidsinto different lipid phases was observed. For a saturated tris-NTA lipid, at least 10-fold preference for the liquid orderedphasewas found. In contrast, anunsaturatedNTA lipid showsacomparable preference for the liquid disordered phase. Parti-tioning into submicroscopicmembranedomains formed in solidsupported membranes was confirmed by superresolutionimaging. Single molecule tracking of His-tagged proteins teth-ered to solid supported membranes revealed clear differencesin the diffusion behavior of the different NTA-lipids. By usingBSA as a carrier, multivalent NTA lipids were efficiently incor-porated into the plasma membrane of live cells. Based on thisapproach, we established versatile methods for probing andmanipulating the spatiotemporal dynamics of membrane nanodomains in live cells.

P-136Biophysical investigation of IL-9R assemblyand function in human T-lymphoma cellsE. Nizsaloczki1, I. Csomos1, G. Mocsar1, N. Szaloki1,T. A. Waldmann2, S. Damjanovich1, G. Vamosi1

and A. Bodnar11HAS-UD Cell Biology and Signaling Research Group,Department of Biophysics and Cell Biology, Universityof Debrecen, Debrecen, Hungary, 2Metabolism Branch, NCI,National Institute of Health, Bethesda, MD, USA

IL-9 is a multifunctional cytokine with pleiotropic effects on Tcells. The IL-9R consists of the cytokine-specific a-subunit andthe cc-chain shared with other cytokines, including IL-2 and -15, important regulators of Tcells. We have previously shown

the preassembly of the heterotrimeric IL-2 and IL-15R, as wellas their participation in common superclusters with MHC gly-coproteins in lipid rafts of human T lymphoma cells. Integrity oflipid raftswasshown tobe important in IL-2 signaling.Wecouldhypothesize that other members of the cc cytokine receptorfamily, such as the IL-9R complex, may also fulfill their tasks ina similar environment, maybe in the same superclusters.Co-localization of IL-9R with lipid rafts as well as with the IL-2R/MHC superclusters was determined by CLSM. Molecularscale interactions of IL-9Ra with IL-2R and MHC moleculeswere determined by microscopic and flow cytometric FRETexperiments. The role of lipid rafts in IL-9R signaling wasassessed by following the effect of cholesterol depletion onIL-9 induced STAT phosphorylation.Our results suggest the possibility that preassembly of thereceptor complexes in common membrane microdomainswith MHC glycoproteins may be a general property of cccytokines in T cells.

P-137Membrane organization of Fas transmembrane domain:Insights for clustering and apoptosis triggeringBruno M. Castro1, Rodrigo F. M. de Almeida2

and Manuel Prieto11CQFM and IN, IST, Av. Rovisco Pais, 1049-001 Lisboa,Portugal 2 CQB, FCUL, Campo Grande, 1749-006 Lisboa,Portugal

To unravel the molecular processes leading to Fas clusteringin lipid rafts, a 21-mer peptide corresponding to the singletransmembrane domain of the death receptor was reconsti-tuted into model membranes that display liquid-disordered/liquid-ordered phase coexistence, i.e.mimicking cells plasmamembranes. Using the intrinsic fluorescence of the peptidetwo tryptophans residues (Trp176 and Trp189), Fas membranelateral organization, conformation and dynamics was studiedby steady-state and time-resolved fluorescence techniques.Our results show that the Fas has preferential localization toliquid disordered membrane regions, and that it undergoesa conformational change from a bilayer inserted state inliquid-disordered membranes to an interfacial state in liquid-orderedmembranes. This is a result of the strong hydrophobicmismatch between the (hydrophobic) peptide length and thehydrophobic thickness of liquid-ordered membranes. In addi-tion, we show that ceramide, a sphingolipid intimately involvedin Fas oligomerization and apoptosis triggering, does notaffect Fas membrane organization. Overall, our results high-light ceramide role as an enhancer of Fas oligomerization, andunravel the protective function of Fas transmembrane domainagainst non-ligand induced Fas apoptosis.

P-138Organization and dynamics of membrane-bound bovinea-lactalbumin: a fluorescence approachArunima Chaudhuri and Amitabha ChattopadhyayCentre for Cellular and Molecular Biology, Hyderabad 500007, IndiaE-mail: amit@ccmb.res.in

Many soluble proteins are known to interact with membranesin partially disordered states, and the mechanism and

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relevance of such interactions in cellular processes arebeginning to be understood. Interestingly, apo-bovine a-lactalbumin (BLA), a soluble protein, specifically interactswith negatively charged membranes and the membrane-bound protein exhibits a molten globule conformation. Wehave used the wavelength-selective fluorescence approachto monitor the molten globule conformation of BLA uponbinding to negatively charged membranes as compared tozwitterionic membranes. Tryptophans in BLA exhibit differ-ential red edge excitation shift (REES) upon binding tonegatively charged and zwitterionic membranes, implyingdifferential rates of solvent relaxation around the tryptophanresidues. Our results utilizing fluorescence anisotropy, life-time and depth analysis by the parallax approach of thetryptophans further support the differential organization anddynamics of the membrane-bound BLA forms. In addition,dipole potential measurements and dye leakage assays arebeing used in our ongoing experiments to explore themechanism of BLA binding to membranes. These resultsassume significance in the light of antimicrobial and tumori-cidal functions of a-lactalbumin.

O-139Role of long-range effective protein-protein forcesin the formation and stability of membrane proteinnano-domainsNicolas DestainvillLaboratoire de Physique Theorique, Universite Paul SabatierToulouse 3 – CNRS, Toulouse, France

We discuss a realistic scenario, accounting for the existenceof sub-micro-metric protein domains in plasma membranes.We propose that proteins embedded in bio-membranes canspontaneously self-organize into stable small clusters, due tothe competition between short-range attractive and interme-diate-range repulsive forces between proteins, specific tothese systems. In addition, membrane domains are sup-posedly specialized, in order to perform a determinedbiological task, in the sense that they gather one or a fewprotein species out of the hundreds of different ones that acell membrane may contain. By analyzing the balancebetween mixing entropy and protein affinities, we proposethat protein sorting in distinct domains, leading to domainspecialization, can be explained without appealing to pre-existing lipidic micro-phase separations, as in the lipid raftscenario. We show that the proposed scenario is compatiblewith known physical interactions between membrane pro-teins, even if thousands of different species coexist.

References

[1] N. Destainville, Cluster phases of membrane proteins,Phys. Rev. E 77, 011905 (2008).

[2] N. Destainville, An alternative scenario for the formationof specialized protein nano-domains (cluster phases) inbiomembranes, Europhys. Lett. 91, 58001 (2010).

P-140Characterization of Brij 58 and 98-Resistant Membranesfrom Human ErythrocytesBruna R. Casadei, Cleyton C. Domingues,Eneida de PaulaDepartamento de Bioquımica / IB, Universidade Estadual deCampinas, SP, Brazil

Lipid rafts are cholesterol and sphingolipid-enriched functionalmicrodomains present in biomembranes. Rafts have beenoperationally defined asmembrane fractions that are detergentinsoluble at low temperature. Here we have characterizedDRMs from erythrocytes treated with the nonionic detergentsBrij58 and Brij98, at 4�C and 37�C, and compared them toDRMs obtained with Triton X-100 (TX100). We have alsoinvestigated the effect of cholesterol depletion in DRMs forma-tion. Brij DRMs were enriched in cholesterol as well as TX100DRMs. HPTLC analysis showed a very similar distribution ofphosphatidylcholine-PC, phosphatidylethanolamine-PE andsphingomyelin-SM in Brij DRMs to that found in ghost mem-branes. SM-enriched DRMs were obtained only with TX100while PE content was decreased in TX100 DRMs, in compari-son to Brij DRMs. Immunoblot essays revealed that raftsmarkers (flotillin-2 and stomatin) were present in all DRMs.Contrary to TX100 DRMs, analysis of electron paramagneticresonance spectra (with 5-doxyl stearate spin label) revealedthat Brij DRMs are not in the liquid-ordered state, evincing thedifferential extraction of membrane lipids promoted by thesedetergents. Supported by FAPESP/CNPq (Brazil).

O-141Magnetic Camemberts and Liposomes: New Toolsfor Structural Biology of Membrane MoleculesErick Dufourc, Nicole Harmouche, Axelle Grelard,Cecile Courreges, Anna DillerInstitute of Chemistry & Biology of Membranes &Nanoobjects, CNRS-University Bordeaux-IPB, Pessac,France

Several biological membrane mimics have been built toinvestigate the topology of molecules in membranes. Amongthem ‘‘bicelles’’, i.e., mixtures of long-chain and short-chainsaturated phospholipids hydrated up to 98%, became verypopular because they orient spontaneously in magneticfields. Disk-shaped systems of 40–80 nm diameter and 4–5nm thickness have been measured by electron microscopyand solid state nmR and can be oriented by magnetic fieldswith the disc-plane normal perpendicular to the field. We havebeen developing recently lipids that contain in one of theirchains a biphenyl group (TBBPC) affording an orientationparallel to the magnetic field, in the absence of lanthanides. Alarge number of hydrophobic molecules including membraneproteins have been successfully embedded and static nmRafforded finding the orientation of protein helices in mem-branes; MAS nmR provided the 3D structure of peptides inbicelles. Biphenyl bicelles keep their macroscopic orientationfor days outside the field, thus leading to combined nmR and

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X-Rays experiments. TBBPC allows also construction of lmvesicles showing a remarkable oblate deformation in mag-netic fields (anisotropy of 3-10) and opens the way toapplications for structural biology or drug delivery under MRI.

O-142Imaging membrane heterogeneities and domainsby super-resolution STED nanoscopyChristian Eggeling, Veronika Mueller, Alf Honigmann,Stefan W. HellDepartment of Nanobiophotonics, Max Planck Institutefor Biophysical Chemistry, Am Fassberg 11, 37077Gottingen, Germany

Cholesterol-assisted lipid and protein interactions such as theintegration into lipid nanodomains are considered to play afunctional part in a whole range of membrane-associatedprocesses, but their direct and non-invasive observation inliving cells is impeded by the resolution limit of[200nm of aconventional far-field optical microscope. We report thedetection of the membrane heterogeneities in nanosizedareas in the plasma membrane of living cells using thesuperior spatial resolution of stimulated emission depletion(STED) far-field nanoscopy. By combining a (tunable) reso-lution of down to 30 nm with tools such as fluorescencecorrelation spectroscopy (FCS), we obtain new details ofmolecular membrane dynamics. Sphingolipids or other pro-teins are transiently (* 10 ms) trapped on the nanoscale incholesterol-mediated molecular complexes, while others dif-fuse freely or show a kind of hopping diffusion. The results arecompared to STED experiments on model membranes, whichhighlight potential influences of the fluorescent tag. The novelobservations shed new light on the role of lipid-protein inter-actions and nanodomains for membrane bioactivity.

O-143Ca2+ controlled all-or-none like recruitmentof synaptotagmin-1 C2AB to membranesSune M. Christensen, Nicky Ehrlich, Dimitrios StamouBio-Nanotechnology Laboratory, Departmentof Neuroscience and Pharmacology, Nano-Science Center,Lundbeck Foundation Center Biomembranes inNanomedicine, University of Copenhagen, 2100 Copenhagen,DenmarkE-mail: ehrlich@nano.ku.dk & stamou@nano.ku.dk

Synaptotagmin-1 (Syt) is the major Ca2+ sensor that triggersthe fast, synchronous fusion of synaptic vesicle with the pre-synaptic membrane upon Ca2+-mediated membranerecruitment of the cytosolic C2AB domain. The Ca2+-depen-dent recruitment of Syts C2AB domain to membranes has sofar been investigated by ensemble assays. Here we revisitedbinding of wild typeC2ABand different C2ABmutants of Syt tolipid membranes using a recently developed single vesicleassay. The hallmark of the single vesicle approach is that itprovides unique information on heterogeneous properties thatwould otherwise be hidden due to ensemble averaging. Wefound that C2AB does not bind to all vesicles in a homogenousmanner, but in an all-or-none like fashion to a fraction of thevesicles. The fraction of vesicles with bound C2AB is regu-lated by the amount of negatively charged lipids in the

membrane as well as by [Ca2+] . This Ca2+ controlled all-or-none like recruitment of Syt to membranes provides a possibleexplanation for the strongly heterogeneous behavior of the invitro model system for neuronal membrane fusion. Further-more, heterogeneity in release probability among synapticvesicles is a critical property in determining the output of aneuronal signaling event.

P-144New insights in the transport mechanismof Ciprofloxacin revealed by fluorescence spectroscopyMariana Ferreira, Sılvia C. Lopes, Paula GameiroREQUIMTE, Faculdade de Ciencias da Universidadedo Porto, Porto, Portugal

Keywords: Fluoroquinolones; Liposomes; Proteoliposomes;OmpF; Ciprofloxacin; Fluorescence; Anisotropy.Fluoroquinolones are antibiotics that have a large spectrumof action against Gram negative and some Gram positivebacteria. The interaction between these species and lipo-somes has been cited as a reference in the understanding oftheir diffusion through phospholipid bilayer and can bequantified by the determination of partition coefficientsbetween a hydrophobic phase (liposomes) and an aqueoussolution. It is also known that some porins of the bacterialmembranes are involved in transport mechanism of manyfluoroquinolones. OmpF, a well characterized membraneprotein characteristic of the outer membrane of Gram nega-tive bacteria assumes the conformation of homo-trimer,whose monomers have two tryptophan residues (one locatedat the interface of monomers and the other at the interfacelipid/protein). Thus, we proceeded to study the interaction ofCiprofloxacin, a second generation fluoroquinolone, withunilamellar liposomal vesicles and OmpF proteoliposomes ofPOPE/POPG, POPE/POPG/Cardiolipin and E. coli total.Partition coefficients (Kp’s) and the association with OmpFproteoliposomes were determined by steady state fluores-cence spectroscopy under physiological conditions (T=37�C;pH 7.4). The membrane mimetic systems used were char-acterized by DLS and fluorescence anisotropy.

P-145CD44-ICAM-1 crosstalk alters MHCI rafts – A flowcytometric FRET and microscopic colocalization studyA. Forgacs, N. Szaloki, L. BeneCell Biology and Signaling Research Group of the HungarianAcademy of Sciences, Research Center for MolecularMedicine, University of Debrecen, Debrecen, HungaryE-mail: forgacsmeister@gmail.com, bene@dote.hu

Motivation is whether there exist differences in the pattern-forming capabilities of two adhesion molecules of differentroles: CD44, mediating ,,dynamic’’ adhesion in cell rolling andICAM-1, mediating ,,static’’ adhesion during the formation ofimmune-synapse. Homo- and hetero-associations of CD44,ICAM-1 and the MHCI is investigated on the nm- and lm-distance levels on LS174T colon carcinoma cells in two dif-ferent conditions of lymphocyte homing: (1) With IFNc andTNFa, both lymphokines up-regulating the expression level ofMHCI and ICAM-1 and down-regulating that of CD44. (2)Crosslinking of CD44 and ICAM-1 representing receptor

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engagement. The observations are explained by assumingthe existence of a kinase cascade-level crosstalk betweenthe CD44 and ICAM-1 molecules which manifests in char-acteristic complementary changes in the properties of cellsurface receptor patterns. For the characterisation of clustermorphology new colocalization approaches were developed:(i) ,,number of first neighbours’’ distribution curves, (ii),,acceptor photobleaching FRET-fluorescence intensity fluc-tuation product’’ correlation diagrams, and (iii) ,,randomgradient-kernel smoothing assisted decay’’ of Pearson-cor-relations, and (iv) K-function formalism.

P-147Analyzing Janus kinases in living cells with single-colorFCS using confocal and TIR-illuminationThomas Weidemann1, Hetvi Gandhi1, Remigiusz Worch1,3,Robert Weinmeister1, Christian Bokel2, and Petra Schwille11Biophysics Research Group, Technische UniversitatDresden, Germany, 2CRTD, Center for RegenerativeTherapies Dresden, Technische Universitat Dresden,Germany, 3Institute of Physics, Polish Academy of Science,Warsaw, Poland

Cytokine receptors of the hematopoietic superfamily transducetheir signal through non-covalently bound Janus kinases. Thereare only 4 such kinases in humans (JAK1, 2, 3 and TYK2),which associate to 46 different cytokine receptor chains. Herewe study the dynamics of GFP-tagged JAK1 and JAK3 in epi-thelial cells with fluorescence correlation spectroscopy (FCS).JAK1 and JAK3 behave differently in various aspects: In theabsence of receptors, JAK1 still binds the membrane, whereasJAK3 diffuses homogeneously in the cytoplasm. We used FCSunder total internal reflection illumination (TIR-FCS) and deter-mined the membrane binding affinity of JAK1 to be 60±36 nm.The association of JAK3 with the common gamma chain (cc) isvery tight as shown by fluorescence recovery after photoble-aching (FRAP). Molecular brightness analysis of single-pointFCS shows that JAK1 diffuses as amonomer in the rather smallcytoplasmic pool, whereas JAK3 diffuses as dimers, whichundergo a defined oligomerization. The degree of oligomeriza-tion decays at higher concentrations, indicating that someunknown, saturable scaffold is involved. Characterizing thebinding and mobility schemes of the Janus kinases may beimportant to further elucidate their specific and redundanteffects in signal transduction.

P-148Brain plasma membrane-enriched fractions containand preserve the activity of mitochondrial membraneMarcelo Ganzella1, Roberto Farina Almeida1, FernandaLopes1, Gustavo da Costa Ferreira2, Moacir Wajner1,Fabio Klamt1, Diogo Onofre Souza11Universidade Federal do Rio Grande do Sul, Porto Alegre,RS, Brasil, 2Universidade do Extremo Sul Catarinense,Criciuma, SC, Brasil

Plasma membrane (PM)-enriched fraction obtained throughsubcellular fractioning protocols are commonly used in stud-ies investigating the ability of a compound to bind to areceptor. However, the presence of mitochondria membranes(MI) in the PM-enriched fraction may compromise several

experimental results because MI may also contain the interestbinding proteins. Aiming to analyze the subcellular fractioningquality of a standard sucrose density based protocol, weinvestigated (a) the Na+K+-ATPase (PM marker) and succi-nate dehydrogenase - SD (MI marker) activities; (b) theimmunocontent of the adenine nucleotide translocator (ANT –MI membrane marker) in both PM- and MI-enriched fractions.Since several binding protocols may require long incubationperiod, we verified the quality of both fractions after 24 hoursof incubation in adequate buffer. Our results show that PM-and MI-enriched fractions exhibit contamination with MI orPM, respectively. We did not observe any effect of incubationon Na+K+-ATPase activity and ANT content in both fractions.Surprisingly, SD activity was preserved in the PM- but not inMI-enriched fraction after incubation. These data suggest theneed of more careful use of PM-enriched fraction preparationin studies involving PM proteins characterization.

P-150Membrane docking mode of the C2 domain of PKCe:an infrared spectroscopy studyAlessio Ausili, Antonio L. Egea, Senena Corbalan-Garcıaand Juan C. Gomez-FernandezDepartamento de Bioquımica y Biologıa Molecular A,Universidad de Murcia, 30100-Murcia, Spain

PKCe is activated by binding to membranes. Its C2 domain, inparticular, binds tonegatively chargedphospholipids.Wehaveused Fourier Transform Infrared Spectroscopy (FTIR) todetermine the membrane docking mode of the C2 domain ofthis PKC by the calculation of the b-sandwich orientation whenthe domain is bound to different types of model membranes,using ATR-FTIR spectroscopy with polarized light. The vesi-cles lipid compositions used were: POPA, POPC/POPA(50:50), POPC/POPS/POPA (65:25:10), POPC/POPE/CL(43:36:21) and POPC/POPG/POPA (65:25:10). Results showthat the interaction with membranes does not strongly affectthe secondary structure of the domain and that some differ-ences were found with respect to the extent of binding as afunction of phospholipid composition. Small changes are onlyevident when the domain is bound to model membranes ofPOPC/POPA and POPC/POPG/POPA. In this case, the per-centage of b-sheet of C2 domain increases if compared withthe secondary structure of the domain in the absence of ves-icles. With respect to the b-sandwich orientation, when thedomain is bound to POPC/POPG/POPA membranes it formsan angle with the surface of the lipid bilayer bigger than thatone observed when the domain interacts with vesicles ofPOPC/POPA and POPC/POPS/POPA.

P-151Lipid selectivity as a determinant factor for HNP1biological activitySonia Goncalves, Alexandre Teixeira, Joao Abade,Nuno C. SantosInstituto de Medicina Molecular, Faculdade de Medicina daUniversidade de Lisboa, Lisbon, Portugal

Human neutrophil peptide 1 (HNP1) is a human cationicdefensin that present microbial activity against various bac-teria (both Gram-positive and negative), fungi and viruses.

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HNP1 is stored in the cytoplasmic azurophilic granules ofneutrophils and epithelial cells. In order to elucidate the modeof action of this antimicrobial peptide (AMP), studies basedon its lipid selectivity were carried out. Large unilamellarvesicles (LUV) with different lipid compositions were used asbiomembranes model systems (mammal, fungal and bacte-rial models). Changes on the intrinsic fluorescence of thetryptophan residues present in HNP1 upon membranebinding/insertion were followed, showing that HNP1 havequite distinct preferences for mammalian and fungal mem-brane model systems. HNP1 showed low interaction withglucosylceramide rich membranes, but high sterol selectivity:it has a high partition for ergosterol-containing membranes(as fungal membranes) and low interaction with cholesterol-containing membranes (as in mammalian cells). Theseresults reveal that lipid selectivity is the first step after inter-action with the membrane. Further insights on the HNP1membrane interaction process were given by fluorescencequenching measurements using acrylamide, 5-doxylstearicacid (5NS) or (16NS).

P-152Study of supported lipid bilayers in interactionwith nanoparticles by AFMRokhaya Faye, Christine Grauby-Heywang,Fabien Morote, Touria Cohen-BouhacinaLaboratoire Ondes et Matiere d’Aquitaine (LOMA) CNRS5798 –Universite Bordeaux 1, Talence, France

Nanoparticles (NP) are currently used in many industrial orresearch applications (paints, cosmetics, drug deliverymaterials…). Recent papers demonstrate clearly their activitywith biological membranes (nanoscale holes, membranethinning, disruption). Different studies of the NP-membraneinteraction suggest that parameters are particularly impor-tant, such as the NP size, their surface properties or theiraggregation state. Composition of biological membranesbeing particularly complex, supported lipid bilayers (SLB)composed of a restricted number of lipids are usually used assimplified membrane model. Moreover, these two-dimen-sional systems are convenient for surface analysistechniques, such as atomic force microscopy (AFM), givinginformation on the morphology of the SLB and its mechanicalproperties. In this work, we study the behaviour of SLBsmade of lipids representative of the membrane fluid phase(POPC) or of the raft phase (sphingomyelin). These SLBsare deposited on planar surfaces (mica or glass) previouslyrecovered with silica beads (10 or 100 nm in diameter) inorder to mimick the NP-membrane interaction. We willpresent in this work our first results obtained by AFM andfluorescence microscopy.

P-153Effect of cisplatin on content of phospholipids in rat liverchromatin fractionE.S. Gevorgyan, Zh.V. Yavroyan, N.R. Hakobyan,A.G. Hovhannisyan, G.N. KhachatryanYerevan State University, 1 Alex Manoogyan St., Yerevan,0025, Armenia, E-mail: nunehakobyan@rambler.ru

It is well known that the eukaryotic nuclei are the sphere oflipids active metabolism. The investigations demonstrated

the existence of numerous enzymes in nuclei which modu-late the changes of nuclear lipids during different cellularprocesses. Although the nuclear membrane is accepted asthe main place of the lipids localization, nearly 10% ofnuclear lipids are discovered in chromatin fraction. The abilityof chromatin phospolipids to regulate DNA replication andtranscription was already demonstrated. The chromatinphospolipids seems to play an important role in cell prolifer-ation and differentiation as well as in apoptosis. It seems alsopossible that chromatin phospholipids may be participated inrealization of cisplatin antitumor effects.The 24-hour in vivo effect of cisplatin on rat liver chromatinphospholipids was investigated. The phospholipids of ratliver chromatin were fractionated by microTLC technique.The quantitative estimation of fractionated phospholipids wascarried out by computer program FUGIFILM Science Lab.2001 Image Gauge V 4.0.The alteration of total phospholipids content as well as thequantitative changes among the individual phospholipidsfractions in rat liver chromatin after in vivo action of cisplatinwas established. The total content of chromatin phospholipidswas significantly decreased after the cisplatin action. Fourfrom five individual phospholipids fractions were markedlychanged after the drug action. Two cholin-content phospho-lipids, particularly phosphatidylcholine and sphingomyelinexhibit diversity in sensitivity to this drug: the increase ofsphingomyelin content accompanied by quantitative decreaseof phosphatidylcholine. The quantity of cardiolipin was mark-edly increased while the amount of phosphatidylinositol wasdecreased after the cisplatin treatment. The phosphatidyleth-anolamin content remained unchanged after the drug action.It seems that high sensitivity of chromatin phospholipidsexhibited to cisplatin action may play an important role inantitumor effects of this drug.

P-154Membrane lipids and drug resistance in StaphylococciR. D. HarveyInstitute of Pharmaceutical Science, King’s College London,150 Stanford Street, London SE1 9NH, UK

Staphylococci express numerous resistance mechanismsagainst common antimicrobials, including peptide compo-nents of the innate immune system which have beentrumpeted as being likely candidates to replace ourincreasingly ineffective antibiotics. The membranephospholipid lysylphosphatidylglycerol (L-PG) appears toplay a key role in Staphylococcal drug resistance, since itsabsence in mutant bacteria renders them susceptible to arange of cationic antimicrobials. The current assumptionabout the role L-PG plays in drug resistance is that of facili-tating charge neutralisation of the plasma membrane, leadingto loss of affinity towards cationic moieties. We have inves-tigated this phenomenon using a range of model membranesystems composed of both synthetic lipids and reconstitutednatural lipid extracts, using such techniques as stopped-flowfluorescence, circular dichroism and neutron scattering. Ourconclusions indicate that the initial assumptions about therole of L-PG in drug resistance are over-simplistic and cer-tainly do not tell the whole story of the physical and biologicalproperties of this fascinating moderator or membranebehaviour. Our findings show that L-PG does not inhibit

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antimicrobial drug action by charge dampening, hinting at adifferent protective mechanism.

P-155Modulation of a-toxin binding by membrane compositionM. Schwiering, A. Brack, C. Beck, H. Decker,N. HellmannInstitute for Molecular Biophysics, University of Mainz,Mainz, Germany

Although the alpha-toxin from S. aureus was the first pore-forming toxin identified, its mode of interaction with mem-branes is still not fully understood. The toxin formsheptameric pores on cellular and artificial membranes. Thepresent hypothesis is that the initial binding to the membraneoccurs with low affinity, and that an efficient oligomerisation,relying on clusters of binding sites, is the reason for theoverall high affinity of the binding process. In order to sep-arate the effects of increasing concentration of binding sitesfrom this topological effect, we investigated the oligomerformation based on pyrene-fluorescence for a series of lipidcompositions, where the fraction of toxin binding lipids (eggphospatidylcholine (ePC) or egg sphingomyelin (eSM)) wasvaried while their concentration remained constant. Theresults indicate that an increased local density of toxinbinding sites occurring due to phase separation facilitatesoligomer formation. Furthermore, the change in local envi-ronment (number of neighboring cholesterol molecules) upondomain formation also enhances oligomer formation.Wethank the DFG (SFB 490) for financial support, S. Bhakdi andA. Valeva for production of the toxin and helpful discussions.

P-156Quercetin effects on Laurdan emission fluorescencein the presence of cholesterolDiana IonescuUniversity of Medicine and Pharmacy ‘‘Carol Davila’’,Bucharest, Romania

We explored quercetin effects on lipid bilayers containingcholesterol using a spectrofluorimetric approach. We usedthe fluorescent probe Laurdan which is able to detect chan-ges in membrane phase properties. When incorporated inlipid bilayers, Laurdan emits from two different excited states,a non-relaxed one when the bilayer packing is tight and arelaxed state when the bilayer packing is loose. Thisbehavior is seen in recorded spectra as a shift of maximumemission fluorescence from 440 nm at temperatures belowlipids phase transition to 490 nm at temperatures above lipidsphase transition values. Emission spectra of Laurdan wereanalyzed as a sum of two Gaussian bands, centered on thetwo emission wavelengths allowing a good evaluation of therelative presence of each population. Our results show thatboth Laurdan emission states are present with differentshares in a wide temperature range for DMPC liposomeswith cholesterol. Quercetin leads to a decrease in the phasetransition temperature of liposomes, acting in the same timeas a quencher on Laurdan fluorescence.This paper is supported by the Sectorial Operational Pro-gramme Human Resources Development, financed from the

European Social Fund and by the Romanian Governmentunder the contract number POSDRU/89/1.5/S/64109

P-157Comparative molecular dynamics simulationsof the caveolin CRAC motif in micelle and bilayerVeronica Beswick1,2, Pierre Nedellec1, Alain Sanson1,Alecu Ciorsac3, Vasile Ostafe4, Adriana Isvoran4

and Nadege Jamin11Commissariat a‘ l0Energie Atomique (CEA), Instituteof Biology and Technology (iBiTecS), Gif-sur-Yvette, France,2Department of Physics and Modeling, Universite0 d’Evry-val-d’Essonne, Evry, France, 3Departament of PhysicalEducation and Sport, Polytechnical University of Timisoara,Timisoara, Romania, 4Department of Chemistry, WestUniversity of Timisoara, Timisoara, Romania

Caveolins are essential membrane proteins found in caveo-lae. The caveolin scaffolding domain of caveolin-1 includes ashort sequence containing a CRAC motif (V94TKYWFYR101)at its C-terminal end. To investigate the role of this motif in thecaveolin–membrane interaction at the atomic level, we per-formed a detailed structural and dynamics characterization ofa cav-1(V94-L102) nonapeptide encompassing this motif andincluding the first residue of cav-1 hydrophobic domain(L102), in dodecylphosphocholine (DPC) micelles and inDMPC/DHPC bicelles, as membrane mimics. nmR datarevealed that this peptide folded as an amphipathic helixlocated in the polar head group region. The two tyrosine side-chains, flanked by arginine and lysine residues, are situatedon one face of this helix, whereas the phenylalanine andtryptophan side-chains are located on the opposite face (LeLan C. et al., 2010, Eur. Biophys. J., 39, 307-325). Toinvestigate the interactions between the CRAC motif and thelipids, we performed molecular dynamics simulations in twodifferent environment: a DPC micelle and a POPC bilayer.The results obtained are in good agreement with nmR dataand the comparison between both systems provided insightinto the orientation of the CRAC motif at the membraneinterface and into its interactions with lipids.This work was partially supported by the strategic grantPOSDRU/21/1.5/G/13798, inside POSDRU Romania2007-2013, co-financed by European Social Found-Investingin People and by the bilateral collaboration project BrancusiRomania France, 3V/21J.

P-158Effect of hydroxyl groups in the sphingolipid backboneon sterol/sphingomyelin interactionShishir Jaikishan and J. Peter SlotteBiochemistry, Department of Biosciences, Abo AkademiUniversity, 20520 Turku, Finland

D-ribo-phytosphingosines are biologically significant naturalsphingolipids possessing two hydroxyl groups at positions 3and 4 in their backbone. These are found in yeasts, plants andmammalian tissues. In this study we observed bilayer proper-ties and interaction with cholesterol of N-16:0 phytosphing-omyelin (PhytoSM) as well as their N-acyl hydroxylated (2-OH,D or L conformations) chain-matched sphingomyelin. Anisot-ropy of diphenylhexatriene suggested that pure N-16:0

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PhytoSM showed higher gel-fluid transition temperature (Tm,47�C) as compared to nonhydroxylated chain matched sphin-gomyelin i.e. palmitoyl sphingomyelin (Tm, 40.5�C). Tm valuesof pure 2-OH(D) PhytoSM and 2-OH(L) PhytoSM bilayers were42�C and 55�C respectively. Cholestatrienol (CTL) quenchingsuggested that PhytoSM and 2-OH(L) PhytoSM appeared toincrease sterol-rich domain stability compared to 2-OH(D)PhytoSM in fluid phospholipid bilayer. DSCdata suggested thatPhytoSM showed good miscibility with PSM. CTL partitioningshowed that sterol affinity of PhytoSM and 2-OH(L) PhytoSMbilayers was higher as compared to 2-OH(D) PhytoSMor PSM.Our study suggests that conformation and positioning ofhydroxyl groups significantly affects thermotropic properties ofsphingolipids and sterol interaction.

P-159Slow relaxation following the thermal hysteresis of newbolaamphiphiles studied by SAXS/WAXSJelena Jeftic1,4, Mathieu Berchel2,4, Cristelle Meriadec3,4

and Thierry Benvegnu1,41Ecole Nationale Superieure de Chimie de Rennes, Rennes,France, 2Universite de Bretagne Occidentale, Brest, France,3Institut de Physique de Rennes, Rennes, France,4Universite Europeenne de Bretagne, Rennes, France

The polymorphism of a new series of bolaamphiphile mole-cules based on N-(12-Betainylamino-dodecane)-octyl b-D-Glucofuranosiduronamide Chloride is investigated. The lengthof the main bridging chain is varied in order to modify thehydrophilic/lipophilic balance. The other chemical modificationwas to introduce a diacetylenic unit in themiddle of the bridgingchain to study the influence of the p – p stacking on thesupramolecular organisation of these molecules. Dry bola-amphiphiles self-organize in supramolecular structures suchas lamellar crystalline structure, lamellar fluid structure andlamellar gel structure. The thermal dependence of thesestructures, as well as the phase transition is followed by small-angle and wide-angle X-ray scattering. Once the thermal cycleis accomplished, the system remains in the kinetically stabi-lized undercooled high-temperature phase at temperature of20 �C. Subsequently, the time dependence of the relaxation tothe thermodynamically stable phase is followed and very slowrelaxation on the order of hours or days is observed. The studyof polymorphism and the stability of various phases of this newseries of bolaamphiphiles is interesting for potential applicationin health, cosmetics or food industry, is undertaken in thiswork.

P-160Interaction of alkylphospholipid liposomes with breastcancer cellsT. Koklic1,2, R. Podlipec1,2, J. Mravljak3, M. Sentjurc1,R. Zeisig41Jozef Stefan Institute, Ljubljana, Slovenia, 2Centerof Excellence NAMASTE, Ljubljana, Slovenia, 3Facultyof Pharmacy, University of Ljubljana, Ljubljana, Slovenia,4Max-Delbruck-Centre for Molecular Medicine, Berlin-Buch,Germany

Alkylphospholipids have shown promising results in severalclinical studies and among them perifosine (OPP) is prom-ising for breast cancer therapy. Antitumor effect was muchbetter in estrogen receptor negative (ER-) than in estrogen

receptor positive (ER+) tumors in vivo. It is believed that APLdo not target DNA, but they insert in the plasma membraneand ultimately lead to cell death.Liposomes made of OPP and different amount of cholesterol(CH) showed diminished hemolytic activity as compared tomicellar OPP, but in most cases cytotoxic activity was lower. Inorder to find optimal liposomal composition and to understandbetter the difference in the response of ER+ and ER- cells theinteractionOPP liposomeswithER+andER- cellswasstudied.For liposomes with high amount of CH both cell types showedslow release of the liposome entrapped spin probe into thecytoplasm. Liposmeswith lowamount ofCH interact betterwithcells but the release is faster for ER- as for ER+ cells at 37�C.Experiments with nitroxide-labeled OPP (SL-OPP) liposomessuggest that the exchange of SL-OPP between liposomes andcellular membranes is fast. However, translocation of SL-OPPacross theplasmamembrane is slow, but seems tobe faster forOPP resistent, ER+ cells as for ER- cells at 37�C.

P-161Estimation of a membrane pore size based on the lawof conservation of massKrystian Kubica1, Artur Wrona1 and Olga Hrydziuszko21Institute of Biomedical Engineering and Instrumentation,Wroclaw University of Technology, Wroclaw, Poland, 2Centrefor Systems Biology, University of Birmingham, Birmingham,UK

The size of biomembrane pores determines which solutes oractive compounds may enter the cell. Here, using a mathe-matical model of a lipid bilayer and the law of conservation ofmass, we calculate the radius of a membrane pore created byrearranging the lipid molecules (the pore wall was formed outof the lipid heads taken from the membrane regions situateddirectly above and below the pore, prior its formation).Assuming a constant number of lipid molecules per bilayer(with or without the pore) and based on the literature data (60%decrease in the area per chain for a fluid-to-gel transition and amatching change of one chain volume not exceeding 4%) wehave shown that the pore radius can measure up to 4.7nm (fora 7nm thick lipid bilayer) without the lipid molecules under-going a phase transition. A further assumption of area perchain being modified as a consequence of the lipids confor-mational changes has resulted in an increase of the calculatedradius up to 7.1nm. Finally, a comparison of the pore volumewith the corresponding volume of the lipid bilayer has led to aconclusion that for the system under consideration the mem-brane pore can only be createdwith the lipids undergoing fluid-to-gel conformational changes.

P-162Transient STAT recruitment into submicroscopicdomains at the membrane skeletonSara Lochte, Stephan Wilmes, Changjiang You,Christian Richter, Jacob PiehlerUniversity of Osnabruck, Department Biology, Divisionof BiophysicsE-mail: sara.loechte@biologie.uni-osnabrueck.de

The type I interferon receptor is comprised of two subunitsIFNAR1 and IFNAR2, which are cross-linked by their ligand.

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The key signaling pathway involves tyrosine phosphoryla-tion of signal transducers and activators of transcription(STAT1 and STAT2) by receptor-associated Janus kinases.We aim to unveil of the very early events of signal activationincluding ligand-induced receptor assembly and therecruitment of the cytoplasmic effector proteins STAT1 andSTAT2 in living cells. To this end, we have explored thespatiotemporal dynamics of STAT recruitment at the mem-brane on a single molecule level. Highly transientinteraction of STATs to membrane-proximal sites wasdetected by TIRF-microscopy, allowing for localizing andtracking individual molecules beyond the diffraction limit.Thus, we obtained a pattern of the spatio-temporal recruit-ment of STAT molecules to the plasma membrane revealingdistinct submicroscopic structures and hotspots of STATinteraction with overlapping recruitment sites for STAT1 andSTAT2. Strikingly, these STAT binding sites were indepen-dent on receptor localization and expression level.Simultaneous superresolution imaging of the cytoskeletonrevealed the organization of STAT recruitment sites withinthe cortical actin skeleton.

P-163Nanoscale fluorescence correlation spectroscopyon intact living cell membranes with NSOM probesCarlo Manzo1,2, Thomas S. van Zanten2,Maria F. Garcia-Parajo1,2,31ICFO-Institute of Photonic Sciences, Castelldefels(Barcelona), Spain, 2IBEC - Institute for Bioengineeringof Catalonia, Barcelona, Spain, 3 CIBER-bbn and ICREA -Institucio Catalana de Recerca i Estudis Avancats,Barcelona, Spain

Characterization of molecular dynamics on living cellmembranes at the nanoscale level is fundamental tounravel the mechanisms of membrane organization and-compartmentalization. We have recently demonstr ated thefeasibility of fluorescence correlation spectroscopy (FCS)based on the nanometric illumination of near-field scanningoptical microscopy (NSOM) probes on intact living cells [1].NSOM-FCS was applied to study the diffusion of fluores-cent lipid analogs on living CHO cells. The experimentsallowed to reveal details of the diffusion hidden by largerillumination areas and associated with nanoscale mem-brane compartmentalization. The technique also offers theunique advantages of straightforward implementation ofmultiple color excitation, opening the possibility to studynanoscale molecular cross-correlation. Furthermore, theNSOM probe evanescent axial illumination allows toextend diffusion study to the membrane-proximal cyto-solic region. As such, NSOM-FCS represents a novelpowerful tool to characterize the details of many biologicalprocesses in which molecular diffusion plays a relevantrole.

[1] Manzo C., T.S. van Zanten and M.F. Garcia-Parajo.2011. Biophys J. 100:L08-L10.

P-164Formation of ternary and quaternary lipid bilayerswith nano/microdomains on bare and modified goldJoaquim T. Marques, Rodrigo F.M. de Almeida,Ana S. VianaCQB, Departamento de Quımica e Bioquımica, Faculdadede Ciencias, Universidade de Lisboa, Campo Grande, Ed.C8, 1749-016 Lisboa, Portugal

The growing interest in supported lipid bilayers (SLBs) onconductive substrates, such as gold, is due to the possibilityof designing lipid-based biosensor interfaces with electro-chemical transduction. Due to the hydrophobicity of gold it isstill a challenge to deposit planar and continuous bilayerswithout previous surface modification. Most studies on goldconcern single-phase SLBs without cholesterol or ganglio-sides, two vital components of biomembranes.In this work the experimental conditions suitable for the for-mation of complex SLBs with phase-separation directly ongold are exploited. The mixtures DOPC/DPPC/cholesterol(4:4:2) with 0 or 10 mol % of ganglioside GM1, which shouldyield lipid raft-like domains according to reported phasediagrams, were studied. SLB with lipid rafts were success-fully formed onto bare Au (111), although surfacemodification with 11-mercapto-undecanoic acid SAM stabi-lized the SLBs due to its charge and hydrophilicity. Thedifferent experimental conditions tested had an impact onnano/microdomains organization observed by atomic forcemicroscopy in buffer solution. Surface characterizationthrough the combined use of ellipsometry, cyclic voltammetryand AFM allowed to optimize the conditions for the formationof more planar and compact SLBs.

P-165Mechanism of amyloid formation by Ab in raft-likemembranes containing ganglioside clustersKeisuke Ikeda, Takahiro Yamaguchi, Saori Fukunaga,Masaru Hoshino, Katsumi MatsuzakiGraduate School of Pharmaceutical Sciences, KyotoUniversity, Kyoto, Japan

It is widely accepted that the conversion of the soluble, nontoxicamyloid b-protein (Ab) monomer to aggregated toxic Ab rich inb-sheet structures is central to the development of Alzheimer’sdisease.However, themechanismof the abnormal aggregationof Ab in vivo is not well understood. We have proposed thatganglioside clusters in lipid rafts mediate the formation ofamyloid fibrils by Ab, the toxicity and physicochemical proper-ties of which are different from those of Ab amyloids formed insolution [1, 2]. In this presentation, we report a detailed mech-anism by which Ab-(1-40) fibrillizes in raft-like lipid bilayerscomposed of GM1/cholesterol/sphingomyelin. At lower con-centrations, Ab formed an a helix-rich structure, which wascooperatively converted to a b sheet-rich structure above athreshold concentration. The structurewas further changed to aseed-prone b structure at higher concentrations. The seedrecruited Ab in solution to form amyloid fibrils.

[1] Int. J. Alzheimers Dis. 95614 (2011).[2] Biochim. Biophys. Acta 1801, 868 (2010).

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P-167Alterations in membrane cholesterol potentiatecarbohydrate-dependent neutrophil degranulationAnn V. Mukhortava, Irina V. GorudkoDepartment of Biophysics, Belarussian State University,prosp. Nezavisimosti, 4, Minsk, 220030 Belarus

At present there is a lot of controversy about lipid raft role –glycolipid- and cholesterol-rich membrane microdomains –in neutrophil signal transduction. As a large fraction ofneutrophil receptors is glycosylated, this study wasdesigned to clarify lipid rafts role in carbohydrate-dependentneutrophil degranulation. The effect of methyl-b-cyclodextrin(MbCD), a well-known lipid raft disrupting agent, on neu-trophil degranulation induced by 8 plant lectins with differentcarbohydrate-binding specificity was examined. Lectinsfrom Phaseolus vulgaris (PHA-E), Triticum vulgaris (WGA),Vicia sativa (VSA), Canavalia ensiformis (ConA), Caraganaarborescens (CABA), Glycine hispida (SBA), Sambucusnigra (SNA) and Urtica dioica (UDA) were used for cellactivation. Activity of lysozyme released from cells wasassayed by measurement of the lysis rate of Micrococuslysodeikticus. Cholesterol extraction by MbCD resulted inpotentiating of neutrophil degranulating response to VSA,SBA, UDA, SNA and PHA-E, but had no effect on WGA-,ConA- and CABA-induced degranulation. Our results dem-onstrate that MbCD effects on neutrophil degranulationdepend on the lectin-induced signal transduction pathwaysand are readily resulted from recruitment of raft domainsfrom the specific granules. The observed synergetic effectMbCD and lectin on neutrophil degranulation apparentlytakes place for those lectins, which receptors are associ-ated with lipid rafts.

P-168The hedgehog receptor patched is involvedin cholesterol transportMichel Bidet1, Olivier Joubert 1, Benoit Lacombe1,Marine Ciantar1, Rony Nehme 1, Patrick Mollat1,Lionel Bretillon2, Helene Faure3, Robert Bittman4,Martial Ruat3, Isabelle Mus-Veteau11Universite de Nice - Sophia Antipolis, CNRS-UMR 6543,Institute of Developmental Biology and Cancer, Parc Valrose,Bat Recherche Sciences Naturelles, F-06108 Nice Cedex 2,France, 2Universite de Bourgogne, CNRS-INRA, Centre desSciences du Gout et de l’Alimentation, 17 rue Sully BP86510F-21065 Dijon France, 3CNRS, UPR-3294, Laboratoire deNeurobiologie et Developpement, Institut de NeurobiologieAlfred Fessard IFR2118, 1 avenue de la Terrasse, F-91198,Gif-sur-Yvette France, 4 Department of Chemistryand Biochemistry, Queens College of the CityUniversity of New York, Flushing, New York11367-1597 USA

Background: Sonic hedgehog (Shh) signaling plays a crucialrole in growth and patterning during embryonic development,but also in stem cell maintenance and tissue regeneration inadults. Aberrant Shh pathway activation is involved in thedevelopment of many tumors, and one of the most affectedShh signaling steps found in these tumors is the regulation of

the signaling receptor Smoothened by the Shh receptor Pat-ched. In thepresentwork,we investigatedPatchedactivity andthe mechanism by which Patched inhibits Smoothened.Methodology / Principal Findings: Using the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we firstobserved that the enhancement of the intracellular choles-terol concentration induces Smoothened enrichment in theplasma membrane, which is a crucial step for the signalingactivation. We found that binding of Shh protein to itsreceptor Patched, which involves Patched internalization,increases the intracellular concentration of cholesterol anddecreases the efflux of a fluorescent cholesterol derivative(BODIPY-Cholesterol) from these cells. Treatment of fibro-blasts with cyclopamine, an antagonist of Shh signaling,inhibits Patched expression and reduces BODIPY-Choles-terol efflux. We also show that the over-expression of humanPatched in the yeast S. cerevisiae results in a significantboost of the BODIPY-Cholesterol efflux. Furthermore, wedemonstrate that purified Patched binds to cholesterol, andthat the interaction of Shh with Patched inhibits the binding ofPatched to cholesterol.Conclusion / Significance: Our results suggest that Patchedmay contribute to the cholesterol efflux from cells, and tomodulation of the intracellular cholesterol concentration. Thisactivity is likely responsible for the inhibition of the enrich-ment of Smoothened in the cell plasma membrane which isan important step in the Shh pathway activation.

P-169SNARE-mediated membrane fusion assay basedon pore-suspending membranes: observing singlefusion eventsHenrik Neubacher, Ines Hofer, Karsten Meyenberg,Ulf Diederichsen and Claudia SteinemGeorg-August-Universitat Gottingen, Institute of Organicand Biomolecular Chemistry, Tammannstr. 2,37077 Gottingen, Germany

We developed a new versatile single-vesicle fusion assay tostudy the molecular mechanisms of membrane fusion, basedon pore-suspending membranes. This system offers advan-tages over other model membrane systems, such as blacklipid membranes (BLMs) or solid supported membranes(SSMs). Specifically, it provides a solvent-free environmentwith aqueous compartments that are accessible on bothsides of the membrane. With this assay, we were able toobserve single fusion events of large unilamellar vesicles(LUVs) with pore-suspending membranes using confocallaser scanning fluorescence microscopy. Membranes wereprepared by spreading of giant unilamellar vesicles (GUVs)on highly ordered porous silicon substrates. GUVs weredoped with the fluorescent dye Oregon Green DHPE, LUVswith Texas Red DHPE. Membrane fusion could be observedthrough Forster resonance energy transfer that occurs if thetwo lipid dyes come in close proximity as a result of lipidmixing. Membrane fusion was mediated using SNARE-derived artificial peptides, consisting of a coiled-coil-formingthree-heptad repeat segment that is linked to a SNARE-transmembrane domain anchoring it to the lipid membrane.

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Currently, a setup is developed to be able to also monitor thecontent release that occurs upon fusion.

P-170Effect of dl-alpha-tocopherol glicosidic derivativeon phospholipids monolayer structureG. Neunert1, J. Makowiecki2, R. Hertmanowski2,T. Martynski2, K. Polewski11Poznan University of Life Sciences, Poznan, Poland,2Poznan University of Technology, Poznan, Poland

Phospholipid dipalmitoyl phosphatidylcholine (DPPC) and itsbinary mixtures with dl-a-tocopheryl b-D-glucopyranoside*(BG) in Langmuir and Langmuir–Blodgett films have beenstudied. Surface pressure versus mean molecular area (p–A)isotherms for Langmuir films were recorded. Langmuir-Blodgett monolayers were visualized with atomic forcemicroscopy (AFM).The plateau surface pressure of BG/DPPC mixturesmonolayers is shifted proportionally with increasing concen-tration of BG. This flat region of the curve disappears formonolayers with mole fraction (MF) of BG greater than 0.2.At higher surface pressures the isotherms exhibit composedcharacter. To obtain information about miscibility meanmolecular area was analyzed. For MFB0.3 the excess areaper molecule parameter, AE, possess value in the range ofmeasuring error what suggests either complete miscibility, orideal phase separation of the components. For MF[0.3 thepositive deviation of AE is observed, indicating partial misci-bility and repulsive interaction between DPPC and BGmolecules.AFM image of mixed BG/DPPC monolayer with mole fractionof BG MF=0.1 shows 10 lm circular forms of liquid con-densed phase which coexist with small grains of liquidexpanded phase. The measured height of pure DPPCmonolayer is greater than for monolayer with MF=0.1 of BG.It may be assigned to larger tilt angle of the molecules to thenormal to the surface or greater elasticity of the monolayer inthe presence of BG molecules. The AFM image for BG/DPPC monolayer shows the randomly distributed precipita-tions on borders of the condensed phase domains. Theyappear in the form of narrow cone with height of 2-5 nm.Since they are seen only in BG/DPPC mixture we assumethat those are aggregated or crystalline forms of BG.During p–A isotherms and AFM studies of monolayer of BG/DPPC mixtures it has been shown that presence of BGmolecules changes the structure of DPPC monolayer in a BGconcentration dependent manner. Additionally, presence ofBG in the monolayer mixture increases its stability comparedto DPPC monolayer.

AcknowledgmentsThis study was supported by the interdisciplinary grant No.12/782/WI/10 from Poznan University of Life Sciencesand Poznan University of Technology.*dl-a-tocopherylb-D-glucopyranoside is the gift from Prof. S.Witkowski fromBiałystok University, Poland.

P-171Interaction of segment H1 (NS4B_H1) from HCV proteinNS4B with model biomembranesM. Francisca Palomares Jerez, Henrique de Castro Nemesioy Jose VillalaınInstituto de Biologıa Molecular y Celular, Universidad MiguelHernandez, E-03202 Elche (Alicante), Spain

Hepatitis C virus (HCV) has a great impact on public health,affecting more than 170million people worldwide since it is thecause of liver-related diseases such as chronic hepatitis, cir-rhosis and hepatocarcinoma. HCV entry into the host cell isachieved by the fusion of viral and cellular membranes, repli-cates its genome in a membrane associated replicationcomplex, and morphogenesis has been suggested to takeplace in the endoplasmic reticulum (ER) or modified ERmembranes. The variability of theHCVproteins gives the virusthe ability to escape the host immune surveillance system andnotably hampers the development of an efficient vaccine. HCVhas a single-stranded genome which encode a polyprotein,cleaved by a combination of cellular and viral proteases toproduce the mature structural proteins (core, E1, E2, and p7)and non-structural ones (NS2, NS3, NS4A, NS4B, NS5A andNS5B), the latter associated with the membrane originatedfrom the ER in the emerging virus. The NS4B protein, a fun-damental player in the HCV replicative process and the leastcharacterized HCV protein, is a highly hydrophobic proteinassociated with ER membranes. It has recently been shownthat the C-terminal is palmitoylated and the N-terminal regionhas potent polymerization activity. The expression of NS4Binduces the formation of the so calledmembranousweb,whichhas been postulated to be the HCV RNA replication complex.Thus, a function of NS4B might be to induce a specific mem-brane alteration that serves as a scaffold for the formation ofthe HCV replication complex and therefore has critical role inthe HCV cycle. Due to the high hydrophobic nature of NS4B, adetailed structure determination of this protein is very difficult.The NS4B protein is an integral membrane protein with four ormore transmembrane domains. The C-terminal region ofNS4B is constituted by two a helices, H1 (approximately fromamino acid 1912 to 1924) and H2 (approximately from aminoacid 1940 to 1960), which have been studied as potential tar-gets for inhibiting HCV replication. Previous studies from ourgroup, based on the study of the effect of NS4B peptidelibraries on model membrane integrity, have allowed us topropose the location of different segments in this protein thatwouldbe implicated in lipid-protein interaction.Additionally, theH1 region could be an essential constituent in the interactionbetween protein and membrane. In this study we show thatpeptides derived from the C-terminal domain of NS4B proteinof HCVare able to interact with high affinity to biomembranes,significantly destabilizing them and affecting their biophysicalproperties. Therewere also differences in the interaction of thepeptide depending on the lipid composition of the membranesstudied. We have also applied fluorescence spectroscopy,infrared spectroscopy and differential scanning calorimetrywhich have given as a detailed biophysical study of the inter-action of the peptide with model biomembranes.This work was supported by grant BFU2008-02617-BMC(Ministerio de Ciencia y Tecnologıa, Spain) to J.V.

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P-172Theoretical and computational models for explainingthe enhancement of the fusion rate of chargedmembranes in presence of water soluble polymerAntonio Raudino1, Martina Pannuzzo1,2, Mikko Karttunen21Dept. of Chemical Science, University of Catania, Catania,Italy, 2Dept. of Applied Mathematics, Western OntarioUniversity, London (ON), Canada,E-mail: mpannuzz@uwo.ca

A semi-quantitative theory describing the adhesion kineticsbetween soft objects as living cells or vesicles was developed.The nucleation-like mechanism has been described in theframework of a non-equilibrium Fokker-Planck approachaccounting for the adhesion patch growth and dissolution (A.Raudino, M. Pannuzzo, J. Chem. Phys. 132, 045103 (2010)).A well known puzzling effect is the dramatic enhancement ofthe adhesion/fusion rate of lipid membranes by water-solublepolymers that do not specifically interact with the membranesurface.We extend the previous approach by Molecular Dynamicssimulations in the framework of a coarse-grained picture of thesystem (lipid+polymer+ions embedded in an explicit watermedium) in order to test and support our previous analyticalresults.Simulations show that the osmotic pressure due to the polymerexclusion from the inter-membrane spacing is partially bal-anced by an electrostatic pressure. However, we alsoevidencedan interesting coupling between osmotic forces andelectrostatic effects. Indeed, when charged membranes areconsidered, polymers of low dielectric permittivity are partiallyexcluded from the inter-membrane space because of theincreased local salt concentration. The increased salt con-centration means also a larger density of divalent ions whichform a bridge at the contact region (stronger adhesion). Theoverall effect is a smaller membrane repulsion. This effectdisappears when neutral membranes are considered.The model could explain the fusion kinetics between lipidvesicles, provided the short-range adhesion transition is therate-limiting step to the whole fusion process.

P-173The role of ceramide acyl chain length and unsaturationon membrane structureSandra N. Pinto1, Liana C. Silva2, Anthony H. Futerman3,Manuel Prieto41Centro de Quımica- Fısica Molecular and IN- Instituteof Nanosciences and Nanotechnology. Instituto SuperiorTecnico, Lisboa, Portugal, 2iMed.UL, Faculdade deFarmacia, Universidade de Lisboa, Lisboa, Portugal,3Department of Biological Chemistry, Weizmann Instituteof Sciences, Rehovot, Israel

Ceramide fatty acid composition selectively regulates distinctcell processes by a yet unknown mechanism. However,evidence suggests that biophysical processes are importantin the activation of signalling pathways. Indeed, ceramidestrongly affects membrane order, induces gel/fluid phaseseparation and forms highly-ordered gel domains. Theimpact of ceramide N-acyl chain in the biophysical propertiesof a fluid membrane was studied in POPC membranes mixed

with distinct ceramides. Our results show that: i) saturatedceramide has a stronger impact on the fluid membrane,increasing its order and promoting gel/fluid phase separation,while their unsaturated counterparts have a lower (C24:1ceramide) or no (C18:1 ceramide) ability to form gel domainsat physiological temperature, ii) differences between distinctsaturated species are smaller and are related mainly todomain morphology, and iii) very long chain ceramide indu-ces the formation of tubular structures probably associatedwith interdigitation. These results suggest that generation ofdifferent ceramide species in cell membranes has a distinctbiophysical impact with acyl chain saturation dictatingmembrane lateral organization, and chain asymmetry gov-erning interdigitation and membrane morphology.

P-174Smoothing effect of cholesterol on a phosphatydylo-choline bilayer. A molecular simulation studyElzbieta Plesnar1, Witold K. Subczynski2,Marta Pasenkiewicz-Gierula31Faculty of Biochemistry, Biophysics and Biotechnology,Jagiellonian University, Krakow, Poland, 2Departmentof Biophysics, Medical College of Wisconsin, Milwaukee,Wisconsin, USA

Extra high content of cholesterol (Chol) in fiber-cell membranesin the eye lens leads to Chol saturation and formation of cho-lesterol bilayer domains (CBDs). It is hypothesized that highenrichment in cholesterol helps to maintain lens transparencyand protect against cataractogenesis. In model studies, theCBD is formed in a phospholipid bilayer when cholesterolcontent exceeding the cholesterol solubility threshold, thus, theCBD is surrounded by phospholipid bilayer saturated withcholesterol. In the present study, we carried out moleculardynamics (MD) simulation of two bilayers: a palmitoyloleoyl-phosphatidylcholine (POPC) bilayer (reference) and a 1:1POPC-Chol bilayer, to investigate the smoothing effect of thesaturating amount of cholesterol on the bilayer. To our knowl-edge, this effect has not been studied so far so this study iscertainly providing new results. Our results indicate that satu-ration with cholesterol significantly narrows the distribution ofvertical positions of the center-of-mass of the POPCmoleculesand the POPC atoms in the bilayer and smoothes the bilayersurface. We hypothesize that this smoothing effect decreaseslight-scattering and helps to maintain lens transparency.

P-175Biophysical characterisation of the Staphylococcusaureus membrane: a neutron diffraction studyR. Rehal1, F. Sebastiani3, K. Homer1, K. Bruce1,G. Fragneto2 and R. Harvey11King’s College London, Franklin-Wilkins Building,150 Stamford Street, London SE1 9NH, UK, 2 InstitutLaue-Langevin, BP 156, 6 rue Jules Horowitz, 38042Grenoble Cedex 9, France, 3 Universita degli Studi di Roma‘‘La Sapienza’’, Piazzale Aldo Moro 5, 00185 Roma, Italy

The phospholipid content of Staphylococcus aureus mem-branes displays a high degree of variability (1-3). The majorphospholipids found in S. aureus are phosphatidylglycerol

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(PG), cardiolipin (CL) and lysylphosphatidylglycerol (LPG),(1) the concentrations of which are environment dependentand see fluctuations on exposure to high concentrations ofpositively charged moieties (4). Up regulation of LPG has asuspected role in neutralisation of the plasma membrane inresponse to cationic threats. Studies have been conductedto probe biomimetic models of this theory however our focusis to look at atomic details of membrane extracts in thepresence of magaininF5W. S. aureus 476 lipid extracts fromcells grown at pH 5.5 and 7.0, studies by neutron diffractionwith and without peptide at two contrasts. D-spacings wereassessed by Vogt area fitting and Bragg’s law. Bilayerseparation at low pH was *1-2 A less than pH 7.0. Withpeptide, bilayer separations of pH 5.5 and 7.0 extracts werereduced by *2 A and *4 A, respectively. Reduced PGcontent of low pH extracts is suggested to reduce D-spac-ing, however presence of peptide further reduces this,possibly by an anion neutralisation effect. AbnormalD-spacing on increased humidity may be due to the break-down of LPG.

(1) Mukhopadhyay K, et al. Microbiology 2007 153:1187.(2) Ernst, CM, et al. PLoS Pathog 2009 11/13;5(11):

e1000660.(3) Peschel, A, et al. J Exp Med 2001 193:1067.(4) Gould, RM, & Lennarz, WJ. J Bacteriol 1970 104:1135.

P-177Chlorohydrins induce oxidative stress, phosphorylationof p38 kinase and apoptosis of HUVEC-ST cellsAgnieszka Robaszkiewicz, Grzegorz Bartosz,Miroslaw SoszynskiDepartment of Molecular Biophysics, University of Lodz,Pomorska 141/143, 90-236 Lodz, Poland

Activation of neutrophils releasing HOCl and apoptosis ofvein endothelial cells are the events documented to occur inthe course of atherosclerosis. As lipid chlorohydrins, whichare the key products of the reaction between HOCl andunsaturated fatty acid residues, were found in atheroscleroticplaques, we decided to check their biological activity in thecontext of their ability to act as the mediators of HOCl-induced oxidative stress and apoptosis in the culture ofimmortalized human umbilical vein endothelial cells (HU-VEC-ST). The concentration of reactive oxygen species wasfound to be elevated after 1 h cell incubation with phos-pahtidylcholine chlorohydrins. This effect was at leastpartially caused by the leakage of superoxide anion frommitochondria and followed by depletion of GSH and totalthiols. The significant decrease of antioxidant capacity of cellextracts was also observed. The intracellular red-ox imbal-ance was accompanied by the increase of the ratio betweenphosphorylated and dephosphorylated forms of p38 MAPkinase. After longer incubation a significant number ofapoptotic cells appeared. Summing up, phosphatidylcholinechlorohydrins may be regarded as signaling molecules, ableto initiate signalling pathways by induction of oxidative stress.

P-179Influence of avidin-coated surface for GUV immobilizationon lipid domain size and distribution.Maria Joao Sarmento, Manuel Prieto, Fabio FernandesCentro de Quımica-Fısica Molecular and Instituteof Nanoscience and Nanotechnology, Instituto SuperiorTecnico, Complexo I, Avenida Rovisco Pais, 1049-001Lisbon, Portugal

Giant unilamellar vesicles (GUVs) are a valuable tool in thestudy of lateral distribution of biological membrane compo-nents. GUV dimensions are comparable to typical cellplasma membranes and lipid phase separation can beobserved through fluorescence microscopy. GUV studiesfrequently require immobilization of the vesicles, and severalmethods are available for that effect. One of the most com-mon methodologies for vesicle immobilization is the use ofavidin/streptavidin coated surfaces and biotin labeled lipidsat very low concentration in the vesicles. Here, we analyzethe effect of using this methodology on lipid domain distri-bution for different lipid compositions. We show that as aresult of non-homogeneous distribution of biotin labeled lip-ids between liquid disordered, liquid ordered and gel phases,distribution of lipid domains inside GUVs can be dramaticallyaffected.

P-180Monitoring membrane permeability: developmentof a SICM approachChristoph Saßen1,2 and Claudia Steinem1

1Institute for Organic and Biomolecular Chemistry, Universityof Gottingen, Tammannstraße 2, 37077 Gottingen, Germany,2GGNB Doctoral Program: IMPRS, Physics of Biologicaland Complex Systems

Scanning ion conductance microscopy (SICM) utilises ananopipette containing an electrode as a probe for surfaceinvestigations with resolutions of 1/3 of the inner pipettediameter. Experiments are conducted under physiologicalconditions, in situ and without mechanical contact of probeand sample. Hence, SICM serves as a well-suited techniquefor the investigation of soft objects such as cells or artificiallipid membranes.Using pore-suspending membranes (PSM) as a model sys-tem, interactions of melittin as an example for cell penetratingpeptides (CPPs) and lipid membranes are investigated bymeans of SICM. Formation of a range of solvent free PSMfrom lipid vesicles has been achieved as confirmed bymeans of fluorescence microscopy and SICM. Application ofmelittin results in rupturing of the lipid bilayer.Putative insights gained from this assay are critical concen-trations of membrane permeabilising CCPs and answers tomechanistic questions, e.g. whether CCPs translocate onlyor form pores within the lipid bilayer.

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P-181Min-waves on confined artificial membranesJakob C. Schweizer1,#, Martin Loose1,3,#,Ingolf Monch2 and Petra Schwille11Biophysics, BIOTEC, Dresden University of Technology,Tatzberg 47-51, 01307 Dresden, Germany, 2Institutefor Integrative Nanosciences, IFW Dresden, HelmholtzStrasse 20, 01069 Dresden, Germany, 3present address:Department of Systems Biology, Harvard Medical School,200 Longwood Avenue, Boston, MA 02115, USA#These authors contributed equally to this workE-mail: petra.schwille@biotec.tu-dresden.de

Positioning of the Z-Ring in Escherichia Coli prior to cell divi-sion is regulated by intracellular pole-to-pole oscillation andmembrane binding of Min proteins, allowing assembly of FtsZfilaments only at the center plane of the cell. In order toinvestigate the influence of membrane geometry on thedynamic behavior of membrane binding of Min proteins, wecombined concepts of synthetic biology and microfabricationtechnology. Glass slides were patterned by a gold coating withmicroscopic windows of different geometries, and supportedlipid bilayers (SLB) were formed on these microstructures. OnSLBs, Min-proteins organize into parallel waves. Confinementof the artificial membranes determined the direction of propa-gation.Min-waves could be guided along curved membranestripes, in circles and even along slalom-geometries. In elon-gated membrane structures, the protein waves alwayspropagate along the longest axis. Coupling of protein wavesacross spatially separated membrane patches was observed,dependent on gap size and viscosity of the aqueous mediaabove the bilayer. This indicates the existence of an inhomo-geneous and dynamic protein gradient above the membrane.

O-182Minimal systems for membrane associated cellularprocessesPetra SchwilleBio Technology Center BIOTEC, Technical Universityof Dresden, Germany

The strive for identifying minimal biological systems, particu-larly of subcellular structures or modules, has in the past yearsbeen very successful, and crucial in vitro experiments withreduced complexity can nowadays be performed, e.g., onreconstituted cytoskeleton and membrane systems. In thisoverview talk, I will first discuss the virtues of minimal mem-brane systems, such as GUVs and supported membranes, inquantitatively understand protein-lipid interactions, in particularlipid domain formation and its relevance on protein function.Membrane transformations, such as vesicle fusion and fission,but also vesicle splitting, can be reconstituted in these simplesubsystems, due to the inherent physical properties of selfas-sembled lipids, and it is compelling question how simple aprotein machinery may be that is still able to regulate thesetransformations. As an exciting example of the power of mini-mal systems, I show how the interplay between a membraneand only two antagonistic proteins from the bacterial celldivision machinery can result in emergence of proteinself-organization and pattern formation, and discuss the pos-sibility of reconstituting a minimal divisome.

P-183Quantitative microscopic analysis reveals cellconfluence regulated divergence of PDGFR-initiatedsignaling pathways with logically streamlined cellularoutputsArpad Szo}or, Laszlo Ujalky-Nagy, Janos Szoll}osi,Gyorgy VerebUniversity of Debrecen, Department of Biophysics and CellBiology, Debrecen, Hungary

Platelet derived growth factor receptors (PDGFR) play animportant role in proliferation and survival of tumor cells.PDGF-BB stimulation caused a redistribution of PDGFreceptors towards GM1 rich domains, which was moreprominent in confluent monolayers. PDGF-BB stimulationsignificantly increased relative receptor phosphorylation ofthe Ras / MAPK pathway specific Tyr716 residues and thePI3-kinase / Akt pathway specific Tyr751 residues in non-confluent cultures. Tyr771 residues that serve as adaptors forRas-GAP which inactivates the MAPK pathway and Tyr1021residues feeding into the PLC-gamma / CaMK-PKC pathwaywere the docking sites significantly hyperphosphorylationfollowing ligand stimulation in confluent cells. We found thatp-Akt facilitated cell survival and pMAPK dependent prolif-eration is more activated in dispersed cells, whilephospholipase C-gamma mediated calcium release andPKC-dependent RhoA activation are the prominent outputfeatures PDGF stimulus achieves in confluent cultures.These observations suggest that the same stimulus is able topromote distinctly relevant signaling outputs, namely, celldivision and survival in sparse cultures and inhibition ofproliferation joined with promotion of migration in confluentmonolayers that appear contact inhibited.

P-184A thermodynamic approach to phase coexistencein ternary cholesterol-phospholipid mixturesJean Wolff, Carlos M. Marques and Fabrice ThalmannInstitut Charles Sadron, Universite de Strasbourg, CNRSUPR, 22, 23 rue du Loess, Strasbourg Cedex, F-67037,FranceE-mail: thalmann@ics-cnrs.unistra.fr

We present a simple and predictive model for describing thephase stability of ternary cholesterol-phospholipid mixtures.Assuming that competition between the liquid and gel orga-nizations of the phospholipids is the main driving forcebehind lipid segregation, we derive a phenomenologicalGibbs free-energy of mixing, based on the calorimetricproperties of the lipids main transition. Gibbs phase diagramsare numerically obtained that reproduces the most importantexperimental features of DPPC-DOPC-Chol membranes,such as regions of triple coexistence and liquid ordered -liquid disordered segregation. Based on this approach, wepresent a scenario for the evolution of the phase diagramwith temperature. Results for other phospholipid species,such as POPC or PSM will also be presented.J. Wolff, C. M. Marques, and F. Thalmann Phys. Rev. Lett.106, 128104 (2011)

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P-185Membrane potential influences mobility, interactionsand signaling of interleukin-2 and -15 receptors in T cellsV. Sebestyen1, E. Nagy1, G. Mocsar1, F. Papp1,P. Hajdu2, G. Panyi2, K. Toth3, T. A. Waldmann4,S. Damjanovich1, A. Bodnar1, G. Vamosi11Cell Biology and Signaling Research Group of the Hung. Acad.Sci., Hungary, 2Dept. of Biophysics and Cell Biology, Medicaland Health Science Center, Univ. of Debrecen, Hungary,3Div. Biophysics of Macromolecules, German Cancer ResearchCenter, Heidelberg, Germany, 4Metabolism Branch, Centerfor Cancer Research, National Cancer Institute, NationalInstitutes of Health, Bethesda, MD, USAE-mail: even@mailbox.hu

Interleukin-2 and -15 receptors play a central role in theactivation, survival and death of T lymphocytes. They formsupramolecular clusters with MHC I and II glycoproteins in Tcells. In damaged or inflamed tissues the extracellular K+concentration increases, which can depolarize the mem-brane. The common signaling beta and gamma chains of IL-2/15R are phosphorylated upon cytokine binding and get apermanent dipole moment, thus their conformation, interac-tions, mobility and activity may be sensitive to the membranepotential. We induced depolarization on FT 7.10 T lymphomacells by increasing the ec. K+ level or by blocking Kv 1.3voltage gated K+ channels with margatoxin. FCS measure-mens showed that the lateral mobility of Fab-labeled IL-2/15R and MHC I and II decreased upon depolarization, whilethat of GPI-linked CD48 did not change. FRET efficiencymeasured between some elements of the IL-receptor/MHCcluster increased, which may reflect an increase of clustersize. IL-2-induced receptor activity, as monitored bymeasuring STAT5-phosphorylation, increased upon depo-larization, whereas IL-15 induced phosphorylation did notchange. Our results may reveal a novel regulatory mecha-nism of receptor function by the membrane potential.

P-186MHC I organizes protein clusters and inhibits IL-2/IL-15signaling in human T cellsJ. Volko1, G. Mocsar1, E. Menczel1, P. Nagy2,K. Toth3, T. A. Waldmann4, S. Damjanovich1,A. Bodnar1, G. Vamosi11Cell Biology and Signaling Research Group of the Hung.Acad. Sci. and 2Dept. of Biophysics and Cell Biology, Medicaland Health Science Center, Univ. of Debrecen, Hungary,3Div. Biophysics of Macromolecules, DKFZ, Heidelberg,Germany, 4Metabolism Branch, Center for Cancer Research,National Cancer Institute, National Institutes of Health,Bethesda, MD, USA

Major histocompatibility class I and II glycoproteins andinterleukin-2 and -15 receptors form supramolecular clustersin lipid rafts of FT7.10 T lymphoma cells. IL-2 and IL-15

cytokines play an important role in T cell activation andimmunological memory, whereas MHCs are known for therole in antigen presentation.We applied RNAi to silence the expression of MHC I in order tostudy its possible role in receptor assembly and function.FRET data indicated that the association of IL-2R and IL-15Rwith MHC I as well as between IL-2R and IL-15R weakened.FCS indicated an increase of receptormobility also suggestingthe partial disassembly of the clusters. MHC I gene silencinglead to a remarkable increase of IL-2/IL-15 induced phos-phorylation of STAT5 transcription factors. In search for themolecular background of this inhibition of signaling by MHC Iwe checked IL-2 binding and the formation of the receptorcomplex (IL-2R alpha - IL-2R beta association), but we did notfind a difference as compared to the control.Our results suggest that MHC I plays an organizing role inmaintaining supramolecular receptor clusters and inhibits IL-2R signaling, revealing a nonclassic new function of MHC Ibeyond its classical role in antigen presentation.

P-187Monitoring Interleukin-4 receptor activation:from the cell surface into endosomesThomas Weidemann1, Remigiusz Worch1,3, KristinaKurgonaite2, Hetvi Gandhi1, Christian Bokel2,Petra Schwille11Biophysics Research Group, BIOTEC, TechnischeUniversitat Dresden, Germany, 2CRTD, Centerfor Regenerative Therapies Dresden, Technische UniversitatDresden, Germany, 3Institute of Physics, Polish Academyof Sciences, Warsaw, Poland

Interleukin-4 (IL-4) is an important cytokine involved inadaptive immunity. IL-4 binds with high affinity the single-pass transmembrane receptor IL-4Ra. The occupied com-plex, IL-4/IL-4Ra then engages either IL-2Rc or IL-13Ra1,to form an activated type I or II receptor, respectively. Thisformation of heterodimers is believed to trigger cross-acti-vation of intracellular Janus kinases. Here we follow afluorescently labeled ligand through various stages ofreceptor activation in HEK293T: Using fluorescence corre-lation spectroscopy (FCS), we see that the receptor chainsdiffuse as monomers within the plasma membrane. Usingdual-color FCCS provides direct evidence for ligandinduced co-diffusion of occupied IL-4Ra and IL-13Ra1. Incontrast, type I complexes containing IL-2Rc could not beobserved. However, ectopic expression of GFP-tagged IL-2Rc/JAK3 induced stable fluorescent speckles in or closeto the plasma membrane. We identified these structures asearly sorting endosomes by colocalization of surfacemarkers like EEA1 and Rab GTPases. The IL-4Ra chain iscontinuously trafficking into these compartments. Theseobservations suggest that the formation of a type I IL-4Rheterodimer may require internalization and that early en-dosomes serve as a platform for IL-4 signaling.

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O-188Spot variable Fluorescence Correlation Spectroscopyreveals fast scouting of K-Ras at the plasma membraneof living cellsTomasz Trombik1?, Verena Ruprecht2?,Sebastien Mailfert1?, Cyrille Billaudeau1,Fabien Conchonaud1, Gerhard J. Schutz2,Yoav Henis3, Stefan Wieser1,2

and Didier Marguet1*1Centre d’Immunologie de Marseille-Luminy,CNRS-INSERM-Universite de la Mediterranee, Marseille,France, 2Biophysics Institute, Johannes-Kepler-UniversityLinz, Austria, 3 Department of Neurobiology,George S Wise Faculty of Life Sciences,Tel Aviv University, Israel

Among the membrane associated proteins, the Ras family,which is lipid-anchored G protein, plays a key role in a largerange of physiological processes and, more importantly, isderegulated in a large variety of cancer. In this context,plasma membrane heterogeneity appears as a central con-cept since it ultimately tunes the specification and regulationof Ras-dependent signaling processes.Therefore, to investigate the dynamic and complex mem-brane lateral organization in living cells, we have developedan original approach based on molecule diffusion measure-ments performed by fluorescence correlation spectroscopyat different spatial scales (spot variable FCS, svFCS) (1).We have shown in a variety of cell types that lipid-based nanodomains are instrumental for cell membranecompartmentalization. We have also observed that these-nanodomains are critically involved in the activation ofsignaling pathways and are essential for physiologicalresponses (2-3).More recently, weextend the application of svFCS tocharacterizethe dynamics of K-Rasproteinat the plasmamembrane. As major result, we demonstrated that the rateof K-Ras association/dissociation from the membrane isfast but vary as a functional of the activation state ofthe molecule as well as of specific intracellular proteininteractions.*Correspondance : D. Marguet, marguet@ciml.univ-mrs.fr,CIML, Parc Scientifique de Luminy, Case 906, F-13288Marseille Cedex 9, France, Phone: +(33)491 269 128, FAX:+(33)491 269 430

(1) He HT, Marguet D ‘‘Detecting nanodomains in living cellmembrane by fluorescence correlation spectroscopy’’Annu Rev PhysChem 2011 62:417-36

(2) Lasserre R et al ‘‘Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation’’NatChemBiol 2008 4:538-47

(3) Guia S et al ‘‘Activating receptor confinement at theplasma membrane controls Natural Killer cell tolerance’’Science Signaling 2011 4:ra21

Aggregated proteins

P-190Insights into the molecular mechanisms of Hsp70-mediated inhibition of amyloid formationFrancesco A. Aprile1,2, Anne Dhulesia2, Florian Stengel3,Tommaso Eliseo2, Cintia Roodvelt2, Benedetta Bolognesi2,Paolo Tortora1, Xavier Salvatella4, Nunilo Cremades2,Christopher M. Dobson21Department of Biotechnology and Biosciences, Universityof Milano-Bicocca, Milan, Italy, 2Department of Chemistry,University of Cambridge, Cambridge, UK, 3Departmentof Chemistry, University of Oxford, Oxford, UK, 4Institutefor Research in Biomedicine, Barcelona, Spain

Molecular chaperones have been recognised as key players inthe avoidance of misfolding and amyloid fibril formation. Inparticular, recent evidencesdemonstrate that lossof chaperoneheat-shock protein 70kDa (Hsp70) activity is strictly relatedwiththe formation of intra-neuronal inclusions of alpha-synuclein(AS), one of the main hallmarks of Parkinson’s disease.Human Hsp70 is composed of two functional domains; the44-kDa N-terminal nucleotide-binding domain (NBD), withATPase activity, and the 30-kDa C-terminal substrate-bindingdomain (SBD). With the aim of gaining insight into Hsp70substrate recognition process of AS in relation with the Hsp-70 mediated inhibitory effect on AS aggregation, we havecharacterized both processes in detail using full-lengthHsp70 and several truncated variants and by means of awide range of biophysical techniques, including nmR.We have so demonstrated that an helical lid sub-domain inthe SBD is essential for monomeric AS binding, but not forthe anti-aggregation activity of the chaperone, suggestingthat Hsp70 is able to interact with pre-fibrillar oligomericspecies formed during AS aggregation and that, then, themechanism of binding for these species is different from thatof the monomeric protein.

P-191Characterizing the amyloidogenic state of theacylphosphatase from Sulfolobus solfataricusFrancesco Bemporad1, Alfonso De Simone1,Fabrizio Chiti2, Christopher M. Dobson11Department of Chemistry, University of Cambridge,Lensfield Road, Cambridge CB2 1EW, UK, 2Dipartimento diScienze Biochimiche, Universitr degli Studi di Firenze, VialeMorgagni 50, 50134, Firenze, Italy

Aggregation of the acylphosphatase from Sulfolobus sol-fataricus (Sso AcP) into amyloid-like protofibrils is inducedby the establishment of an intermolecular interactionbetween a 11-residue unfolded segment at the N-terminusand the globular unit of another molecule. We have useddata from hydrogen/deuterium exchange experiments,intermolecular paramagnetic relaxation enhancements and

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isothermal titration calorimetry measurements on anaggregation-resistant Sso AcP variant lacking the 11-resi-due N-terminus to characterize the initial steps of theaggregation reaction. Under solution conditions that favouraggregation of the wild-type protein, the truncated proteinwas found to interact with a peptide corresponding to theN-terminal residues of the full length protein. This interac-tion involves the fourth strand of the main b-sheet structureof the protein and the loop following this region andinduces a slight decrease in protein flexibility. We suggestthat the amyloidogenic state populated by Sso AcP prior toaggregation does not present local unfolding but is char-acterized by increased dynamics throughout the sequencethat allow the protein to establish new interactions, leadingto the aggregation reaction.

O-192Amyloid-like aggregates alter the membrane mobilityof GM1 gangliosidesMartino Calamai and Francesco PavoneUniversity of Florence, LENS - European Laboratory for Non-Linear Spectroscopy, Sesto Fiorentino, Florence, Italy

Neuronal dysfunction in neurodegenerative pathologies suchas Alzheimer’s disease is currently attributed to the interac-tion of amyloid aggregates with the plasma membrane.Amongst the variety of toxic mechanisms proposed, oneinvolves the binding of amyloid species to GM1 gangliosides.GM1 takes part into the formation of membrane rafts, andexerts antineurotoxic, neuroprotective, and neurorestorativeeffects on various central neurotransmitter systems. In thisstudy, we investigated the effects of amyloid-like aggregatesformed by the highly amyloidogenic structural motif of theyeast prion Sup35 (Sup35NM) on the mobility of GM1 on theplasmamembrane of living cells.Preformed Sup35NM aggregates were incubated with cellsand GM1 molecules were subsequently labeled with biotin-ylated CTX-B and streptavidin quantum dots (QDs). SingleQDs bound to GM1 were then tracked. The mobility of GM1was found to decrease dramatically in the presence ofSup35NM aggregates, switching from Brownian to mainlyconfined motion.The considerable interference of amyloid-like aggregateswith the lateral diffusion of GM1 might imply a consequentloss of function of GM1, thus contributing to explain the toxicmechanism ascribed to this particular interaction.

P-193Insights into the early stages of fibrillogenesis of insulinusing mass spectrometryHarriet L. Cole1,2, Massimiliano Porrini2,Cait E. MacPhee2 and Perdita E. Barran11School of Chemistry, University of Edinburgh, Joseph BlackBuilding The Kings Buildings, Edinburgh, EH9 3JJ, UK2School of Physics, University of Edinburgh, James ClerkMaxwell Building The Kings Buildings, Edinburgh,EH9 3JZ, UK

Insulin is a vital hormone in metabolic processes as it regu-lates the glucose levels in the body. Insulin is stored in the b

cells of the pancreas as a hexamer, however its biologicallyactive form is the monomer. The formation of fibrillar aggre-gates of insulin rarely occurs in the body; however localisedamyloidosis at the site of injection for diabetes patients andaggregation of pharmaceutical insulin stocks presentproblems.In the current study oligomers formed early in the process offibril assembly in vitro are observed by mass spectrometry(MS). MS is the only technique which allows early species tobe characterised as it can identify different oligomeric ordersby mass to charge ratio and show protein abundance andaggregation propensity.on mobility MS is used to examine rotationally averagedcollision cross sections of oligomers in the aggregatingsolution. A wide array of oligomers is observed and thestability of specific species is remarked. The presence ofmultiple conformations for the highly charged oligomers isparticularly noted and their assignment confirmed usingFourier Transform Ion Cyclotron Resonance MS and collisioninduced dissociation. Molecular modelling has been used tofurther explore the conformational space the oligomersinhabit.

P-194Viscometric detection of conformational transitionsand aggregation of polylysineD. Fedunova, J. Bagelova, Z. Gazova, M. AntalikDepartment of Biophysics, Institute of Experimental PhysicsSAS, Kosice, Slovakia

Amyloid fibrils consisting of different proteins have beenrecognized as an accompanying feature of several neuro-degenerative diseases. Many proteins without knownconnection to any diseases have been found to form amy-loid fibrils in vitro, leading to suggestion that the ability toform fibrils is the inherent property of polypeptide chain. Theobserved common character of protein amyloid formationenables to seek further clues of fibrillation mechanism bystudying generalized sequenceless polypeptide models, e.g.polylysine. We have studied conformational transitions ofpolylysine, with different chain length at various pH, ionicstrength and temperature by means of novel approach -viscometric method. This polypeptide undergoes alfa-helixto beta-sheet transition and forms amyloid fibrils in specialconditions. Temperature induced a-helix to b-sheet transi-tions occurs at pH interval form 10 to 11.8 and withincreasing chain length is slightly shifted to the lower pH. Wehave found narrow pH interval, in which the thermal transi-tion is fully reversible, suggesting the high sensitivity ofpolypeptide conformation on subtle changes in charge on itsside chains.

This work was supported within the projects VEGA0079,0038 and 0155, CEX SAS Nanofluid and by ESF pro-ject No. 26220120033.

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P-195Flow-induced polymorphism of protein aggregatesin micro channelsVito Fodera, Stefano Pagliara, Ulrich F. Keyserand Athene M. DonaldSector of Biological and Soft Systems, Cavendish LaboratoryUniversity of Cambridge JJ Thomson Avenue, CambridgeCB3 0HE, UK

Understanding the mechanisms of the conversion from thenative state of a protein to the amyloidal state represents afundamental step in improving the purification, storage anddelivery of protein-based drugs and it is also of great rele-vance for developing strategies to prevent in vivo proteinaggregation. Amyloid fibrils have a structural arrangement ofcross b-sheet but they can also experience different packinginto three dimensional superstructures, i.e. polymorphism. Itis well known that, among others, both the geometric con-finement of the molecules and shear forces can affect thefinal morphology of the aggregates. Importantly, due to thecomplexity and crowding of the cellular region, suchparameters also play a crucial role in in vivo processes. Wepresent an experimental approach to study in vitro amyloidaggregation in a controlled and uniform shear force field andwithin microscale environments. In particular we focus onthe effect of these two parameters on the formation ofspherical aggregates, known as spherulites. Using microchannels of different cross-sections from 5 to1000 lm x 12lm and flow rates in the range of hundreds of ll/min, thenumber and diameter of spherulites within the channelshave been characterized using crossed polarizers opticalmicroscopy.

P-196Inhibition of insulin amyloid fibrillization by albuminmagnetic fluidK. Siposova1,2, Z. Bednarikova2, M. Kubovcikova1,A. Antosova1, M. Koneracka1, V. Zavisova1, P. Kopcansky1,Z. Gazova11Institute of Experimental Physics, Slovak Academyof Sciences, Kosice, Slovakia, 2Institute of ChemicalSciences, Faculty of Science, P. J. Safarik University, Kosice,Slovakia

Insulin amyloid aggregation causes serious problems forpatient with insulin dependent diabetes undergoing long-termtreatment by injection, in production and storage of this drugand in application of insulin pumps. Recent studies indicatethat protein amyloid aggregation causes the cell impairmentand death; however, the prevention of amyloid aggregation isbeneficial. We have investigated ability of albumin magneticfluid (AMF) to inhibit insulin amyloid aggregation by spec-troscopic and microscopic techniques. Albumin magneticfluid consists of magnetic Fe3O4 nanoparticles stericallystabilized by sodium oleate and functionalized with bovineserum albumin (BSA) at various weight ratios BSA/Fe3O4.We have found the positive correlation between inhibitingactivity of AFM and nanoparticle diameter and zeta potential.The ability of AMF to inhibit formation of amyloid fibrilsexhibits concentration dependence with IC50 values

comparable to insulin concentration. The observed featuresmake AMF of potential interest as agents effective in thesolving of problems associated with insulin amyloidaggregation.

(This work was supported within the projects VEGA 0079,0155 and 0077, CEX SAS Nanofluid, APVV-0171-10, SK-RO-0012-10 and ESF project 26220220005).

P-197Beta-sheet Structure Formation at the Air-WaterInterface: Triggers, Transitions and OrientationsMaria Hornke1, Jessica Falenski2, Beate Koksch2,Gerald Brezesinski11Max-Planck-Institute of Colloids and Interfaces, AmMuhlenberg 1, 14476 Potsdam, Germany, 2Free UniversityBerlin, Organic Chemistry, Takustr. 3 14195 Berlin, Germany

Amyloid formation of peptides causes diseases like Alzhei-mer’s and Parkinson’s disease. However, the conditions forthe onset of the neurotoxic beta-sheet formation are poorlyunderstood.We focus on aggregation triggers and their interplay: Inter-actions with hydrophobic-hydrophilic interfaces, orientation ofpeptides in 2D, metal ion complexation and lipid layers. Thetailor-made model peptides exhibit defined secondarystructure propensities and metal ion binding sites.The interactions of the peptide with the air-water interfaceand with metal ions are studied using surface sensitivemethods connected to film balance measurements. X-raydiffraction, X-ray reflection, infrared reflection-absorptionspectroscopy and total reflection X-ray fluorescence wereapplied to reveal the layer structure, peptide conformationsand metal ion binding at the interface.We found that amyloid formation in 2D is dominated by thehydrophobic-hydrophilic interface and not comparable to thebulk behaviour. The interface can enhance or inhibit beta-sheet formation. The effect ofmetal ion complexation dependson the arrangement of the binding sites in the peptide andthe preferred metal complexation geometry. The two trig-gers interface and metal ion complexation, can oppose eachother.

P-198Effect of ApoE isoform and lipidation status onproteolytic clearance of the Amyloid-beta peptideHubin, E.1,2,3, Van Nuland, N.1,2,3, Broersen, K.3,41Department of Molecular and Cellular Interactions, FlandersInstitute for biotechnology (VIB), Brussels, Belgium, 2JeanJeener nmR Centre, Structural Biology Brussels, Brussels,Belgium, 3Vrije Universiteit Brussel (VUB), Brussels,Belgium, 4MIRA Institute for Biomedical Technologyand Technical Medicine, Nanobiophysics Group, Universityof Twente, Enschede, The Netherlands

Alzheimer’s disease (AD) is the most common type ofdementia in the elderly. The most important genetic riskfactor identified for AD is the isoform, e2, e3 or e4, of apoli-poprotein E (ApoE), a lipid-carrying protein. One hallmark of

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AD is the accumulation of amyloid-beta peptide (Ab) in thebrain which is thought to result from an imbalance betweenthe production of Ab and its clearance. Previous studiesreport an important role for ApoE in Ab degradation. Wesought to determine the effect of ApoE isoform and lipidationstatus on the degradation of soluble Ab by proteinases suchas insulin-degrading enzyme and neprilysin. In this study anin vitro Ab clearance assay based on the competitionbetween Ab and a fluorogenic peptide substrate is developedto quantify Ab degradation. To elucidate the proteolyticclearance mechanism, the fragments resulting from cleavageare identified by mass spectrometry and further analyzed toidentify the interacting stretch of the Ab sequence with thedifferent ApoE isoforms. The results suggest that ApoEinfluences the rate of Ab degradation.

P-199The role of protein context of polyglutaminein misfolding and aggregation: a combined biophysicaland computational approachG. Invernizzi, E. Papaleo, M. Lambrughi,M. Bonanomi, A.M. Frana, A. Natalello, S.M. Doglia,M.E. Regonesi, P. TortoraUniversity of Milano-Bicocca, Department of Biotechnologyand Biosciences, Milano, Italy

Polyglutamine (polyQ) diseases are a class of neurologicalamyloid pathologies triggered by an aberrant aggregation ofproteins bearing an expanded polyQ stretch. Proteinsinvolved in polyQ diseases do not share any functional andstructural similarity besides the polyQ tract which has beenaccounted as the causative agent of the pathology. Never-theless, it has been demonstrated that the aminoacidiccontext in which polyQ is inserted can greatly influenceprotein aggregation and, consequently, cytotoxicity. In thisview, a deep knowledge of the structural and dynamic fea-tures of polyQ flanking sequences and their interplay withinthe misfolding and aggregation events are crucial to under-stand the molecular mechanisms leading to amyloidformation. In the present contribution, we used the multi-domain polyQ protein ataxin-3 as a model for polyQ contextstudies, integrating computational approaches, as atomisticmolecular dynamics simulations, and experimental dataderived from fluorescence, FT-IR and CD spectroscopies,ESI-MS and SEC. Besides the well-known role of the N-termstructured Josephine domain (JD) in the protein aggregationpathway, our studies highlighted a crucial role for the disor-dered tract between JD and polyQ, as well as for theC-terminal tract beyond the polyQ.

O-200Kinetics of protein aggregationTuomas Knowles, Michele Vendruscolo, Chris DobsonDepartment of Chemistry, University of Cambridge, LensfieldRoad, Cambridge CB2 1EW, UK

The aggregation of proteins into fibrillar nanostructures is ageneral form of behaviour encountered for a range of

different polypeptide chains. The formation of these struc-tures is associated with pathological processes in the contextof Alzheimer’s and Parkinson’s diseases but is also involvedin biologically beneficial roles which include functional coat-ings and catalytical scaffolds. This talk focuses on recentwork directed at understanding the kinetics of this processthrough the development and application of experimentalbiophysical methods and their combination with kinetic the-ories for linear growth phenomena.

P-201Effect of 14-3-3(eta) on alpha-synuclein aggregationintermediatesDhruv Kumar1, Nicoletta Plotegher2, Isabella Tessari2,Bruno Samorı1, 3, Marco Brucale1, 3, Luigi Bubacco21University of Bologna, Dept. of Biochemistry, Via Irnerio 48,Bologna, Italy, 2University of Padova, Dept. of Biology, ViaUgo Bassi 58/B, Padova, Italy, 3S3 Center of Nanostructuresand Biosystems at Surfaces, Istituto di Nanoscienze – CNR,ItalyE-mail: dhruv.kumar2@unibo.it

Alpha-synuclein (AS) is a neuronal protein of unknownfunction which is mainly involved in the pathophysiology ofneurodegenerative diseases, including Parkinson’s disease,Alzheimer’s disease and Dementia with Lewy Bodies (LBs).LBs contains not only AS, but also other proteins including14-3-3 proteins. 14-3-3 proteins exist mainly as a dimer andits exact functions are remain unclear. However, recent workhas shown that the association of 14-3-3(eta) with AS in LBs.Herein we show how 14-3-3(eta) can modulate AS in vitroaggregation behavior, by rerouting it toward the formation ofstable non-fibrillar aggregates. We also show that theresulting populations of fibrillar and pre-fibrillar aggregatesexhibit a modified toxicity in vivo with respect to the unper-turbed aggregates. Interestingly, 14-3-3(eta) does not showany binding affinity for monomeric AS, nor for the maturefibrillar aggregates. We provide evidence that it acts on theoligomeric species which form during the amyloidogenesisprocess of aggregation. Since 14-3-3(eta) can influence thetoxicity of amyloidogenesis without perturbing the functionalAS monomers, we are convinced that once fully understood,its mode of action could represent a promising model tomimic with synthetic drugs and peptides.

P-202What makes an amyloid toxic: morphology, structureor interaction with membranes?K. Berthelot1, H.P. Ta2, B. Coulary-Salin1, C. Cullin1,S. Lecomte1Institut de Biochimie et Genetique Cellulaires, UMR 5095,CNRS/Universite de Bordeaux, France, 2Chimie Biologie desMembranes et Nano-objets, UMR 5248, CNRS/Universite deBordeaux, France

More than 40 human diseases are related to amyloids. Inorder to understand why some amyloids may become toxicto their host and some others are not, we first developed agenetical approach in the yeast Saccharomyces cerevisiae.We have chosen the amyloid/prion protein HET-s Prion

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Forming Domain (218-289) from the fungi Podospora anse-rina, which is not toxic in yeast. Some toxic amyloids mutantswere generated by random mutagenesis. In vitro the mosttoxic mutant called ‘‘M8’’ displays very peculiar nanofibers,which polymerized mainly in amyloid antiparallel b-sheetswhereas the non-toxic WT exhibits a parallel polymerization.We further established the dynamic of assembling of the M8toxic amyloid, in comparison to the WT non-toxic amyloid,and showed the presence of specific oligomeric intermedi-ates also organized in antiparallel b-sheet structures. A moreglobal structure/toxicity study on more than 40 mutantsclearly identified an antiparallel b-sheet signature for all thetoxic mutants. Therefore size, intermediates and antiparallelstructures may account for amyloid toxicity in yeast but westill wonder what their cellular targets are. Recently, weestablished the first evidences that toxic mutants may spe-cifically bind in vitro to lipids, particularly negatively charged.

P-203Interconnected mechanisms in Abeta(1-40) peptide fibrilformationMaurizio Leone, Giovanna Di Carlo, Michele D’Amico,Valeria Militello, Valeria VetriUniversity of Palermo - Dept. of Physics, Palermo, Italy

We present an experimental study on the fibril formation ofAb(1-40) peptide at pH 7.4. The kinetics of this process ischaracterized by the occurrence of multiple transient spe-cies that give rise to final aggregates whose morphologyand molecular structure are strongly affected by the growthconditions. To observe in details the aggregation pathway asa function of solution conditions, we have used differentexperimental techniques as Light scattering, Thioflavin Tfluorescence, Circular Dichroism and two-photon fluores-cence microscopy. This approach gives information on thetime evolution of conformational changes at molecular level,on the aggregates/fibrils growth and on their morphologies.The selected experimental conditions allowed us to highlightthe existence of at least three different aggregation mecha-nisms acting in competition. A first assembly stage, whichimplies conformational conversion of native peptides, leads tothe formation of small ordered oligomers representing anactivated conformation to proceed towards fibril growth. Thisprocess constitutes the rate limiting step for two distinct fibrilnucleation mechanisms that probably implicate spatially het-erogeneous mechanisms.

P-204Amyloid formations and interactionsDmitrijs Lapidus1, Salvador Ventura2, Cezary Czaplewski3,Adam Liwo3, Inta Liepina11Latvian Institute of Organic Synthesis, Aizkrauklesstr.21,Riga LV1006, Latvia, 2Institut de Biotecnologia i deBiomedicina, Universitat Autonoma de Barcelona, E-08193Bellaterra, Spain, 3Department of Chemistry, and Universityof Gdansk, ul. Sobieskiego 18, 80-952 Gdansk, Poland

The formation of amyloid fibrils of amylin 10-29 was studiedby means of molecular dynamics (MD) and energy partition

on three peptide ß-sheet stack systems with the same aminoacid composition: wild type amylin 10-29 (Amyl 10-29),reverse amylin 10-29 (Rev-Amyl 10-29) and scrambledamylin version Scr-Amyl 10-29. The results show that forAmylin10-29 peptides, amino acid composition determinesthe propensity of a peptide to form amyloid fibrils indepen-dent of their sequence. The sequence of amino acids definesthe shape and the strength of amyloid protofibril, whichconforms with the atom force microscope (AFM) data [1]. MDshow that the 6x6RevAmyl-10-29 stack has looser self-assembly than the 6x6Amyl-10-29 stack, which conformswith the results of Fourier transform infrared spectroscopy(FTIR) measurements for the peptides studied [1]. Theresults of MD show that 6x6Amyl-10-29 could have a turn,which consists with FTIR data [1].

AcknowledgmentsSupported by ESF project 2009/0197/1DP /1.1.1.2.0/09/APIA/VIAA/014, European Economic Area block grantLV0015.EEZ09AP–68, Gdansk Academic Computer CenterTASK.

Reference

1. Sabate, R, Espargaro A, de Groot NS, Valle-Delgado JJ,Fernandez-Busquets X, Ventura S. J. Mol. Biol. 40,337-352 (2010).

O-205Mechanistic insights into Ab42 aggregation and effectsof inhibitorsSara Linse1, Erik Hellstrand2, Olga Szepankiewicz2, RistoCukalevski1, Celia Cabaleiro-Lago1, Birgitta Frohm1, BjornLinse1, Emma Sparr31Department of Biochemistry, Lund University, Sweden,2Department of Biophysical Chemistry, Lund University,Sweden, 3Department of Physical Chemistry, LundUniversity, Sweden

Data on Ab aggregation kinetics have been characterized bya large spread between experiments on identical samplesthat contain so many molecules that stochastic behaviour isdifficult to explain unless caused by uncontrolled amounts ofimpurities or interfaces. We have therefore spent consider-able effort to eliminate sources of inhomogeneity andreached a level of reproducibility between identical samplesand between experiments on separate occasions that wecan now collect data that can lead to mechanistic insightsinto the aggregation process per se, and into the mechanismof action of inhibitors. Data on Ab42 aggregation will beshown that give insight into the influence of physicalparameters like peptide concentration, shear and ionicstrength, as well as the effect of inhibitory proteins, modelmembranes and the effects of sequence variations. MonteCarlo simulations of amyloid formation from model peptidescorroborate the finding from experiments and underscorethat the very high level of predictability and reproducibilitycomes from multiple parallel processes.References: Hellstrand et al ACS Chem Neurosci. 1, 13-18(2010); Hellstrand et al, Biophys J. 98:2206-2214 (2010);Cabaleiro-Lago et al, ACSChem Neurosc., 1, 279-287 (2010)

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P-206Lipid-protein interaction and amyloid-like fiberformation: FLIM and FCS studiesAna M. Melo1, Ana Coutinho1,2, Manuel Prieto11Centro de Quımica-Fısica Molecular and IN- Instituteof Nanosciences and Nanotechnology, Instituto SuperiorTecnico, Lisbon, Portugal, 2Departamento de Quımica eBioquımica, FCUL, Lisbon, Portugal

Negatively-charged membranes were reported to catalyze‘‘amyloid-like’’ fiber formation by non-amyloidogenic proteins[1]. Our study aims to elucidate the factors that govern theformation of these amyloid-like fibers. Lysozymewas selectedas a model of non-amyloidogenic protein and was fluores-cently-labeled with Alexa Fluor 488 (A488-Lz). First, A488-Lzpartition towards phosphatidylserine-containing liposomeswas characterized quantitatively using fluorescence correla-tion spectroscopy (FCS), in order to calculate the proteincoverage of liposomes. Secondly, the interaction betweenA488-Lz and negatively-charged lipid membranes was stud-ied using both steady-state and time-resolved fluorescencetechniques. This interaction was found to switch from aperipheral binding to the anionic headgroups, at high lipid/protein molar ratio (L/P), to a partial insertion of protein into thehydrophobic coreof themembrane, at lowL/P. Finally, the lipid-protein supramolecular complexes formed at low L/P werecharacterized by fluorescence lifetime imaging microscopy(FLIM). Themean lifetime of A488-Lz in these supramolecularstructures is much lower compared to the values obtained forthe free and bound A488-Lz at high L/P. The fiber character-ization will be complemented by FCS studies.

[1] Zhao et al. 2004 Biochemistry 43: 10302–10307

P-207Zinc modulates copper coordination mode in prionprotein octa-repeat subdomainsFrancesco Stellato 1,2, Ann Spevacek3, Olivier Proux4,Velia Minicozzi1, Glenn Millhauser3, Silvia Morante11Dipartimento di Fisica, Universita di Roma ‘Tor Vergata’,via della Ricerca Scientifica, 1, 00133, Roma, Italy, 2Centrefor Free-Electron Laser Science – DESY, Notkestrasse 85,22607, Hamburg, Germany, 3Department of Chemistryand Biochemistry, University of California, Santa Cruz, CA95064, USA, 4Observatoire des Sciences de l’Univers deGrenoble, CNRS & Universite Joseph Fourier, BP 53 38041,Grenoble Cedex 9, France

We present the results of recent XAS measurements carriedout on various portions of Prion-protein tetra-octarepeatpeptides in complex with metals with the aim of elucidatingthe mechanism underlying the competition for peptide bind-ing when two distinct metal ions (in our case Cu2+ and Zn2+)are simultaneously present in solution. Thanks to the abilityof the XAS technique in providing detailed local structuralinformation of the metal site, we are able to demonstratethat Cu and Zn interact with the peptide, in the sense thatincreased Zn (Cu) concentration progressively changes theway Cu (Zn) is bound to the tetra-octarepeat, even if none

of the two metals is ever able to completely displace theother.We propose a structural model in which the Cu2+(Zn2+)coordination mode depends on the [Cu2+]([Zn2+]):[octare-peat] concentration ratio. The model is in agreement withthe EPR results published by one of the authors (G.M.). Ourpicture suggest a mechanism of cross-regulation betweenCu2+ and Zn2+ that is worth further investigating in otherbiological systems, like for instance in the case of the Ab-peptides involved in the Alzheimer disease. A competitionmechanism of the kind we propose may represent anadditional general cell strategy for fine metal bindingregulation.

O-208Membrane permeabilization by purified solubleb-enriched oligomers of prion proteinSylvie Noinville1, Stephanie Prigent1, Alain Brisson2,Celine Chapuis1, Human Rezaei1

INRA, Virologie et Immunologie Moleculaires, Jouy-en-Josas, France, 2CNRS, IECB, Bordeaux, France

The conversion of normal PrPC to its pathological isoformPrPSc is a key event in prion diseases and is proposed tooccur at the cell surface or more probably in acidic lateendosomes. A convergence of evidence strongly suggeststhat the early events leading to the structural conversion ofthe PrP seem to be in relation with more or less stablesoluble oligomers, which could mediate neurotoxicity. Ascommonly shared by other amyloidogenic proteins, mem-brane-bound monomers undergo a series of lipid-dependentconformational changes, leading to the formation of oligo-mers of varying toxicity rich in b-sheet structures (annularpores, amyloid fibrils). Here, we have used a combination ofbiophysical techniques (Dynamic and Static Light Scatter-ing, Fluorescence techniques, and Quartz CrystralMicrobalance) to elucidate the interaction of native mono-meric PrP and that of purified b-rich oligomeric PrP onmodel lipid membranes. Under well established conditions,three b-sheet-rich oligomers were generated from the partialunfolding of the monomer in solution, which were found toform in parallel. From single mutation and/or truncation ofthe full length PrP, the polymerization pathway is stronglyaffected, revealing the high conformational diversity of PrP.In our previous work, we identify the minimal region of thePrP protein leading to the same polymerization pattern ofthe full length PrP. Soluble 12-subunits and 36-subunitsoligomers were obtained depending on the single mutationor truncation and purified. We compare their structuralproperties (FTIR, CD) when associated with anionic lipidbilayers and study their propensities to permeabilize themembrane. Fluorescence kinetics suggest different mech-anisms of membrane perturbation for the monomer and thePrP oligomers. Deciphering this complex network of lipidinteractions and conformational diversity of the PrP proteinwill help for understanding of how amyloidogenic proteinsinduce neurotoxicity.

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P-209Towards a dynamic vision of amyloid-membraneassembliesMartina Pannuzzo1, Antonio Raudino1, Danilo Milardi2,Carmelo La Rosa1, Mikko Karttunen31Dept. of Chemical Science, University of Catania, Catania,Italy, 2Istituto di Biostrutture e Bioimmagini, CNR, Catania,Italy, 3Dept. of Applied Mathematics, Western OntarioUniversity, London (ON), CanadaE-mail: mpannuzz@uwo.ca

The traditional view of the lipid bilayer described as a ‘‘sea’’ oflipids where proteins may float freely, is going to be inade-quate to describe the increasingly large number of complexphenomena which are known to take place in biologicalmembranes. Membrane-assisted protein-protein interac-tions, formation of lipid clusters, protein-induced variation ofthe membrane shape, abnormal membrane permeabilitiesand conformational transitions of membrane-embeddedproteins are only a few examples of the variegated ensembleof events whose tightly regulated cross-talk is essential forcell structure and function.Experimental work on the above mentionated problems isvery difficult and some time not accessible, especially whenthe studied systems have a fast dynamics.Due to the large size of the systems usually involved in thismultifaceted framework, a detailed molecular description ofthese phenomena is beyond the possibilities of conventionalMolecular Dynamics techniques. It is thus necessary toapproach new computational models on a mesoscopic scalethat if, on one hand, overlook atomistic details, allow tocapture phenomena that are thought to occur in spatial andtime scales far beyond what is normally sufficient to describesmall molecules. In this light we have endeavored in our laban extensive campaign focused on coarse grained (CG)models of different lipid-protein assemblies. Simulationshave been performed by the GROMACS simulation packageand the standard MARTINI force field.Several examples of lipid-protein assemblies involved insome amyloidoses are presented with the aim of evidencingthe potential of this multiscale approach.

P-212Phytoalexins in prevention of amyloid aggregationK. Siposova1,2, A. Antosova1, P. Kutschy2, Z. Daxnerova3,D. Fedunova1, J. Bagelova1, Z. Gazova11Department of Biophysics, Institute of Experimental PhysicsSlovak Academy of Sciences, Kosice, Slovakia, 2Instituteof Chemistry, Faculty of Science, P. J. Safarik University,Kosice, Slovakia, 3Institute of Biology and Ecology, Facultyof Science, P. J. Safarik University, Kosice, Slovakia

Amyloid aggregation, a generic behavior of proteins, isrelated to incurable human pathologies - amyloid-relateddiseases, associated with formation of amyloid deposits inthe body. All types of amyloid aggregates possess rich b-sheet structural motif. The recent data confirm the toxic effectof aggregates on the cells, however, it was found thatreduction of amyloid aggregates plays important role in pre-vention as well as therapy of amyloidosis. We have

investigated effect of phytoalexin derivatives on amyloid fi-brillization of two proteins, human insulin and chicken egglysozyme, by ThT and ANS fluorescence assays. We havefound that amyloid aggregation of both studied proteins wassignificantly inhibited by phytoalexin derivates cyclobrassininand benzocamalexin. For most effective phytoalexins theestimated IC50 values were at low micromolar concentration.The observed inhibiting activity was confirmed by transmis-sion electron microscopy. Our data suggest the potentialtherapeutic use of the most effective phytoalexins in thereduction of amyloid aggregation.(This work was supported within the projects VEGA 0079,0155, CEX SAS Nanofluid, APVV-0171-10, SK-RO-0012-10and ESF project 26220220005).

O-213Mechanistic insights into oligomericalpha-synuclein/membrane interactionsAnja N. D. Stefanovic, Martin T. Stockl, Mireille M. A. E.Claessens, Vinod SubramaniamNanobiophysics, MESA+ Institute for Nanotechnology &MIRA Institute for Biomedical Technology and TechnicalMedicine, University of Twente, Enschede, The Netherlands

The amyloid pore hypothesis suggests that interactions ofoligomeric alpha-synuclein (aS) with membranes play animportant role in Parkinson’s disease. Oligomers arethought to permeabilize membranes and interfere withCa2+ pathways. Permeabilization by aS requires the pres-ence of negatively charged phospholipids. Whether aS canbind and permeabilize membranes with physiologicallyrelevant lipid compositions has not been extensivelyexplored.Here we report on the binding of aS to giant unilamellarvesicles (GUVs) with physiologically relevant lipid compo-sitions. Comparing different protocols of oligomerpreparation, leakage assays on both large unilamellarvesicles (calcein release) and GUVs (HPTS efflux assay)show that aS is not able to permeabilize these mem-branes. The presence of cholesterol has a stabilizing effecton these membrane systems. In agreement with thesefindings, we do not observe concentration dependent aStoxicity using in vivo MTS assays. However, in the calceinrelease assay, different aS preparations show differencesin kinetics and aS concentrations that cause 100% leak-age. These results motivate us to critically reassess theamyloid pore hypothesis, and suggest that membranepermeabilization may be attributable only to a very specificas species.

P-214Alpha-synuclein oligomers impair membraneintegrity—a mechanistic viewMartin T. Stockl, Mireille M. A. E. Claessens,Vinod SubramaniamNanobiophysics, MESA+ Institute for Nanotechnology,University of Twente, Enschede, The Netherlands

One of the most prevalent neurodegenerative diseases isParkinson’s disease (PD), which is accompanied with the

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loss of dopaminergic neurons. Although the mechanismsleading to the death of these cells are still unclear, the proteinalpha-synuclein (aS) is one of the pivotal factors. Previousstudies indicate that especially oligomeric forms of aS showa detrimental effect on membrane integrity. As an intactmembrane is crucial to many cellular processes, the impair-ment of the membrane integrity is a likely pathway forneuronal death.We use different phospholipid bilayer model systems toinvestigate the mechanisms underlying this process. Atomicforce microscopy in combination with suspended asymmetricphospholipid bilayers, which closely mimic the plasmamembrane, allows the identification of the binding sites, themeasurement of penetration depths of the aS oligomers intothe phospholipid bilayer, and the detection of membranethinning or creation of membrane defects. Using an approachbased on phospholipid vesicles we were able to observe forthe first time that aS oligomers cause an enhanced lipid-flipflop. Suggesting that the loss of lipid asymmetry is a novelmechanism which may contribute to or trigger neuronaldeath in PD.

P-215Amyloid protein-membrane interactions and structure-toxicity relationship studyH.P. Ta1; K. Berthelot2; B. Coulary-Salin2, B. Desbat1;L. Servant3; C. Cullin2; S. Lecomte11Chimie Biologie des Membranes et Nano-objets, UMR5248, CNRS/Universite Bordeaux 1, France, 2Institut deBiochimie et Genetique Cellulaires, UMR 5095, CNRS/Universite Bordeaux 2, France, 3Institut des SciencesMoleculaires, UMR 5255, CNRS/Universite Bordeaux 1,France

A better characterization of toxic amyloids would allow get-ting better insights into neurodegenerative diseases. Whatmakes an amyloid toxic is, however, still unclear.Our study aims to investigate the membrane interaction withtoxic and non-toxic amyloids. We used the Prion FormingDomain PFD218-289 of the protein HET-s (WT) (Podosporaanserina) and its toxic mutant M8 (generated by PCR ran-dom mutagenesis) as amyloid models. In vitro, they exhibitdifferent fibril characterizations and secondary structures(Berthelot et al. FASEB J. 2009, 23, 2254).At first, the interactions between WT and M8 with differentcharged phospholipid (DOPI, DOPG, DOPC, DOPS andDOPE) monolayers were highlighted. Results indicated thatM8 (toxic) has a much higher and more specific effect onnegatively charged phospholipids (DOPS, DOPI and DOPG)than in the case of WT (non-toxic). Therefore the insertion ofprotein into phospholipid monolayers, which occurred simi-larly for both WT and M8, is not a key factor for these effects(H.P. Ta et al. Langmuir, in press).We are now using unilamellar vesicles as a membranemodel to investigate the amyloid protein (toxic and non-toxic) – phospholipid interactions. Results confirmed thehigh specific and strong effects of M8 on negatively-chargedmembrane.

P-216Investigations on alpha-synuclein pore formingpropertiesL. Tosatto1,2, A. O. Andrighetti1, B. Cronin3, I. Tessari4,N. Plotegher4, K. Ganzinger2, J. McColl2, V. Antonini1,L. Bubacco4, D. Klenerman2, M. Wallace3,M. Dalla Serra11Istituto di Biofisica, Consiglio Nazionale delle Ricerche,Trento, Italy, 2Department of Chemistry, Universityof Cambridge, Cambridge, UK, 3Department of Chemistry,University of Oxford, Oxford, UK, 4Dipartimento di Biologia,Universita di Padova, Padova, Italy

Alpha-synuclein (aS) is a natively unfolded protein widelyexpressed in neurons at the presynaptic level, partially boundto synaptic vesicles. It is linked to Parkinson’s disease (PD)by two lines of evidence: aS amyloid fibrils accumulate inpatients’ brains and 4 genetic mutants cause autosomaldominant forms of PD. The biological role of the protein andthe mechanisms involved in the pathogenesis of PD are stillunknown. Membrane binding of aS monomers causes theacquisition of an amphipatic alpha-helix, which lies on thesurface without crossing the bilayer. Recent observationsreported that a pore-like activity of aS can be recorded uponthe application of a potential. This activity is characterized bydefined conductance levels with a determined fingerprint inPlanar Lipid Membrane (PLM) experiments. As severalstudies found aS oligomers are the most toxic species in cellcultures, we aim to use TIRF imaging and the PLM techniqueto give more information about the pore activity of aS. Par-ticular effort is put into understanding whether monomericand oligomeric aS share a similar mechanism for membranepermeabilization and to find possible implications for theethiopatogenesis of PD. (Sponsored by Provincia Autonomadi Trento, Marie Curie Action COFUND, project SingleSyn)

P-217Encapsulation and study of fibrillar networksin nanocompartmentsR. Urbani1, H. M. Evans2, T. Pfohl1,21Department of Chemistry, University Basel, Switzerland,2Max Planck Institute for Dynamics and Self-Organization,Gottingen, Germany

In this project, we study the chemical, physical and biologicalproperties of fibrillar networks. The formation and the networkmechanics are investigated by combining droplet-based mi-crofluidics with optical microscopy and small angle x-rayscattering (SAXS).The chosen system, fibrin network formation, plays animportant role in blood coagulation processes. Crosslinkingof fibrinogen induced by an enzymatic reaction with thrombinleads to 3D fibrin network formation.The fibrillar networks are formed within picoliter droplets ofaqueous solutions in an continuous oil phase. Dropletscontaining fibrinogen and thrombin can be produced of dif-ferent sizes and stored for fibrin network formation. Theformation of the fibrillar networks is imaged by fluorescencemicroscopy. To analyze the elastic properties of the net-works, the droplets flow through a microchannel device ofalternating width in order to squeeze and stretch the

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networks. Additionally, SAXS experiments will give structuralinformation about the molecular dimensions of the networks.

O-218Biophysical consideration of gamma-secretasemodulation as potential target for Alzheimer’s diseaseAnnelies Vandersteen1,2, Iryna Benilova4,Wim Jonckheere2, Frederic Rousseau1,2,Joost Schymkowitz1,2, and Kerensa Broersen2,31Switch Laboratory, Flanders Institute for Biotechnology(VIB), Brussels, Belgium, 2Vrije Universiteit Brussel (VUB),Brussels, Belgium, 3MIRA Institute for BiomedicalTechnology and Technical Medicine, Universiteit Twente,Enschede, The Netherlands, 4IMEC Bioelectronic SystemsGroup, Heverlee, Belgium

The amyloid beta peptide (Ab), implicated in Alzheimer’sdisease (AD), is released from the Amyloid Precursor Pro-tein (APP) by secretase-induced cleavage. This processresults in the release of a range of Ab peptides varying inlength. The brains of AD patients often contain longer Abpeptides while the total concentration of Ab is unaffected.Longer peptides are more hydrophobic having far-reachingconsequences for their toxicity and aggregation. As Ab isnecessary for normal neuronal function, research activitiesinto AD therapeutic development currently explore thepossibilities of modulating c-secretase activity to produceshort Ab peptides. Whether such an approach effectivelyameliorates the toxic effect of Ab has not been exploredyet. To answer this question, we studied the impact ofheterogeneity in Ab pools in an in vitro biophysical and incellulo context using MicroElectrode Array to assay thesynaptic activity of primary neurons. We show that variouslengths of the Ab peptide and mixtures thereof aggregatewith distinct kinetics and notoriously affect synaptotoxic andcytotoxic response. We also show that small amounts ofless abundant peptides Ab38 and Ab43 induce aggregationand toxicity of Ab40 while the behavior of Ab42 isunaffected.

O-219Microscopic analysis of protein oxidative damage: effectof carbonylation on structure, dynamicsand aggregability of villin headpieceDrazen Petrov and Bojan ZagrovicDepartment of Structural and Computational Biology, Max F.Perutz Laboratories, University of Vienna, Campus ViennaBiocenter 5, Vienna, AT-1030, Austria

One of the most important irreversible oxidative modificationsof proteins is carbonylation, a process of introducing the car-bonyl group in reaction with reactive oxygen species.Importantly, carbonylation increases with the age of cells and isassociatedwith the formation of intracellular protein aggregatesand the pathogenesis of age-related disorders such as

neurodegenerative diseases and cancer. However, it is stilllargely unclear how carbonylation affects protein structure,dynamics and aggregability on the atomic level. Here, we useclassical molecular dynamics simulations to study structureand dynamics of the carbonylated headpiece domain of villin, akey actin-organizing protein. We perform an exhaustive set ofmolecular dynamics simulations of native villin headpiecetogether with every single combination containing carbonylatedversions of its seven lysine, arginine and proline residues, thequantitatively most important carbonylable amino acids. Sur-prisingly, our results suggest that high levels of carbonylation,far above those associated with cell death in vivo, may berequired to destabilize and unfold protein structure through thedisruption of specific stabilizing elements, such as salt bridgesor proline kinks, or tampering with the hydrophobic effect. Onthe other hand, by using thermodynamic integration andmolecular hydrophobicity potential approaches, we quantita-tively show that carbonylation of hydrophilic lysine and arginineresidues is equivalent to introducing hydrophobic, charge-neutral mutations in their place, and, by comparison withexperimental results, demonstrate that this by itself significantlyincreases intrinsic aggregation propensity of both structured,native proteins and their unfolded states. Finally, our resultsprovide a foundation for a novel experimental strategy to studythe effects of carbonylation on protein structure, dynamics andaggregability using site-directed mutagenesis.

P-220Conformational and aggregation studies of Schistosomamansoni septinsA.E. Zeraik, A.P.U. Araujo, R. DeMarcoSergio Mascarenhas Molecular Biophysics Group, Instituteof Physics of Sao Carlos, University of Sao Paulo,Sao Carlos, Brazil

Septins are an evolutionarily conserved family of GTP-bind-ing proteins involved in important cellular processes, such ascytokinesis and exocytosis, and have been implicated inneurological diseases, such as Alzheimer’s and Parkinson’sdiseases. The focus of this study was two septins of Schis-tosoma mansoni, (the causative agent of Schistosomiasis inSouth America) named Smsept5 and Smsept10, which wereproduced in a recombinant system. Our objective was toverify if these septins from a simpler organism display similarcharacteristics to human septins. Analysis of protein struc-ture by Circular Dichroism showed that both recombinantSmSeptins produced were folded. The GTPase activityassay showed that Smsept5 was able to hydrolyze GTP,whereas Smsept10 was not. Aggregation studies for amyloidfibril detection by right angle light scattering and Thioflavin Tfluorescence assay were performed. Both proteins showed atemperature dependent increase in light scattering and fluo-rescence emission of ThT probe. This indicated that S.mansoni septins tend to aggregate into amyloid-like fibers inhigh temperatures, with thresholds of 30 �C for Smsept5 and37 �C for Smsept10. These results are in accordance to thatpreviously reported for human septins.

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Nucleic acid and chromatin structure &function

P-222Efficacy of DNA repair mechanisms in lymphocytesof melanoma patients as prognostic factorof chemotherapyD.N. Artamonov, V.A. Tronov. L.B. GorbachevaInstitute of Biochemical Physics, Moscow, Russia

In our work we investigated the response to standard che-motherapy of blood lymphocytes of patients suffering withmelanoma. DNA single and double strand breaks weredetermined using comet assay; intracellular levels of markerproteins were detected using immunocytochemistry. Ulti-mately this set of parameters allows to characterize twomechanisms of DNA repair (base excision repair, BER andmismatch repair, MMR) which together with apoptosisproneness underlie response of tumor cells to chemotherapy.Cell death caused by O6mG adducts is promoted by MMRsystem by inducing unrepaired double strand breaks in DNA.There was a linear correlation between the level of dsDNAbreaks in lymphocytes after 1-st cycle of chemotherapy andMMR efficiency in them.The level of double strand breaks in DNA after 1-st cycle ofchemotherapy is predictive of clinical outcome. Otherwisedamage at the level of ssDNA (AP-sites and single strandbreaks) and BER mechanism associated with it couldn’t be agood prognostic factor of chemotherapy. High level of doublestrand breaks in DNA in blood lymphocytes of melanomapatients 48 hours after 1-st cycle of chemotherapy appears tobe a marker of a good prognosis.

P-223Self-assembly and stability of g-quadruplex: counter-ions, pressure and temperature effectsE. Baldassarri Jr., P. Mariani, F. Spinozzi, M. G. OrtoreSAIFET Dept. & CNISM, Marche Polytechnic University,Ancona, Italy

The important role of G-Quadruplex in biological systems isbased on two main features: composition and stability oftelomeres, and activity of telomerase. The G-quadruplexstructures are formed by supramolecular organization ofbasic units called G-quartets that are planar rings constitutedby four guanosines linked by Hoogsten hydrogen bonds.GQuadruplex requires the presence of monovalent cationsplaying a key role in stabilizing these structures, since theygive rise to coordination bonds needed for the stacking ofmore tetrads. We performed X-ray diffraction experiments atdifferent pressures (ranging from 1 to 2000 bar), and smallangle X-ray scattering (SAXS) changing the temperature(between 20-60�C). Diffraction results concerning sampleswith an intermediate water content (50-40% H2O) evidencethe presence of two phases (Cholesteric and Hexagonalphase), while more concentrated samples (30-20% H2O)present just the hexagonal phase. High-pressure experi-ments provided the evidence that compression increases thesystem disorder without phase transitions. Data obtained

from SAXS measurements show that increasing concentra-tion of KCl we obtain more stable and larger particles. Alsothe temperature stability of the aggregates increases atincreasing counter-ions content.

O-225Nucleosome accessibility investigated using singlemolecule fluorescenceVera Bohm1, Aaron Hieb2, Andrew Andrews2,Katalin Toth1, Karolin Luger2, Jorg Langowski11German Cancer Research Center, Heidelberg, Germany,2Colorado State University, Fort Collins, USA

Nucleosomes are the smallest compaction unit of chromatinin the cell nucleus. They consist of 150 bp DNA wrappedaround a protein core of two (H2A-H2B) histone dimers and a(H3-H4)2 tetramer. DNA in nucleosomes is sterically occludedand nucleosomes must open to allow full DNA access. Westudied this process using FCS and single molecule FRET.Here we show evidence for a previously uncharacterizedintermediate structural state that preceeds histone dissocia-tion, in which the distance between H2B and the nucleosomaldyad is increased. Our data suggest that the first step innucleosome disassembly is the opening of the (H3-H4)2 tet-ramer/(H2A-H2B) dimer interface, followed by H2A-H2Bdimer release from the DNA and, lastly, (H3-H4)2 tetramerremoval. We estimate that the open intermediate state ispopulated at 0.2 - 3 % under physiological conditions, andcould have significant in vivo implications for factor-mediatedhistone exchange, as well as for regulating DNA accessibilityto the transcription and replication machinery. Additionally,histone variants and histone modifications could substantiallychange the stability of the nucleosome. Our most recentspFRET data indicate that such effects may occur both at thelevel of DNA-histone and histone-histone interactions.

O-226Dissecting the role of coregulator exchangeand chromatin binding in retinoic acid receptor (RAR)mobility by live cell FCSPeter Brazda1, Tibor Szekeres2, Balazs Bravics2,Katalin Toth3, Laszlo Nagy1,4,* and Gyorgy Vamosi2,*1Department of Biochemistry and Molecular Biology,2CellBiology and Signaling Research Group of the HungarianAcademy of Sciences, Department of Biophysics and CellBiology and 4Apoptosis and Genomics Research Groupof the Hungarian Academy of Sciences; Medical and HealthScience Centre, Research Centre for Molecular Medicine,University of Debrecen, Debrecen, Hungary 3DivisionBiophysics of Macromolecules, German Cancer ResearchCenter, Heidelberg, Germany*equal senior authors

Retinoic acid receptor (RAR) is a member of the nuclearreceptor superfamily. This ligand-inducible transcription factorbinds to DNA as a heterodimer with the retinoid X receptor(RXR) in the nucleus. The nucleus is a dynamic compartmentand live-cell imaging techniques make it possible to investigatetranscription factor action in real-time. We studied the diffusionof EGFP-RAR by fluorescence correlation spectroscopy (FCS)in order to uncover the molecular interactions determining

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receptor mobility. In the absence of ligand we identified twodistinct species with different mobilities. The fast componenthas a diffusion coefficient of D1 = 1.8-6 lm2/s corresponding tosmall oligomeric forms, whereas the slow component with D2 =0.06-0.12 lm2/s corresponds to interactions of RAR with thechromatin or other large structures. The RAR ligand bindingdomain fragment also has a slow component probably as aresult of indirect DNA-binding via RXR, with lower affinity thanthe intact RAR:RXR complex. Importantly, RAR-agonist treat-ment shifts the equilibrium towards the slow population of thewild type receptor, but without significantly changing themobility of either the fast or the slow population. By using aseries of mutant forms of the receptor with altered DNA- orcoregulator-binding capacity we found that the slow compo-nent is probably related to chromatin binding, and thatcoregulator exchange, specifically the binding of the coacti-vator complex, is the main determinant contributing to theredistribution of RAR during ligand activation.

P-227Compactization of DNA induced by sperm-specificproteins of H1 familyElena V. Chikhirzhina1, Tatiana Ju. Rodionova1,2,Elena I. Kostyleva1, Alexander M. Polyanichko1,21Institute of Cytology, RAS, Saint-Petersburg, Russia, 2Saint-Petersburg State University, Saint-Petersburg, RussiaE-mail: chikhir@gmail.com

Formation of inactive nuclear with high level of DNA com-paction in sperm cells is accompanied by a substitution oflinker histones H1 by a number of other proteins. Amongthem sperm-specific histones (SSH), which are character-ized by elongated arginine-rich polypeptide chain comparedto the somatic H1. The secondary and tertiary structure of theSSH and their interactions with DNA were studied usingspectroscopic and thermodynamic approaches. The histoneswere isolated from sperm of marine invertebrates and ratthymus. All studied SSH demonstrate no considerablecompaction of DNA in solutions of low ionic strength. How-ever, at physiological conditions, SSH H1 from S.intermediusand A.japonica compact DNA more intensively than otherSSH. The somatic H1 from rat thymus revealed a minimalability to compact the DNA. We suggest that the SSH H1 areable to interact with DNA not only in the major groove butalso in the minor groove of the double helix inducing con-siderable structural changes in DNA and facilitating theformation of the supercompact sperm chromatin.The authors are grateful for the financial support from theRussian Foundation for Basic Research (grants § 10-04-00092 and 09-08-01119) and from Administration of Saint-Petersburg.

P-228Investigation of radiation-induced damages in DNAbases and secondary structureO.A. Dommes, S.V. PastonSaint Petersburg State University, Faculty of Physics,St.-Petersburg, Russia

Ionizing radiation causes modification and destruction ofnitrogenous bases in DNA molecule. There are also local

breakages of hydrogen bonds both in the lesion sitesmentioned above and in other sites of the macromolecule.To reveal the amount of some of these damages we appliedCD and UV absorption spectroscopy. Radiation-inducedchanges in DNA structure influence its UV absorptionspectrum in different ways: partial denaturation causeshyperchromic effect, while destruction of the bases resultsin hypochromic shift. At the same time both of them result inthe same changes in DNA CD spectra: the decrease inintensity. We attempted to segregate the described dam-ages in DNA structure and studied the influence of DNAionic surroundings on the radiation effect. It is shown thatthe radiation efficiency of base destruction and partialdenaturation increases with decreasing concentration ofNaCl in irradiated solution. The substitution of a portion ofNa+ ions on Mg2+ with the total ionic strength remainingconstant (0.005 M) does not influence the radiationefficiency.This work is supported by the Russian Foundation for BasicResearch (project No 09-08-01119-a).

P-229Udu and its homologs, Udu2 and Gon4l, may havesimilar functions in DNA replicationTzu-Min Chan1, Kuo-Chang Chu1, Chiaw-Hwee Lim2

and Yun-Jin Jiang1,21Division of Molecular and Genomic Medicine, NationalHealth Research Institutes, Miaoli County, Taiwan,2Laboratory of Developmental Signalling and Patterning,Genes and Development Division, Institute of Molecularand Cell Biology, Singapore

Udu (Ugly duckling) has been first identified from a zeb-rafish mutant and shown to play an essential role duringerythroid development; however, its roles in other cellularprocesses remain largely unexplored. FACS analysisshowed that the loss of udu function resulted in defectivecell cycle progression and comet assay indicated thepresence of increased DNA damage in udu mutants. Wefurther showed that the extensive p53-dependent apoptosisfound in udu mutants is a consequence of activation in theAtm-Chk2 pathway. Udu appears not to be required forDNA repair, because both wild-type and udu embryossimilarly respond to and recover from UV treatment. Yeasttwo-hybrid and coimmunoprecipitation data demonstratedthat PAH-L repeats and SANT-L domain of Udu interactswith MCM3 and MCM4. Furthermore, Udu was colocalizedwith BrdU and heterochromatin during DNA replication,suggesting a role in maintaining genome integrity.Recently, we started to work on the second zebrafishhomolog, Udu2, and its mammalian counterpart, Gon4l.Preliminary data showed that udu2 and Gon4l mRNA injec-tion can rescue zebrafish udu mutant phenotypes.Furthermore, PAH-L and SANT-L domains of Udu2 andGon4l can bind to MCM3 and MCM4 and they are localizedin the nucleus. These data suggest that Udu2 and Gon4l arefunctionally equivalent to zebrafish Udu. Their molecularmechanism leading to udu phenotypes is currently underinvestigation.

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O-230Chromatin condensation: general polyelectrolyteassociation and histone-tail specific foldingNikolay Korolev, Nikolay Berezhnoy, Abdollah Allahverdi,Renliang Yang, Chuan-Fa Liu, James P. Tam,Lars NordenskioldSchool of Biological Sciences, Nanyang TechnologicalUniversity, 60 Nanyang Drive, 637551, Singapore

Themajor component of chromatin,DNA, is adensely chargedpolyanion. Electrostatic interactions between DNA and DNA-packaging proteins contribute decisively to formation of itselementary unit, the nucleosome, and are also important inchromatin folding into higher-order structures. We investigatecondensation of DNA and chromatin and find that electro-statics and polyelectrolyte character of DNA play dominantrole in both DNA and chromatin condensation. By compre-hensive experimental studies and using novel oligocationicligands,we suggest simple universal equation describingDNAcondensation as a function of oligocation, DNA and monova-lent salt concentrations and including the ligand-DNA bindingconstant. We found that similar dependence was alsoobserved in condensation of the nucleosome arrays. Next, westudied how general electrostatic and specific structuralalterations caused by lysine acetylations in the histone tailsinfluence formation of 30-nm chromatin fibre and inter-molecular nucleosome array association. For the first time, astructural model is suggested which explains critical depen-dence of chromatin fibre folding on acetylation of the singlelysine at position 16 of the histone H4. Exceptional importanceof the H4 Lys 16 acetylation in general and gene specifictranscriptional activation has been known for many years butno structural basis for this effect has yet been proposed.

O-232Lambda genetic switch sensitivity depends on complexlooping kinetics driven by nonspecific bindingCarlo Manzo1,3, Chiara Zurla1,4, David Dunlap2,Laura Finzi11Physics Department and 2Cell Biology Department, EmoryUniversity, Atlanta, GA, USA, 3current address: ICFO -Institute of Photonic Sciences, Castelldefels (Barcelona),Spain, 4 current address: Department of BiomedicalEngineering, Georgia Institute of Technology, Atlanta, GA,USA

The kinetics of DNA loop formation and breakdown by the lrepressor protein (CI) have been characterized at variousconcentrations of protein using the tether particle motion(TPM) technique and a novel method of analysis withincreased time resolution. Comparison of the kinetics oflooping in wild-type DNA or DNA with mutated operators,showed that repressor bound at these sites may nucleateadjacent CI binding to further stabilize loops and broaden thedistribution of rate constants for loop formation and break-down. The average activation energies were calculated fromthe rate constant distributions and are consistent with a modelin which nonspecific binding of CI along the segment betweenthe operators shortens their effective separation, thereby

lowering the energy barrier for loop formation. The interplay ofthis effect with the enhanced loop stability at increased CIconcentration is crucial for loop efficiency since it allows tokeep constant the frequency of transition from the looped tothe unlooped DNA conformation at varying CI concentration.Thus at increased CI concentrations, although the loopbecame more stable thermodynamically, it periodically opensand thus remains sensitive to changing environmental con-ditions which may require switching to lytic conditions.

P-233Surface-enhanced Raman scattering of genomic DNAfrom apple leaf tissuesC. M. Muntean1, N. Leopold2, A. Halmagyi3,and S. Valimareanu31National Institute for Research & Development of Isotopicand Molecular Technologies, P.O. 5, Box 700, R-400293Cluj-Napoca, Romania, 2Babes-Bolyai University, Facultyof Physics, Kogalniceanu 1, RO-400084 Cluj-Napoca,Romania, 3Institute of Biological Research, Republici. 48,400015 Cluj-Napoca, RomaniaE-mail: cmuntean@itim-cj.ro

Detection of specific DNA sequences is central to modernmolecular diagnostic. Ultrasensitive Raman measurementsof nucleic acids are possible through exploiting the effect ofsurface-enhanced Raman scattering (SERS). In this work,the SERS spectra of genomic DNAs from leaves of differentapple trees grown in the field, have been analyzed [1].A detailed comparative analysis of SERS signatures of geno-mic DNAs is given. SERS wavenumbers (cm-1) are reportedhere for all typesof vibrationsof plant genomicDNAs, includingbands assigned to localized vibrations of the purine andpyrimidine residues, localized vibrations of the deoxyribose-phosphatemoiety, etc. Proposedbandassignments are given.A strong dependence of the SERS spectra on DNA con-centration and on time have been observed.In biochemical fields, nucleic acids might be used to explorethe interaction between DNA and small molecules, which isimportant in connection with probing the accurate localstructure of DNA and with understanding the natural DNA-mediated biological mechanisms [2].

References

1. C. M. Muntean, N. Leopold, A. Halmagyi, S. Valimareanu(2011) Spectroscopy-An International Journal, Vol. 25,Nr. 1, 33-43.

2. L. Sun, Y. Song, L. Wang, C. Guo, Y. Sun, Z. Liu, Z. Li, J.Phys. Chem. C 112 (2008) 1415-1422.

P-234The pH-dependent structure of DNA studied by FourierTransform Infrared SpectroscopyT.M. Partskhaladze, T.D. Mdzinarashvili, N.I. ShengeliaIv. Javakhishvili Tbilisi State University, Tbilisi, Georgia

Fourier transform infrared (FT-IR) spectra of deoxyribonu-cleic acid have been obtained and analyzed in the widerange of pH (2.9-10.2) environmental conditions.

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The region of the infrared spectrum studied covered thewave number range from 4000 cm-1 to 700 cm-1. IRspectra show that in pH 8.0-9.0 interval carbonyl (C=O)band at 1689 -1cm (assigned to guanine) is reduced inintensity and slightly shifted to lower frequencies. At the pH10.0 significantly decreases band intensity at 1712 cm-1

due to unbounded C2=O of Thymine and shifts to lowerfrequencies, indicating at the transition of this group inbounded form, supposedly by means of excess polarizedhydroxyl ions. Together this, in basic region a new intenseabsorption band has been observed in 1500-1300cm-1

frequency interval, corresponding to O-H group in-planebending vibration (1410-1310cm-1).As for acidic conditions, it was observed that under theextreme pH (*2.8) value carbonyl absorption region shifts tohigher frequencies and absorption intensity significantlyincreased, indicated at releasing of C=O groups fromH-bonding between base pairs. Moreover, bands intensity at1418cm-1 and 1022cm-1 corresponding to out-of-plandeformation of NH2 groups increased due to rupture ofconnections between the DNA strands.

P-235Structural order in complexes of DNA with chromosomalproteins HMGB1 and H1Alexander Polyanichko1,2, Tatyana Rodionova2,Elena Chikhirzhina21Faculty of Physics of Saint-Petersburg State University,Saint-Petersburg, Russia, 2Institute of Cytologyof the Russian Academy of Sciences, Saint-Petersburg,Russia

During the last decade it was found that in many casesspecific structural organization of multi-molecular proteinand DNA-protein complexes determines their functioningin living cells. Although these functioning structures areusually unique, it is often possible to identify their com-mon structural elements. One of the interesting examplesof such universal elements are HMGB domains: struc-turally conservative functional domains of non-histoneproteins HMGB1/2 also identified in many nuclear pro-teins. Using AFM, thermodynamic approaches, circulardichroism and molecular absorption spectroscopy in far-UV and mid-IR regions we have studied structural orga-nization of the complexes between DNA and differentproteins, including HMGB1, HMGB-domain recombinantproteins and linker histone H1. We have demonstrated,that interaction with DNA leads to increasing both a-he-licity of the proteins and thermal stability of DNA. Also,this interaction may result in formation of highly orderedsupramolecular complexes facilitated by HMGB-domains.The C-terminal sequence of HMGB1/2 regulates affinity ofthe proteins to DNA and can be ‘‘inactivated’’ by inter-action with histone H1. Based on the data obtained amodel of the interaction of multy-domain HMGB-proteinswith DNA is suggested.

O-236DNA replication machinery clamps down on chromatinmobilityJeffrey H. Stear*1, Dinah Loerke*2, Vadim Chagin3, HeinrichLeonhardt4 M. Cristina Cardoso31Humboldt Universitat; Berlin, Germany, 2Denver University;Denver, USA, 3Technische Universitat Darmstadt;Darmstadt, Germany, 4LMU Biozentrum; Munich, Germany*These authors contributed equally to this workChromatin in living cells displays considerable mobility on alocal scale. This movement is consistent with a constraineddiffusion model, in that individual loci execute multiple, randomjumps. To investigate the connection between local chromatindiffusion (LCD) and the changes in nuclear organization, weestablisheda stableHeLacell line expressingGFP-PCNA.Thisprotein, a core component of the replication machinery, servesasa cell-cyclemarker andallowsus tovisualize sites of ongoingDNA synthesis within the nucleus. To monitor LCD, we labeleddiscrete genomic loci through incorporation of Cy3-dUTP. Thisexperimental system, in conjunction with particle trackinganalysis, has enabled us to quantitatively measure chromatinmobility throughout the cell cycle.Our results demonstrate that LCD is significantly decreased inS-phase. To explore the connection between DNA replicationand reduced chromatin movement, we undertook a moredetailed examination of LCD in S-phase nuclei, correlatingchromatin mobility with sites of replication. Our results demon-strate that labeled chromatin in close proximity to GFP-PCNAfoci exhibit significantly decreased mobility. We therefore con-clude that presence of active replication forks constrains themovement of adjacent chromatin.

O-238Single-molecule studies of DNA replicationAntoine M. van OijenZernike Institute for Advanced Materials, GroningenUniversity, Nijenborgh 4. 9747 AG Groningen,The NetherlandsE-mail: a.m.van.oijen@rug.nl

Advances in optical imaging and molecular manipulation tech-niques have made it possible to observe individual enzymesand record molecular movies that provide new insight into theirdynamics and reaction mechanisms. In a biological context,most of these enzymes function in concert with other enzymesin multi-protein complexes, so an important future direction willbe the utilization of single-molecule techniques to unravel theorchestration of large macromolecular assemblies.We are applying a single-molecule approach to study DNAreplication. I will present recent results of single-moleculestudies of replication in bacterial and eukaryotic systems. Bycombining the stretching of individual DNA molecules withthe fluorescence observation of individual proteins, wevisualize the dynamic interaction of replication factors withthe fork. In the bacteriophage T7 replication system, we showthat DNA polymerases dynamically associate with and dis-sociate from the fork during replication. Further, I will present

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new data from single-molecule replication studies in X. laevisoocyte extracts. We have developed a novel imagingscheme that permits single-molecule fluorescence experi-ments at concentrations of labeled protein that were hithertoinaccessible. Using this method, we visualize, in real time,origin firing and fork movement.

O-239In silico single molecule manipulation with rigid bodydynamics: an efficient tool for modeling the mechanicalproperties of DNA-protein complexesPascal Carrivain and Jean-Marc VictorLaboratoire de Physique Theorique de la MatiereCondensee, Universite Pierre et Marie Curie, Case courrier121, 4 Place Jussieu, 75252 Paris Cedex 05, FranceE-mail: victor@lptmc.jussieu.fr

Keywords: Single molecule manipulation, magnetic twee-zers, rigid body dynamics, nucleosome, chromatin fiber.

Whereas Magnetic Tweezers (MT) and Optical Tweezers (OT)provide a unique tool for probing the mechanical properties ofDNA-protein complexes, in particular those of nucleosomes,they remain of little help in characterizing chromatin fiberstructure. As a matter of fact, force-extension diagrams requirean underlying model for their interpretation.We therefore have developed a new and powerful method formanipulating chromatin fibers under MT or OT in silico. Ouralgorithm is based on an open source library called ODE(Open Dynamics Engine, http://www.ode.org/) with which wehave implemented a Langevin dynamics.Force-extension diagrams of reference models of naked DNA(freely jointed chain, wormlike chain) as well as extension-rotation diagrams of naked DNA have been successfullyrecovered. Of note, plectonemic structures are most efficientlysimulated thanks to ODE’s collision detection code.New insights into nucleosome and chromatin fiber structureand dynamics will be presented.

P-240The study of the pKM.101 plasmid effect on the repairof DNAJ. Vincze1, I, Francia2, G. Vincze–Tiszay11HHEIF, Budapest, Hungary, 2Univ. Debrecen, Debrecen,Hungary

In our experiments was studied the effect of pKM.101 plasmidon repair of single strand breaks in DNA induced by 60Co-gamma irradiation in E.coli K 12 AB 1157 (wild type) and itsdifferent rec mutant cells. The pKM.101 resistant-factor incase of the control decreases the sensitivity of radiation,which as we suppose, is reached by the help of DNA con-formation change. It can be supposed from the well knowneffect of radiation biology that by the effect of pKM.101, theratio of DNA radiation sensitive volumes by appearing its newconformation decreases. The pKM.101 R-factor in rec

mutants in case of gamma irradiation shows effects in twoways. One is the ‘‘chemical’’ connection between the R-factorand DNA, though the other relate to positive and negative‘‘induced’’ radiation resistance from the local type effect of theconnection of an R-factor and a rec mutant, and the tworadiation resistant effects are added algebraically. As a resultfrom the view of biology we have to categorize the radiationresistance and the connected repair processes as two dif-ferent classes according to the change either in the chemicalor in the induced radiation resistant effect.

Computational biophysics and simulation

P-241Dependence on membrane composition and amino acidsequence of antimicrobial peptides activity. A moleculardynamics simulation studyJoao A. S. Almeida,1 Margarida Bastos,2 Luıs M. S. Loura31Chemistry Department, University of Evora, 7000-671Evora, Portugal, 2Chemistry Department, Facultyof Sciences, University of Porto, 4169-007 Porto, Portugal,3Faculty of Pharmacy, University of Coimbra, 3000-548Coimbra, PortugalE-mail: jasalmeida@qui.uc.pt

Important antimicrobial properties are recognized in severalpeptides, for which the ability to interact and disrupt bacteriamembranes have been reported. However, different mecha-nisms of action have been identified, suggesting that acomplex interplay of different factors are in the origin of suchbehavior.Recent studies have indicated that two trimethylated pep-tides (K6, K7), derived from the parental hybrid peptide CA(1-7)M(2-9), strongly interact with a bacterial membrane model(mixture of zwitterionic and negatively charged lipids), but notwith a membrane model of mammalian erythrocytes (zwit-terionic lipids) [1]. A reduction of the cytotoxicity effect and animprovement of the therapeutic index have also beenreported for the derivatives when compared with the parentalCA(1-7)M(2-9) [2].In this work, with the aim of providing insight on the interactionphenomena of the indicated peptides with zwitterionic andnegatively charged membrane models, a systematic molec-ular dynamics study was carried out. Full hydrated bilayers ofDMPC:DMPG (3:1) and POPE:POPG(3:1) were studied inthe presence of each peptide, and results analyzed in termsof peptide structure and membrane composition.Funding by FEDER (COMPETE program), and by FCT –Fundacao para a Ciencia e a Tecnologia, project referenceFCOMP-01-0124-FEDER-010787 (FCT PTDC/QUI-QUI/098198/2008) is acknowledged. J. A. S. Almeida gratefullyacknowledgesFCTand the sameProject for the researchgrant.

[1] J. Phys. Chem. B, (2010) 114, 16198-16208.[2] J. Phys. Chem. B. (2005) 109, 17311-17219.

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P-243Interlipid links in galactolipid and phospholipid bilayers.A comparative molecular modeling studyKrzysztof Baczynski, Marta Pasenkiewicz-GierulaFaculty of Biochemistry, Biophysics and Biotechnology,Jagiellonian University, Krakow, Poland

Lipid-water and lipid-lipid interactions at the membrane/waterinterface play important role in maintaining the bilayerstructure, however, this region is not easily available forexperimental studies. We performed molecular dynamicssimulations of two bilayers composed of two different types oflipids: (1) dioleoylphosphatidylcholine (DOPC); (2) galactoli-pid monogalactosyldiacylglycerol (MGDG). To investigate theproperties of the membrane/water interface region, we per-formed analysis of lipid-lipid interactions: direct, via chargepairs (DOPC) and hydrogen bonds (MGDG) as well asindirect, via water bridges. We also examined water-lipidinteractions. Existence of well-defined entities (lipids) linkedby different types of interactions (hydrogen bonds, chargepairs, water bridges) makes the analysis of the membrane/water interface region a suitable for a graph theoreticaldescription. We applied a network analysis approach forcomparative analysis of simulated systems. We note amarked difference between the organization as well as thedynamics of the interfacial region of the two bilayers.

P-244Ligand chirality effects the dynamics of human3-phosphoglycerate kinaseZoltan Palmai1, David Perahia2, Corinne Lionne3,Judit Fidy1,4, Erika Balog1, Laurent Chaloin31Department of Biophysics and Radiation Biology, Facultyof Medicine, Semmelweis University, Budapest, Hungary,2Institut de Biochimie et Biophysique Moleculaire etCellulaire, Universite Paris-Sud, Orsay, France, 3Centred’etudes d’agents Pathogenes et Biotechnologies pour laSante (CPBS), UMR 5236, CNRS - Universite Montpellier1–Universite Montpellier 2, Institut de Biologie, Montpellier,France, 4Research Group for Membrane Biology of HAS,Budapest, Hungary

L-nucleoside analogues form an important class of antiviraland anticancer drug candidates. To be pharmacologicallyactive, they need to be phosphorylated in multiple steps bycellular kinases. Human phosphoglycerate kinase (hPGK)was shown to exhibit low specificity for nucleotide diphos-phate analogues and its catalytic efficiency inphosphorylation was also affected. To elucidate the effect ofligand chirality on dynamics and catalytic efficiency, molec-ular dynamics simulations were performed on four differentnucleotides (D-/L-ADP and D-/L-CDP) in complex withhPGK and 1,3-bisphospho-D-glycerate (bPG). The simula-tion results confirm high affinity for the natural substrate (D-ADP), while L-ADP shows only moderate affinity for hPGK.The observed short residence time of both CDP enantiomersat the active site suggests very weak binding affinity whichmay result in poor catalytic efficiency shown for hPGK withD-/L-CDP. Analysis of the simulations unravels importantdynamic conditions for efficient phosphorylation replacingthe single requirement of a tight binding.

P-246The electron correlation effects on the energy bandstructures of double stranded DNACristian Morari and Attila BendeMolecular and Biomolecular Physics Department, NationalInstitute of R&D of Isotopic and Molecular Technology(INCDTIM), Donath Street, No. 65-103, Cluj Napoca,Romania

Using the van der Waals density functional based on thesemilocal exchange functional PW86 together with a long-range component of the correlation energy [1] implementedin the SIESTA program code, we have calculated the bandstructure of the double stranded DNA. The unit cell was builtby taking together 10 GC (or AT) homogenous base pairsand we have considered the translational symmetry as theperiodic boundary condition. The results obtained are com-pared with the oligomer calculations taking up to seven basepairs. The band structure obtained with this van der Waalsdensity functional is also compared with results obtained withother exchange-correlation functionals as well as with bandstructure obtained by the Hartree-Fock crystal-orbital methodtaking into account the helical symmetry of the doublestranded DNA. The role of different parts of DNA (base pairs,sugar-phosphate backbone, Na ions) is also presented.

[1] K. Lee, E. D. Murray, L. Kong, B. I. Lundqvist, D. C. Lang-reth, Phys Rev B, 82:081101, (2010).

O-247Using low-resolution structural data to model structure,function and motion in transmembrane proteinsNir Ben-TalDepartment of Biochemistry & Molecular Biology, George S.Wise Faculty of Life Sciences, Tel Aviv University, Israel 69978Web: http://ashtoret.tau.ac.ilEmail: bental@ashtoret.tau.ac.il; NirB@tauex.tau.ac.il

Transmembrane (TM) proteins comprise some 15% to 30% ofthe proteome but owing to technical difficulties, relatively fewof these structures have been determined experimentally.Computational modeling techniques can be used to providethe essential structural data needed to shed light on structure-function relationships in TM proteins. Low-resolution electron-density maps, obtained from cryo-electron microscopy(cryo-EM) or preliminary X-ray diffraction studies, can be usedto restrict the search in conformational space. At the rightresolution, the locations of TM helices can be roughly deter-mined even when the amino acids are not visible. When thesedata are combined with physicochemical characteristics ofamino acids (such as their hydrophobicity) and with evolu-tionary conservation analysis of the protein family, thelocation of the amino acids can be modeled. The model-structure may provide molecular interpretations of the effectsof mutations. Moreover, it can be used to suggest molecularmechanisms and to design new mutations to examine them.The overall approach will be demonstrated using two humanproteins: copper transporter 1 (CTR1), which is the maincopper transporter in the human cell, and the 18 kDa trans-locator protein (TSPO) of the outer mitochondria. Model-structures of these proteins and their functional implications inhealth and disease will be discussed.

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P-248Mechanism of conductance and selectivity in a modelcalcium channel from Monte Carlo simulationsDezs}o Boda1,3, Tamas Kristof1, Dirk Gillespie2, BobEisenberg2, Douglas Henderson3, Wolfgang Nonner41Department of Physical Chemistry, University of Pannonia,H-8201 Veszprem, P.O. Box 158, Hungary, 2Departmentof Molecular Biophysics and Physiology, Rush UniversityMedical Center, Chicago, USA, 3Department of Chemistryand Biochemistry, Brigham Young University, Provo, USA,4Department of Physiology and Biophysics, Miller Schoolof Medicine, University of Miami, Miami, USA

Calcium channels play a crucial role in many physiologicalfunctions and their selectivity mechanism is still an unre-solved question and a subject of debate. A physical modelof selective ‘‘ion binding’’ in the L-type calcium channel isconstructed, and consequences of the model are comparedwith experimental data. This reduced model treats only ionsand the carboxylate oxygens of the EEEE locus explicitlyand restricts interactions to hard-core repulsion and ion-ionand ion-dielectric electrostatic forces. According to thecharge/space competition mechanism, the charge ofstructural ions attracts cations into the filter, while excludedvolume effects are trying to keep them out. This is acompetition between energy and entropy, where the bal-ance of these terms minimizes free energy and determinesselectivity. Experimental conditions involving binary mix-tures of alkali and/or alkaline earth metal ions arecomputed. The model pore rejects alkali metal ions in thepresence of biological concentrations of Ca2+ and predictsthe blockade of alkali metal ion currents by micromolarCa2+. Conductance patterns observed in varied mixturescontaining Na+ and Li+, or Ba2+ and Ca2+, are predicted.Ca2+ is substantially more potent in blocking Na+ currentthan Ba2+. In apparent contrast to experiments sing buf-fered Ca2+ solutions, the predicted potency of Ca2+ inblocking alkali metal ion currents depends on the speciesand concentration of the alkali metal ion, as is expected ifthese ions compete with Ca2+ for the pore. These exper-iments depend on the problematic estimation of Ca2+activity in solutions buffered for Ca2+ and pH in a varyingbackground of ulk salt. Equilibrium binding affinity(expressed as the occupancy of the selectivity filter byvarious ions) is computed by equilibrium grand canonicalMonte Carlo (GCMC) simulations. The conductivity of thechannel is estimated from the equilibrium concentrationprofiles using the integrated Nernst-Planck equation. Oursimulations show that the selectivity of L-type calciumchannels can arise from an interplay of electrostatic andhard-core repulsion forces among ions and a few crucialchannel atoms. The reduced system selects for the cationthat delivers the largest charge in the smallest ion volume.We have also performed dynamic Monte Carlo (DMC)simulations for a model Ca channel to simulate currentdirectly and present our results for the dynamical selectivity(expressed as the flux carried by various ions). We showthat the binding affinity of Ca2+ versus Na+ is alwayslarger than the dynamical selectivity because Ca2+ ionsare tightly bound to the binding site of the selectivity filter of

the channel and, at the same time, their mobility and driftvelocity is smaller in this region.

1. D. Boda, M. Valisk0o, B. Eisenberg, W. Nonner, D.Henderson, and D. Gillespie, J. Chem. Phys., 125,034901 (2006).

2. D. Boda, M. Valisk0o, B. Eisenberg, W. Nonner, D.Henderson, and D. Gillespie, Phys. Rev. Lett., 98,168102, (2007).

3. D. Gillespie and D. Boda, Biophys. J., 95, 2658, (2008).4. D. Boda, M. Valisk0o, D. Henderson, B. Eisenberg, D.

Gillespie, and W. Nonner. J. Gen. Physiol., 133, 497,(2009).

5. G. Rutkai, D. Boda, and T. Krist0of, J. Phys. Chem. Lett.,1, 2179, (2010).

P-249Vibrational Spectroscopy of Biomolecules by mixedQuantum / Classical Molecular DynamicsDaniele Bovi1, Riccardo Spezia2, Alberto Mezzetti3,Rodolphe Vuilleumier4, Marie-Pierre Gaigeot2 and LeonardoGuidoni11Dipartimento di Fisica, La Sapienza - Universita di Roma,Italy and Dipartimento di Chimica, Ingegneria Chimica eMateriali, Universita degli Studi dell’Aquila, Italy, 2LAMBEUMR 8587 CNRS, Universite d’Evry Val d’Essonne, 91025Evry Cedex, France, 3Laboratoire de SpectrochimieInfrarouge et Raman UMR CNRS 8516, Universite deSciences et Technologies de Lille, 59655 Villeneuve d’Ascq,France, 4Departement de Chimie, Ecole NormaleSuperieure, 24 rue Lhomond, 75005 Paris, France

Carotenoids are used in light-harvesting complexes with thetwofold aim to extend the spectral range of the antenna andto avoid radiation damages. The effect of the polarity andconformation of the environment is supposed to be respon-sible for the tuning of the electronic, optical and vibrationalproperties of peridinin carotenoid both in solution and inprotein matrix. We investigate the effect of vibrational prop-erties of peridinin in different solvents by means of vibrationalspectroscopies and QM/MM molecular dynamics simula-tions1. The shift of vibrational fingerprints in the 1500-2000cm-1 frequency region, due to the solvent polarity and pro-ticity, is studied in three cases: cyclohexane (apolar/aprotic),deuterated acetonitrile (polar/aprotic) and methanol (polar/protic).The frequencies and vibrational modes of the carbonyl, theallene, and the polyene chain were identified using effectivenormal mode analysis2 and compared with the present andprevious experimental data3. On the basis of our calcula-tions and experiments in different solvents, we propose aclassification of the four peridinins of the high-salt PCPform.

[1] L. Guidoni et al., Quant. Struct.-Act. Relat. 21,119.(2002)

[2] M. Martinez et al., J. Chem. Phys. 125, 144106. (2006)[3] A. Mezzetti et al., Spectroscopy: Int. J., 22, 235. (2008)

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P-250Adsorbtion of cysteine on gold (111) surfaces:a DFT studyLuiza Buimaga-Iarinca, Cristian Morari, Nicoleta TosaNational Institute of Research and Development for Isotopicand Molecular Technologies, Department of Molecularand Biomolecular Physics, 65-103 Donath Str.,400293, Cluj-Napoca, RomaniaE-mail: iarinca@itim-cj.ro

The controlled self-assembly of functional molecular specieson well defined surfaces is a promising approach towardthe design of nanoscale architectures. By using this meth-odology, regular low-dimensional systems such assupramolecular clusters, chains, or nanoporous arrays canbe fabricated. Small biological molecules such as aminoacids represent an important class of building blocks that areof interest for molecular architectonic on surfaces becausethey inherently qualify for molecular recognition and self-assembly [1]. The interaction between amino acids and solidsurfaces is decisive for the development of bioanalyticaldevices or biocompatible materials as well as for a funda-mental understanding of protein-surface bonding.We investigate the adsorbtion mechanism of the cysteine onAu(111) surfaces bymeans of theDFT [2]. Ourmain concern is todescribe the molecule-metal bonding mechanism. Therefore wepresent a complex study, including the full determination of thedensity of states for the free and adsorbed molecule, the deter-mination of molecule-surface bonding energy. The method ofcrystal orbital overlappopulations is used inorder todetermine thecontributionof specificatomicorbitals to themolecule-metal bond.

[1] Barlow SM, Raval R (2003) Surf Sci Rep 50:201–341.[2] Soler JM, Artacho E, Gale JD, Garcıa A, Junquera P,

Ordejon P, Sanchez-Portal D (2002) J Phys Cond Mat14(11):2745–2779.

P-251Human myoglobins: paradigm of structure-functionrelationM. A. Scorciapino, M. Casu, E. Spiga, M. CeccarelliDepartment of Physics and Department of Chemistry,University of Cagliari, Cagliari, Italy

It isnowwidelyaccepted thatmyoglobin (Mb) isnotsimplyanO2

storage/delivery system but, depending on oxygen concentra-tion, it exerts other fundamental physiological roles. Recentstudies revealed a widespread expression and, in particular, anover-expression in response to hypoxia, in various non-muscletissues, including tumor cells. In Human five different Mb iso-forms are present. The two most expressed ([90%) differ onlyat the 54th position, K54 (Mb-I) and E54 (Mb-II) respectively.Since high-altitude natives from Tibet are characterized by ahigher Mb concentration and locomotion efficiency, togetherwith the observation that the Mb overexpression is totallyattributable to Mb-II, the idea that the latter might be one of theresponses to high-altitude evolutionary adaptation, i.e. hypoxicenvironment, started to emerge. However, this is not yet sup-ported by any structure/function investigation.We performed hundred nanoseconds MD simulations onhuman Mbs to investigate the structure and dynamics of bothprotein and surface water. Important differences have been

found at the histidine gate region, suggesting that the twomore expressed human myoglobins might have a distinctand complementary role. While 1H and 129Xe nmR experi-ments do not show structural/dynamical differences, or noton longer time-scale than those offered by MD simulations,EPR experiments pointed out a different affinity of the twoisoforms for NO, thus confirming a potential diverse role aspredicted by simulations.R. Anedda et al. J. Phys. Chem. B, 112: 15856, 2008; M.A. Scorciapino et al. J. Am. Chem. Soc., 131, 11825, 2009; M.A. Scorciapino et al. J. Am. Chem. Soc. 132, 5156-5163, 2010;M. A. Scorciapino et al., Submitted; Gussoni et al., Submitted.

P-252Structural, dynamic and thermodynamic effects of KITmutations: a computational multi-approach studyI. Chauvot de Beauchene, E. Laine, C. Auclairand L. TchertanovLBPA, CNRS - ENS de Cachan, France

Protein kinases play key roles in cell signaling and constitutecrucial therapeutic targets. In normal cell, upon substrate bind-ing, tyrosine kinase receptor KITundergoes extensive structuralrearrangement leading to receptor dimerization and activation.This process is initiated by the departure of the juxta membraneregion (JMR) from the active site, allowing the activation loop (A-loop) deployment. The deregulation of KITactivity is associatedwith various forms of cancer provoked by abnormalities in signaltransduction pathways. Mutations V560G (JMR) and D816H/V(A-loop) havebeen reportedasoncogenic and/or drug-resistant.To contribute further in the understanding of KIT activation/deactivationmechanisms, we applied amulti-approach protocolcombining molecular dynamics (MD), normal modes analysis(NMA) and pocket detection. Disturbing structural effects, bothlocal (A-loop) and long-range (JMR), were evidenced for KITD816H/V in the inactive state. nmA showed that JMR is able todepart itspositionmoreeasily in themutants than in thewild type.Pockets analysis revealed that this detachment is sufficient toopen an access to the ATP binding site. Our results provided aplausible conceptionofmutantdimerizationandaway toexploreputative allosteric binding sites.

P-253Transmembrane association of leukocyte integrinheterodimer might be mediated by a polar interactionChoon-Peng Chng1 and Suet-Mien Tan21Biophysics Group, A*STAR Institute of High PerformanceComputing, and, 2School of Biological Sciences, NanyangTechnological University, Republic of Singapore

The lateral association of transmembrane (TM) helices isimportant to the folding of membrane proteins as well as ameans for signaling across the cell membrane. For integrin, ahetero-dimeric protein important for cell adhesion andmigration, the association of its a- and b-subunits’ TM helicesplays a key role in mediating bi-directional mechanical sig-naling across the membrane. We found evidence fromexperiment and simulation for a polar interaction (hydrogen-bond) across leukocyte integrin aLb2 TM that is absent in thebetter-studied platelet integrin aIIbb3 [1]. Our coarse-grained

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molecular dynamics simulations of TM helix-helix self-assembly showed more native-like packing achieved byaLb2 within the simulation timescale as compared to its ‘loss-of-function’ b2T686G mutant or aIIbb3 [2]. Association freeenergy profiles also showed a deeper minimum at a smallerhelix-helix separation for aLb2, suggestive of tighter packing.The likely conservation of this polar interaction across the b2integrin family further reinforces its importance to the properfunctioning of leukocyte integrins.

1. Vararattanavech, C-P. Chng, K. Parthasarathy, X-Y.Tang, J. Torres and S-M. Tan. J. Mol. Biol. 38:569-83,2010.

2. C-P. Chng and S-M. Tan. Proteins, 2011. Accepted.

P-254Meropenem vs. Imipenem interacting with MexB:structural and dynamical determinants of the effluxaction on two substratesFrancesca Collu1, Attilio V. Vargiu2,3, Michele Cascella1,Paolo Ruggerone2,31Department of Chemistry and Biochemistry, UniversitatBern, Freiestrasse 3, CH-3012 Bern, Switzerland, 2CNR-IOM, Unita SLACS, S.P. Monserrato-Sestu Km 0.700,I-09042 Monserrato (CA), Italy, 3Department of Physics,University of Cagliari, S.P. Monserrato-Sestu Km 0.700,I-09042 Monserrato (CA), Italy

Active extrusion of drugs through efflux pumps constitutesone of the main mechanisms of multidrug resistance in cells.In recent years, large efforts have been devoted to the bio-chemical and structural characterization of RND effluxpumps in Gram-negative bacteria, in particular the AcrB/A-TolC system of E.Coli. Specific attention has been addressedto the active part of the efflux system, constituted by the AcrBunit. Despite the presence of several data, crucial questionsconcerning its functioning are still open. The understandingof the structure-dynamics-function relationship of MexB, theanalogous transporter in P. Aeruginosa, encounters evenmore difficulties, because of the lack of structural data of thetransporter in complex with substrates. To shade some lighton the activity of MexB, we performed computational studieson MexB interacting with two compounds, meropenem andimipenem, the first known to be a good substrate, and thesecond a modest one. Several techniques were used in thepresent work, ranging from flexible docking [1] to standardand targeted molecular dynamics (MD) simulations. Startingfrom the published crystal structure [2] we identified the mostprobable poses of the two compounds in both the originalexperimental and in the MD-equilibrated structures. We usedinformation from AcrB binding pocket in order to find relevantbinding sites of the two compounds in the analogous bindingpocket of MexB.Meropenem frequently lies with appropriate orientation in apocket similar to the one identified for doxorubicin in AcrB [3],while the occurrence of imipenem poses in the same pocketis very scarce. Additionally, when present in the pocket,imipenem is located in a position that renders very unlikely itsextrusion toward the OprM docking domain during the sim-ulation of the functional peristalsis. The analysis of thetrajectories has provided a complete inventory of the

transporter and antibiotic hot spots, which is key informationin terms of screening and design of antibiotics and inhibitors.

[1] Zacharias M., Protein Sci 2003, 12, 1271-82.[2] Sennhauser G., et al., J Mol Biol 2009, 389, 134-145.[3] Murakami S., et al., Nature 2006, 443, 173-9.

P-255Simulations of clathrin self-assembly into polyhedralcagesW. K. den Otter and W. J. BrielsComputational BioPhysics, University of Twente, Enschede,P.O. Box 217, 7500 AE Enschede, The Netherlands

Clathrins are three-legged proteins with the intriguing ability toself-assemble into a wide variety of polyhedral cages. Thenucleation and growth of a clathrin lattice against the cytosolicface of a cell membrane enables the endocytosis of membraneproteins and various external molecules, by wrapping themembrane around the cargo to produce a coated transportvesicle within the cell. Clathrins can also self-assemble, inslightly acidic solutions devoid of auxiliary proteins, into emptycages. Our simulations of this process, using a highly coarse-grained model, indicate that the key to self-assembly is neithercalthrin’s characteristic puckered triskelion shape, nor thealignment of four legs along all cage edges, but an asymmetricdistribution of interaction sites around the leg’s circumference.Based on the critical assembly concentration, the bindingstrength in these cages is estimated at 25 to 40 kBT per clathrin.The simulations also answer the long-standing conundrum ofhow flat patches of purely hexagonal clathrin lattices, which incryo electron microscopy are frequently seen to decorate cellmembranes, canproducehighlycurvedcagescontaining twelvepentagonal faces interdispersed between hexagonal faces. Wepresent experimental evidence supporting this pathway.

P-256Allostery in nuclear export: a hybrid methods approachN. Dolker1,4, C. Blanchet2, T. Monecke3, D. Svergun2,R. Ficner3, H. Grubmuller1, A. Dickmanns31Department of Theoretical and Computational Biophysics,Max Planck Institute for Biophysical Chemistry, 37077Gottingen, Germany, 2European Molecular BiologyLaboratory, Notkestrae 85, Geb. 25A, 22603 Hamburg,Germany, 3Institut f ur Mikrobiologie und Genetik & GZMB,Georg-August-Universitat Gottingen, Justus-von-Liebig-Weg11, 37077 Gottingen, Germany, 4Present address:Computational Biophysics Group, Spanish National CancerResearch Centre (CNIO), Melchor Fern0andez Almagro, 3,28029 Madrid, Spain

In eukaryotic cells, the exchange of macromoleculesbetween the cytoplasm and the nucleus is mediated byspecialized transport factors. By binding to these transport-ers, cargo molecules, which are otherwise excluded fromentering the nucleus, can traverse the nuclear pore complexefficiently. Most of the proteins mediating nuclear import andexport exhibit a characteristic _-solenoid fold, which providesthem with an unusual intrinsic flexibility. CRM1 is an essentialnuclear export receptor, which recognizes a very broad range

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of export cargoes. CRM1-dependent nuclear export is Ran-GTPase-driven, and recognition of RanGTP and cargo ishighly cooperative. However, recent crystal structures showthat the binding sites for export cargos and RanGTP arelocated at distant parts of CRM1 [1-3]. We have used acombined approach of all-atom molecular dynamics simula-tions and small-angle X-ray scattering to study RanGTP andcargo binding to CRM1. We have found that the allostericeffect in CRM1-dependent nuclear export arises from acombination of subtle structural rearrangements and chan-ges in the dynamic properties of CRM1.

[1] Monecke, T.; G uttler, T.; Neumann, P.; Dickmanns, A.;G orlich, D. & Ficner, R. Science 2009, 324, 1087-1091.

[2] Dong, X.; Biswas, A.; S uel, K. E.; Jackson, L. K.;Martinez, R.; Gu, H. & Chook, Y. M. Nature 2009, 458,1136-1141.

[3] Koyama, M. & Matsuura, Y. EMBO J. 2010, 29, 2002-13.

P-2573D-Structural model of Channelrhodopsin-2Kirstin Eisenhauer1, Steffen Wolf1,2, Erik Freier1,Peter Hegemann3, Klaus Gerwert1,21Department of Biophysics, Ruhr-University Bochum, ND 04North, Universitatstr. 150, 44780 Bochum, Germany, 2CAS-MPG Partner Institute for Computational Biology, 320 YueYang Road, 200031 Shanghai, China, 3Departmentof experimental Biophysics, Humboldt-University Berlin,Invalidenstraße 42, 10115 Berlin, Germany

Light-induced phototactic responds in green algae Chlamydo-monas reinhardtii are mediated via microbial-type rhodopsins,termed Channelrhodopsin-1 (ChR1) and Channelrhodopsin-2(ChR2)1, which carry the chromophore retinal covalently linkedto lysin viaaSchiff baseandwereshown tobedirectly light-gatedion-channels2. The N-terminal putative seven-transmembraneregionofChR2wasshown tobe responsible for thegenerationofphotocurrents and exhibits sequence similarity to the wellunderstood proton pump bacteriorhodopsin (BR) and the sen-sory rhodopsinAnabaena sensory rhodopsin (ASR)3. As for themajority of membrane proteins, there is no 3D-structural data forChR2 available yet. Here we present homologymodels of ChR2using two high-resolution X-ray template structures of BR(1QHJ4) respectively ASR (1XIO5) in order to get structural andfunctional insights into ChR2. With both homology models weperformed molecular dynamics (MD) simulations in a nativemembrane/solvent environment using GROMACS 4.0.36.Comparison of energetic and structural results revealed obviousadvantages of theBR-basedhomologymodel ofChR2.Hereweshow that the BR-based homology model is a reliable model ofChR2 exhibiting structural features already found experimen-tally7. Our BR-based homology model of ChR2 allowspredictions of putative crucial residues within ChR2. So we pro-posed several mutations within the ChR2 sequence which arealready accomplished. Electrophysiologic and spectroscopicstudies of these mutations are underway in order to confirm thefunctional relevance of these residues and to contribute to anoptimized usage of ChR2 as a powerful tool in optogenetics.

(1) Sineshchekov O. et al. Two rhodopsins mediate photo-taxis to low- and high-intensity light in Chlamydomonasreinhardtii, PNAS, 99, 8689-94 (2002)

(2) Nagel G. et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel, PNAS, 100,13940-45 (2003)

(3) Nagel G. et al. Channelrhodopsins: directly light-gatedcation channels, Biochem. Soc. Trans, 33, 863-66 (2005)

(4) Belrhali H. et al. Protein, lipid and water organization inbacteriorhodopsin crystals: a molecular view of the purplemembran at 1,9 L resolution, Structure, 7, 909-17 (1999)

(5) Vogeley L. et al. Anabaena sensory rhodopsin: A photo-chromic color sensor at 2.0 L,Science 306, 1390-3 (2004)

(6) Hess B. et al. GROMACS 4: Algorithms for highlyefficient, load-balanced and scalable molecular simula-tion, J.Chem.

(7) Nack M. et al. The DC-gate in Channelrhodopsin-2:crucial hydrogen bonding interaction between C128 andD156, Photochem. Photobiol. Sci. 9, 194-8 (2010)

P-258Cooperativity in a multidomain proteinMarta Enciso, Gael Radou, Sergei Krivov andEmanuele PaciInstitute of Molecular and Cellular Biology, Faculty of BiologicalSciences, University of Leeds, UK

Cooperativity is an important feature in the folding of mostproteins. Recently, in a two domain protein such as T4 lyso-zyme the interdomain cooperativity has been preciselyquantified at single molecule level. Such cooperativity hasbeen shown to be dramatically attenuated in a circular per-mutant which folds to the same native structure. In this paperwe exploit this single molecule measurement of cooperativityand a simple molecular model to infer the relationshipbetween the interdomain cooperativity, chain connectivity andfree energy landscapes. In particular we show that bothspecies have an onpathway intermediate where one of thedomains is completely formed. The change in cooperativitycan be ascribed to a change in the rate limiting step for fold-ing. This occurs before the intermediate in the wild type andafter in the circular permutant. We suggest that the evolutionof discontinuous subdomains may have been facilitatedthrough the exploitation of features of the free energy land-scape of an ancestor with continuous subdomains.

P-260Flash photolysis experiments and molecular dynamicssimulation of ligand binding to neuroglobinZofia Gasik1, Stefania Abbruzzetti2, Stefano Bruno2, SylviaDewilde3, Jan Antosiewicz1 and Cristiano Viappiani21University of Warsaw, Warsaw, Poland, 2Universityof Parma, Parma, Italy, 3University of Antwerp, Antwerp,Belgium

Neuroglobin is a recently discovered globin protein pre-dominantly expressed in brain. Its biological function is stillelusive. Despite the fact that neuroglobin shares very littlesequence homology to the well- known globins as mioglobinand hemoglobin, they all have a characteristic globin fold withHEME molecule bound to the distal pocket. The structuralinvestigations and CO binding kinetics revealed existence ofcavities and tunnels within the protein matrix, where small

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ligands can be stored even for hundreds of microseconds [1].In human neuroglobin there is one internal disulfide bondpossible which existence is proved to have significant effecton ligand affinity [2]. In this study effects of temperature, pH,distal histidine mutation and presence of disulfide bond onCO rebinding to neuroglobin are investigated by flash pho-tolysis experiments. In parallel, the molecular dynamicssimulations are performed in corresponding conditions inorder to investigate structural change of neuroglobin andespecially changes in distribution of internal tunnels andcavities able to bind diatomic ligands in response to differentphysical conditions listed above.

1. Abbruzzetti et al., PNAS, 2009, 106, No. 45,18984-18989

2. Hamdane et al., J. Biol. Chem., 2003, 278, No.51,51713-51721

P-261Ligand Entry Pathways in PPARc Explored UsingTargeted Molecular Dynamics SimulationsSamia Aci-Seche, Monique Genest, Norbert GarnierCentre de Biophysique Moleculaire, UPR 4301, CNRS, rueCharles Sadron, 45071 Orleans Cedex 02, affiliatedto the University of Orleans, France

Computational methods based on molecular dynamics (MD)simulations suite very well for objective exploration of pos-sible ligand entry/exit pathways from protein Ligand BindingDomains (LBDs). Identifying ligand entry/exit routes providesfundamental understanding of protein function and addssubstantial information to rational drug design process.In this area of active research, we focus on PPARs family ofnuclear receptors that emerged as therapeutic targets.Agonists have established therapeutics benefits in treatingdiabetes and cardiovascular diseases.In this work, we explore possible ligand entry pathways ofGW0072, a PPARc modulator that appears to function as aninsulin sensitizer, by using Targeted Molecular Dynamics(TMD) simulations. Starting from the apo-form of PPARc withGW0072 distal from probable exit gates, TMD simulationsconverge successfully towards the targeted holo-form of thecomplex. The simulation results showed that i) entrancepaths depend on the protonation state of the ligand; ii) ligandapproach suggests that hydrophobic interactions guide thepenetration process iii) the routes for ligand access to thebinding site closely superimpose the most probable ligandexit pathway described in our previous studies(Genest & al. Eur Biophys J 2008).

P-262Molecular recognition at atomic level: interactionof autism related protein MCP-1 with an antibodyAnna Gogolinska1,2, Wieslaw Nowak11Faculty of Physics, Astronomy and Informatics, NicolausCopernicus University, Torun, Poland, 2Facultyof Mathematics and Computer Science, Nicolaus CopernicusUniversity, Torun, Poland

Autism is often related to infections of autistic children’smothers during pregnancy. The higher activity of the immune

system perturbs developing brain and may result in thisdisorder. It has been found that Monocyte ChemoattractantProtein (MCP-1) levels in brains of autistic children are ele-vated. Interactions of chemokine MCP-1 with antibodies areimportant for diagnosis and possible treatment . We presentresults of classical Molecular Dynamics (MD) studies ofMCP-1 complex with a murine Fab antibody fragment [1].Regions important for molecular recognition (MR) aredescribed and their dynamics is critically analyzed. Theprocess of MCP-1 binding/dissociation is modeled on all-atoms level using the steered MD method by adding a virtualforce to increase probability of rare transition in antibody-antigen complexes. The detailed path of MCP-1- antibodyMR is analyzed. The interaction mechanism is compared toIgE-grass Phl p2 allergen recognition process studied earlier.These results should help to interpret AFM experimentsexploiting lateral force microscopy – a promising techniquefor MR tracking and medical diagnosis [2].ACKN: Supported in part by MNiSZW grant no. N519 578138

[1] C. Reid et al. Prot Eng 19 (2006) 317[2] M. Lekka et al. J Chem Phys 123 (2005) 014702

P-264Atomistic simulation of single molecule fluorescenceexperiments: FRET beyond j2=2/3Martin Hoefling1, Nicola Lima1, Dominik Haenni2, Claus A.M.Seidel3, Benjamin Schuler2, Helmut Grubmuller11Max Planck Institute for Biophysical Chemistry, Gottingen,Germany, 2University of Zurich, Zurich, Switzerland,3Heinrich Heine University, Dusseldorf, Germany

Forster Resonance Energy Transfer (FRET) experimentsprobe molecular distances via distance dependent energytransfer from an excited donor dye to an acceptor dye.Single molecule experiments not only probe average dis-tances, but also distance distributions or even fluctuations,and thus provide a powerful tool to study biomolecularstructure and dynamics. However, the measured energytransfer efficiency depends not only on the distancebetween the dyes, but also on their mutual orientation, whichis typically inaccessible to experiments. Thus, assumptionson the orientation distributions and averages are usuallymade, limiting the accuracy of the distance distributionsextracted from FRET experiments. Here, we demonstratethat by combining FRET experiments with the mutual dyeorientation statistics obtained from Molecular Dynamics(MD) simulations, improved estimates of distances anddistributions are obtained. From the simulated time-depen-dent mutual orientations, FRET efficiencies are calculatedand the full statistics of individual photon absorption, FRETtransfer, and photon emission events is obtained from sub-sequent Monte Carlo (MC) simulations of the FRET transferkinetics. All recorded emission events are collected to burstsfrom which efficiency distributions are calculated in closeresemblance to the actual FRET experiment, taking shotnoise fully into account. Using polyproline chains withattached Alexa 488 and Alexa 594 dyes as a test system,we demonstrate the feasibility of this approach by directcomparison to experimental data. We identified cis-isomersand different static local environments as sources of the

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experimentally observed heterogeneity. Reconstructions ofdistance distributions from experimental data at differentlevels of theory demonstrate how the respective underlyingassumptions and approximations affect the obtained accu-racy. Our results show that dye fluctuations obtained fromMD simulations, combined with MCsingle photon kinetics,provide a versatile tool to improve the accuracy of distancedistributions that can be extracted from measured singlemolecule FRET efficiencies.

P-265Molecular Simulations of Thrombospondin’s C-TerminalRegionT. Haschka1,3, C. Etchebest1, E. Henon2, L. Martiny3,M. Dauchez31Inserm UMRS 665 - Laboratoire Dynamique des Structureset Interactions des Macromolecules Biologiques (DSIMB) 6,rue Alexandre Cabanel, 75739 Paris Cedex 15, France,2Institut de chimie moleculaire de Reims, CNRS UMR 6229Universite de Reims Champagne-Ardennes, Pole Sciences,Moulin de la Housse - BP1039, 51687 Reims Cedex 2,France, 3SiRMa Laboratory CNRS UMR6237 - MatriceExtracellulaire et Dynamique Cellulaire (MEDyC) Universitede Reims Champagne-Ardennes, Pole Sciences, Moulin dela Housse - BP1039, 51687 Reims Cedex 2, France

The thrombospondin family, being extracelluar proteins, isknown to be implicated in various physiological processessuch as wound healing, inflammation, angiogenesis andneoplasia. The signature domain of thrombospondins showshigh sequence identities and thus allows us to transferresults obtained, studying this complex calcium reach part ofthe proteins, from one member of the family to the other. Thedomain is known to play a key role in hereditary diseasessuch as PSACH or MED. In this part of thrombospondins liesa binding site to integrins, important for cell attachment. It isfurther known that the lectin like globe binds to CD-47, afeature known to be important in cancer research. As thetheoretical unit we are trying to resolve these problems bynumerical means and are constantly challenged by the size,where thrombospondin can be a huge trimeric protein as onestrand can measure 430 kDa, and the large variety of sub-domains found in this proteins. We are thus facing amultiscale problem which can range from solving, by meansof quantum mechanics a specific ion binding site, to largescale abstraction by continuum mechanics. In our talk we willshow you our newest results that we obtained by simulatingcalcium rich C-terminal domain which is known to be con-served across the entire family, and give you an outlook intothe future of our research.

http://upload.…28logo%29.svg 6/7/10 2:38 PM Page 1 of 3http://upload.wikimedia.org/wikipedia/fr/0/0f/CNRS_fr_quadri.Corresponding Bibliography:C. B. Carlson, J. Lawler and D. F. Mosher: Structures ofthrombospondins (Cell. Mol. Life Sci. 65, 672-686, 2008)D. S. Annis, K. A. Gunderson and D. F. Mosher: Immuno-chemical Analysis of the Structure of the Signature Domainsof Thrombospondin-1 and Thrombospondin-2 in Low

Calcium Concentrations (Journal of Bio. Chem., 282, 37,27067-27075, 2007)M. D. Briggs and K. L. Chapman: Pseudoachondroplasia andMultiple Epiphyseal Dysplasia: Mutation Review, MolecularInteractions, and Genotype to Phenotype Correlations(Human Mutation, 19, 465-478, 2002)M. Kavansakul,J. C. Adams and E. Hohenester: Structure ofa thrombospondin C- terminal fragment reveals a novel cal-cium core in the type 3 repeats (EMBO Journal, 23,1223-1233, 2004)C. B. Carlson, D. A. Bernstein, D. S. Annis, T. M. Misenhei-mer, B. A. Hannah, D. F. Mosher and J. L. Keck: Structure ofthe calcium-rich signature domain of human thrombospon-din-2 (Nature Struc. Mol. Bio., 12, 10, 910-914, 2005)N. Floquet, S. Dedieu, L. Martiny, M. Dauchez, D. Perahia:Human thrombospondin&s (TSP-1) C-terminal domain opensto interact with the CD-47 receptor: A molecular modelingstudy (Archives of Biochemistry and Biophysics 478,103–109, 2008)

P-266MD-based Modeling of Swift Heavy Ion BeamNanostructuring of DielectricsD.S. Ivanov and W. ShultzInstitute of Laser Technique Fraunhofer, Aachen, Germany

The process of swift heavy ions energy deposition whilepenetrating a solid or scattering on its surface can result in astrong and nonequilibrium excitation of matter. An extremelylocalized character of this excitation, meanwhile, can makepossible both selective changes in chemistry of matter1 andits surface nanomodifications2. Since possible applicationshave been found in Bio- and IT- technologies (cancer curingand nanostructuring respectively) in the last decade, theheavy ion bombardment technique has attracted a lot ofscientific interest3,4. The processes of fast energy depositioninto the solid and its further dissipation, however, areessentially perturbed with highly excited and nonequilibriumstate of both lattice and electron systems. At such conditionstherefore, the precision in treatment of processes of electronthermalization, fast electron heat conduction, and phasetransformation of the overheated solid becomes crucial.Having several physical models to handle the mentionedprocesses, it is nevertheless difficult to describe all of themwithin a scale of a single computational approach. Our workis aimed on elaboration of the atomistic-continuum model5 ofheavy ion bombardment of solids. In particular, the model willbe applied to study the formation of nanohillocks in theexperiments on swift heavy ion Xe+ scattering on SrTiO3

surface2.

References

[1] M. Bellia, A. Camp, V. Dini, G. Esposito, Y. Furusawa, G.Simone, E. Sorrentino, and M. A. Tabocchini, ‘‘DNAFragmentation Induced in Human Fibroblasts by Accel-erated 56Fe Ions of Differing Energies’’, Radiat. Res.165, 713 (2006).

[2] E. Akcoeltekin, S. Akcoeltekin, O. Osmani, A. Duven-beck, H. Lebius,and M. Schleberger, ‘‘Swift Heavy IonIrradiation of SrTiO3 under Grazing Incidence’’, New J.Phys. 10, 053007 (2008).

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[3] G. Kraft, M. Scholz and U. Bechthold, ‘‘Tumor Therapyand Track Structure’’, Radiat. Environ. Bioph. 38, 229(1999).

[4] H.-G. Gehrke, A.-K. Nix, H. Hofsass, J. Krauser, C.Trautmann, and A. Weidinger, ‘‘Self-Aligned Nanostruc-tures Created by Swift Heavy Ion Irradiation’’, J. App.Phys. 107, 094305 (2010).

[5] D.S. Ivanov and L.V. Zhigilei, ‘‘Combined Atomistic-Continuum Modeling of Short-Pulse Laser Melting andDisintegration of Metal Films’’, Phys. Rev. B 68, 064114(2003).

P-267Transport properties of the human aquaporin HsAQP5Lorant Janosi and Matteo CeccarelliUniversity of Cagliari, Cagliari, Italy

Aquaporins are protein channels located across the cellmembrane with the role of conducting water or other smallsugar alcohol molecules (aquaglyceroporins). The presenceof the human aquaporin 5 (HsAQP5) in cells proximal to air-interacting surfaces (eyes, lacrimal glands, salivary glands,lungs, stomach etc.) suggest its potentially important role in‘‘wetting’’ these surfaces. The high-resolution X-ray structureof the HsAQP5 tetramer (PDB code 3D9S) exhibits twoimportant features: (i) lack of the four fold symmetry, commonin most of the aquaporins, and (ii) occlusion of the centralpore by a phosphatidylserine lipid tail. In this study weinvestigate the importance of these two features on thetransport properties of the human AQP5 by means ofmolecular dynamics simulations. We found that the asym-metry in the tetramer leads to a distribution of monomericchannel structures characterized by different free energylandscapes felt by the water molecules passing through thechannel. Furthermore, the structures’ distribution is influ-enced both by the presence/absence of the lipid tail in thecentral pore, and by the lipid composition of the bilayer thatsolvates the HsAQP5 tetramer.

P-268Elucidating the modular structure of the protein G C2fragment and human IgG Fc domain binding site usingcomputer simulationsHiqmet KamberajFaculty of Technical Sciences, International BalkanUniversity, Skopje, R. of Macedonia

Protein–protein recognition plays an important role in mostbiological processes. Although the structures of many pro-tein–protein complexes have been solved in molecular detail,general rules describing affinity and selectivity of protein–protein interactions break down when applied to a larger setof protein-protein complexes with extremely diverse nature ofthe interfaces.In this work, we will analyze the non-linear clustering of theresidues at the interface between proteins. The boundariesbetween clusters are defined by clustering the Mutual Infor-mation of the protein-protein interface. We will show that themutations in one module do not affect residues located in a

neighboring module by studying the structural and energeticconsequences of the mutation. To the contrary, within theirmodule, we will show that the mutations cause complexenergetic and structural consequences.In this study, this is shown on the interaction between proteinGC2 fragment and human IgG Fc domain by combiningmolecular dynamics simulations and mutual information the-ory, and computational alanine scanning technique. Themodular architecture of binding sites, which resembles humanengineering design, greatly simplifies the design of new pro-tein interactions and provides a feasible view of how theseinteractions evolved. The results test our understanding of thedominant contributions to the free energy of protein–proteininteractions, can guide experiments aimed at the design ofprotein interaction inhibitors, and provide a stepping-stone toimportant applications such as interface redesign.

P-269Entropy estimates of protein-induced lipid perturbationsby molecular dynamics simulationsJonathan Khao, James Sturgis, Jean-Pierre DuneauLaboratoire d’Ingenierie des Systemes Macromoleculaires,CNRS, Aix-Marseille Universite, France

Membrane proteins can form large multimeric assemblies innative membranes that are implicated in a wide range ofbiological processes, from signal transduction to organellestructure. Hydrophobic mismatch and membrane curvatureare involved in long range forces largely contributing to suchsegregation. However, the existing assembly specificity isthought to be coded in the atomic details of protein surfaceand topology. These are best described in high resolutionstructures and atomistic molecular dynamics simulations. Inorder to explore more systematically such forces and ener-getics arising at intermediate time scales and resolution, weuse coarse grained molecular dynamics simulations appliedto 20 membrane systems spanning over 5 to 15us. As a firstglimpse we study how proteins induce lipid perturbationsusing a previously developed conformational entropy esti-mator. We show that in the model membrane wherehydrophobic mismatch is present, lipid perturbations extendup to *40A from the protein surface. However, significantvariations in perturbation profiles are seen. Parameters suchas protein shape, surface topology, and amino acid physi-cochemical properties are studied to discover the parametersgoverning such perturbations.

P-270Crossing energy barriers with self-guided LangevindynamicsGerhard Konig, Xiongwu Wu, Bernard BrooksNational Institutes of Health, National Heart, Lung and BloodInstitute, Laboratory of Computational Biology, Rockville,MD, USA

Even with modern computer power, the applicability ofmolecular dynamics simulations is restricted by their ability tosample the conformational space of biomolecules. Often highenergy barriers cause normal molecular dynamics

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simulations to stay trapped in local energy minima, leading tobiased results. To address this problem, Self-guided Lange-vin dynamics (SGLD) were developed. It enhancesconformational transitions by accelerating the slow system-atic motions in the system. This is achieved by calculatingthe the local average of velocities and adding a guiding forcealong the direction of systematic motions. Thus, the effi-ciency of SGLD is governed by three factors: a.) the frictionconstant involved in the Langevin Dynamics b.) the localaveraging time and c.) the guiding factor that determines theguiding force. However, the guiding force also causes devi-ations from the original ensemble that have to be correctedby reweighting the data, thus decreasing the efficiency. Here,we explore the three-dimensional parameter space of SGLDfor several benchmark systems with particularly roughenergy surfaces. Based on our data, we supply guidelines forthe optimal selection of SGLD parameters, to allow theextension of our method to other biological problems ofinterest.

P-271Propagation of D816V/H mutation effects across KITreceptorE. Laine, I. C. de Beauchene, C. Auclair andL. TchertanovLBPA, CNRS - ENS de Cachan, France

Receptor tyrosine kinases (RTKs) regulate critical biologicalprocesses. Constitutive activation of RTKs provokes cancers,inflammatory diseases and neuronal disorders. Biologicaldata evidenced that oncogenic mutations of the RTK KIT,located either in the juxtamembrane region (JMR) or in theactivation loop (A-loop) - as is the case of D816V/H, displacethe equilibrium of conformational states toward the activeform. We present a multi-approach study that combinesmolecular dynamics (MD), normal modes (NM) and pocketdetection to characterize and compare the impact of D816V/Hon the structure, dynamics and thermodynamics of KIT. Wehave evidenced a local structural destabilization of A-loopinduced by the mutation and a long-range effect on thestructure and position of JMR. We have further correlatedthese observations with experimental data and deciphersome details about the activation mechanisms of the mutants,involving leverage of the JMR negative regulation and releaseof an access to the catalytic site. Through the identification of‘‘local dynamic domains’’ and the recording of interactionswithin the protein, we propose a model of the mutationaleffects propagation, which highlights the importance of bothstructural distortion and local conformational fluctuations.

P-272Comparative molecular dynamics studyof the stereoisomeric forms of indolicidin and itsanalogsBalazs LeitgebInstitute of Biophysics, Biological Research Centre,Hungarian Academy of Sciences, Szeged, Hungary

In this study, molecular dynamics (MD) calculations werecarried out on the stereoisomeric forms of indolicidin and

its analogs with the substitutions of one or more Pro res-idues by Ala amino acids. According to the cis-transisomerism about the Xaa-Pro peptide bonds, variousnumbers of stereoisomers could be distinguished for theparent peptide and its Ala-containing derivatives. On thebasis of MD simulations, the appearance of differentstructural properties was investigated, as well as theiralterations were studied as a function of time. For eachstereoisomeric form of peptides, the presence of second-ary structural elements (i.e. b-turn and helical structures)and intramolecular interactions (i.e. H-bonds, aromatic-aromatic, proline-aromatic and cation-p) was examined.The MD calculations performed on the stereoisomers ofindolicidin and its Ala-containing analogs led to theobservation that the stereoisomeric forms showed charac-teristic conformational features. Nevertheless, theirappearance proved to be dependent on the cis or transnature of Xaa-Pro peptide bonds, as well as on thePro?Ala substitutions.This research was supported by the Hungarian ScientificResearch Fund (OTKA PD 78554), and by the Janos BolyaiResearch Scholarship of the Hungarian Academy ofSciences.

P-273Rational design of potential anticancer ‘‘drug-like’’molecules based on 4-azoldinone scaffoldRoman Lesyk, Borys Zimenkovsky, Danylo Kaminskyy,Dmytro Havryluk, Dmytro Atamanyuk, Anna Kryshchyshyn,Ivanna Subtel’na, Dmytro KhylukDanylo Halytsky Lviv National Medical University, Lviv,Ukraine

Investigation of biological active azolidinones and relatedheterocycles refer to one of the most successful scientificprojects of DH LNMU. It is based on three strategic vectors:organic synthesis, pharmacological research, rational designof ‘‘drug-like’’ molecules (including in silico approaches).While applying the research strategy we succeeded ingaining a number of interesting results that make possible toextend the field, especially in the scope of ‘‘drug-like’’ mole-cules design, notably it has focused on the search of newanticancer agents.Anticancer activity screening was carried out for more than1000 compounds (US NCI protocol (DTP) based on obtaineddirected library over 5000 new compounds, among them 167compounds showed high activity level. For the purpose ofoptimization and rational design directions of highly activemolecules with optimal ‘‘drug-like’’ characteristics and dis-covering of possible mechanism of action SAR, COMPAREanalysis, molecular docking and QSA(P)R were carriedout. Obtained results allowed to form main directions ofpossible anticancer activity mechanisms, which probableare apoptosis-related. Nowadays the investigation ofcellular and molecular aspects of anticancer effects is inprogress.

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P-274New insights into the mechanism of mechanicalregulation on thiol/disulfide exchangeWenjin Li1,2, Scott Edwards1, Frauke Graeter1,3,Gerrit Groenhof21CAS-MPG Partner Institute for Computational Biology,Shanghai, China, 2Max-Plank-Institute for BiophysicalChemistry, Goettingen, Germany, 3Heidelberg Institutefor Theoretical Studies, Heidelberg, Germany

Regulation of (Bio)chemical reactions by mechanical forcehas been proposed to be fundamental to cellular func-tions[1,2,3]. Atomic force microscopy experiments haveidentified the effect of mechanical force on the reactivity ofthiol/disulfide exchange, a biologically important reaction [4].In order to understand the influence of the force at an atom-istic level, we have performed hybrid quantum mechanics -molecular mechanics (QM/MM) transition path samplingsimulation of the reaction under external forces. The results ofthe simulations supported the experimental findings anddemonstrated that the location of the transition state on thefree energy surface was shifted due to force [5].In contrast to our findings, however, a recent experimentalstudy suggests only a weak coupling between the mechanicalforce and the reaction rate[6]. In this study, the reactants werecovalently linked to a stilbene molecule. In this system a forcecan be applied by photo-isomerization from the relaxed transto the strained cis configuration. A drawback of this system isthat one cannot easily determine the forces that acting on thereaction coordinate. Therefore, we have developed a forcedistribution analysis method for quantum mechanical molec-ular dynamics simulations. The results of the analysis showhow isomerization of stilbene alters the forces acting on thereacting atoms. The force distribution is essential for under-standing how chemistry is controlled by external forces.

[1] Beyer MK, Clausen-Schaumann H. Chem. ReV. (2005)105: 2921-2948.

[2] Bustamante C, Chemla Y, Forde N, Izhaky D. Annu.Rev. Biochem. (2004) 73: 705-748.

[3] Vogel V, Sheetz M. Nat. Rev. Mol. Cell Biol. (2006) 7:265-275.

[4] Wiita AP, Ainavarapu SR, Huang HH, Fernandez JM.Proc.Natl. Acad. Sci. U.S.A. (2006) 103: 7222-7227.

[5] Li W, Graeter F. J. Amer. Chem. Soc. (2010) 132:16790-5.

[6] Kucharski T, Huang Z, Yang QZ, Tian Y, Rubin N,Concepcion C, Boulatov R. Angew. Chem. (2009) 121:7174-7177.

P-275How lipids affect the side-chain conformational spaceof a spin label on a protein: an ABF MD studyAjasja Ljubetic, Janez Strancar‘‘Jozef Stefan’’ Institute, Condensed Matter Department,Laboratory of Biophysics Jamova 39, SI-1000 Ljubljana,SloveniaEmail: ajasja.ljubetic@ijs.si

Conformational space modelling (CSM) is a promisingmethod for membrane protein structure determination. It is

based on the concept of the side-chain conformational space(SCCS), which is formed by the allowed side-chain confor-mations of a given residue. Each SCCS can be calculatedfrom a 3D structure or measured via EPR-SDSL experi-ments. For structure determination a set of singly spin-labelled mutants is needed. The final structure is obtained byaltering an initial (possibly random) 3D structure until the bestfit between the calculated and measured SCCS for the wholeset is found.Such optimization is computationally intensive; thereforeCSM includes several empirical approximations. One of themdescribes the effect of the lipid tails on the SCCS. Theimplementation is not trivial as lipids diffuse in the plane ofthe membrane and the lipid tails behave differently at differ-ent membrane depths.To unravel this relationship adaptive biasing force MD sim-ulations were used. An alanine peptide helix was made insilico, spin-labelled at the middle and inserted perpendicu-larly into a DMPC membrane. The free energy of thespin-label orientation at various membrane depths was cal-culated. A 3D free energy surface describing the membrane‘‘depth’’ effect was obtained.

P-276A nano-scavenger in action: the molecularb-cyclodextrin-mediated cholesterol extractionmechanismCesar A. Lopez, Alex H. de Vries, Siewert J. MarrinkGroningen Biomolecular Sciences and BiotechnologyInstitute & Zernike Institute for Advanced Materials,University of Groningen, Nijenborgh 7, 9747 AG Groningen,The Netherlands

It is known that b-cyclodextrins (bCDs) are able to modify thecholesterol content of cell or model membranes. Howeverthe molecular mechanism of this process is still not resolved.Using molecular dynamics simulations, we have been able tostudy the bCD-mediated cholesterol extraction from choles-terol monolayers and lipid-cholesterol monolayer mixtures.We have investigated many conditions that would affect thisprocess (e.g. lipid-cholesterol ratio, lipid chain unsaturationlevel)Our results can be summarized as follow: i) Dimerization ofbCDs, ii) binding of the dimers to the membrane surfaceassuming either a tilted (parallel to the membrane normal) oruntilted (90� respect to the normal of the membrane) configu-ration, iii) the latter configuration is suitable to extractcholesterol at a reasonable computational time scale (100-200ns), however, this process may be affected by unfavorableenergy barriers (from 10 to 80 kJ mol-1), iv) desorption of thecomplex brings cholesterol in solution, v) the bCD-cholesterolcomplex is able to transfer cholesterol to other membranes.With a clearer understanding of the basic molecular mech-anism of the bCD mediated process of cholesterol extraction,we can begin to rationalize the design of more efficient bCDsin numerous applications.

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P-277Molecular dynamics simulations of pyrene in POPCand POPC/cholesterol bilayersLuıs M. S. Loura1,2, Antonio do Canto3,41Faculdade de Farmacia, Universidade de Coimbra,Portugal, 2Centro de Quımica de Coimbra, Universidade deCoimbra, Portugal, 3Departamento de Quımica,Universidade de Evora, Portugal, 4Centro de Quımica deEvora, Universidade de Evora, Portugal

We use molecular dynamics simulations to study bilay-ers composed of 128 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 120 POPC:30 cholesterol(POPC:Chol 4:1) molecules, with 0, 2 or 4 inserted pyrenemolecules. Fluorophore properties such as mass distributionprofile along the bilayer normal, orientation, rotational andtranslational dynamics are monitored, as well as bilayerparameters including area/lipid, mass distribution profiles,translational and rotational dynamics and order parameters.In agreement with recent results reported for fluid 1,2-di-palmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, wefound that pyrene is preferentially located in the acyl chainregion close to the glycerol group of lipid molecules andcauses ordering of the lipid acyl chains. On the other hand,and similarly to DPPC gel bilayers (but unlike DPH inDPPC:Chol 4:1), incorporation of pyrene in binary POPC:-Chol 4:1 bilayers produces a disordering effect, increasingthe area/lipid molecule and decreasing the bilayer thicknessand acyl chain order, thus opposing the ordering effect ofcholesterol. Funding by FEDER (COMPETE program), andby FCT – Fundacao para a Ciencia e a Tecnologia, projectreference FCOMP-01-0124-FEDER-010787 (FCT PTDC/QUI-QUI/098198/2008) is acknowledged.

P-278DFT and FTIR-spectroscopic study of polypeptide-ligandcomplexesO. MakshakovaKazan Institute of Biochemistry and Biophysics of RussianAcademy of Science, Lobachevsky str. 2/31, P. O. box 30,Kazan, 420111, Russia

The mechanism of the complex formation of biopolymerswith ligands including the solvent molecules is an actualproblem of modern biophysical and biological science.Polypeptides form a secondary structure and mimic themotifs of the protein architecture. The study of complexationbetween polypeptides and solvent molecules leads to deeperunderstanding of the basic interaction of proteins with envi-ronment at atomic level. Besides polypeptides are promisingfor the development of applications which encompass someof the following desirable features: anti-fouling, biocompati-bility, biodegradability, specific biomolecular sensitivity. Onthis account polypeptides have a great significance for avariety modern applications ranging from the nanoscalemedicine devises up to food technology and others. Wecompare the results of calculations of complexes betweenhelical polypeptides (polyglutamic acid in neutral form andpoly-c-benzyl-l-glutamate) and water molecules at DFT PBElevel and the results of FTIR-spectroscopic study of the filmof wetted polypeptides. Vibrational spectroscopy is one of the

most useful experimental tools to study non-covalent bondedcomplexes, and calculated spectra in comparison withexperimental data are reliable test for reality of simulatedcomplexes.

P-279DNA bubbles and bending: how conformationalfluctuations modify base pairingManoel Manghi, J. Palmeri, N. DestainvilleUniversite de Toulouse, UPS and CNRS, Laboratoire dePhysique Theorique (IRSAMC) F-31062 Toulouse, France

DNA denaturation is a physical process in the course ofwhich the double strand can open locally thanks to thermalfluctuations. Within a denaturation bubble the two fluctuatingsingle strands have a bending rigidity 50 times weaker thanthat of the unopened helix, which increase its conformationalentropy. The DNA conformation will in turn influence thebubble creation process. This mutual influence naturallyleads to a theoretical model coupling the local internal DNAstates (open or closed base pairs) and the local DNA elas-ticity [1-2].This internal-external coupling allows us to address andanswer still open questions, such as how conformationalfluctuations modify its thermal denaturation, and why DNAchains are kinked when observed on surfaces by AFM [3].Finally, we will discuss, by comparing experiments to simu-lations, the relevance of Tethered Particle Motionexperiments to observe molecular events such as denatur-ation bubble closure in real time [4].

[1] J. Palmeri, M. Manghi, N. Destainville, PRL 99 088103(2007); PRE 77 011913 (2008)

[2] M. Manghi, J. Palmeri, N. Destainville, JPCM 21 034104(2009)

[3] N. Destainville, M. Manghi, J. Palmeri, BJ 96 4464(2009)

[4] M. Manghi et al., Physical Biology 7 046003 (2010)

P-280Kinetic tools for quantifying platelet aggregationBogdan-Nicolae Marincu, Monica Neagu,Oana Munteanu, Adrian NeaguVictor Babes University of Medicine and PharmacyTimisoara, Romania

Platelet aggregation at the site of vascular injury is vital toprevent bleeding. Excessive platelet function, however, maylead to thrombus formation after surgery. Therefore, anaccurate measure and control of platelet aggregation isimportant. In vitro platelet aggregometry monitors aggregateformation in platelet rich plasma triggered by agonists suchas ADP, epinephrine or collagen. The fraction of aggregatedplatelets is plotted versus time, and platelet function isassessed by analyzing the plot’s morphology. We proposenew measures of platelet function based on a compart-mental kinetic model of platelet aggregation induced byADP. Our model includes three compartments: single,aggregated and deaggregated platelets. It is simpler thanearlier models and agrees with experimental data. The

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model parameters were determined by non-linear leastsquares fitting of published data. We associated the kineticparameters with the activity of the ADP receptors P2Y1 andP2Y12. To this end, we studied published data obtained inthe presence and in the absence of specific antagonists ofthese receptors. Comparison of kinetic parameters ofhealthy subjects with those of patients with myeloprolifera-tive disorder (MPD) shows that the function of P2Y12 issignificantly reduced in MPD.

P-281Coarse-grained modeling of drug-membraneinteractionsManuel N. Melo, Durba Sengupta, Siewert-Jan MarrinkGroningen Biomolecular Sciences and BiotechnologyInstitute, University of Groningen, Groningen, TheNetherlands

The MARTINI coarse-grained (CG) forcefield was used tosimulate the actions of the antimicrobial peptide alamethicinand of the anti-tumoral drug doxorubicin. Both drugs wereshown to interact strongly with a fluid phospholipid bilayer,and aggregate there, in agreement with experimental results.Because doxorubicin may establish intermolecular h-bond-ing, and thus lower its dipole moment, the CG representationof a doxorubicin dimer was adjusted. This less polar dimerwas then tested for translocation and/or pore formation.Contrary to results of atomistic simulations, alamethicinaggregates did not spontaneously open pores. They did so,however, when the size of the water beads was decreased.Several small independent pores could then be observed. Themagnitude of the permeability of these pores is analyzed andcompared to experimental values. The occurrence of multiplesmall pores could indicate that thedifferent conductance levelsexperimentally observed for alamethicin might simply resultfrom the association of different numbers of these small pores.

1. Leontiadou H, Mark AE, Marrink SJ. J Am Chem Soc.2006 Sep 20;128(37):12156-61.

2. Marrink SJ, Risselada HJ, Yefimov S, Tieleman DP, deVries AH. J Phys Chem B. 2007 Jul 12;111(27):7812-24

P-282Neuronal (bi)polarity as a self-organized processwith membrane growthSilvia A. Menchon1, Annette Gartner1, Pablo Roman2,Carlos G. Dotti1,31Department of Molecular and Developmental Genetics, VIBand Center for Human Genetics, KULeuven, Leuven,Belgium, 2Department of Mathematics, KULeuven, Leuven,Belgium, 3Centro de Biologıa Molecular Severo Ochoa,Universidad Autonoma de Madrid, Madrid, Spain

Polarization refers to the asymmetric changes in cellularorganization in response to external or internal signals. Neu-ronal polarization begins with the growth of a single neuriteshortly after cell division, followed by the growth of a secondneurite at the opposite pole. This early bipolar shape is criticalfor brain function, as it defines axis of migration and conse-quently proper three dimensional organization and nervecircuitry. However, it is not known if a direct relationship exists

between the formation of the second, opposite, neurite and themechanisms involved in the formation of the first. We tackledthis issue through mathematical modeling, based on mem-brane traffic (exocytosis-endocytosis), and lateral diffusion.With this approach, we demonstrated that a single pole ofmolecular asymmetry is sufficient to induce a second one atthe opposite side, upon induction of growth from the first pole.Our work gives mathematical proof that the occurrence of asingle asymmetry in a round cell is sufficient to warrant mor-phological bipolarism.

P-283QM/MM study of serine protease enzyme acylation usingDFTB methodJesus I. Mendieta-Moreno1, Fernando Martın-Garcıa1,2, InigoMarcos-Alcalde1, Paulino Gomez-Puertas1 and JesusMendieta1,21Molecular Modelling Group, Centro de Biologıa Molecular‘‘Severo Ochoa’’ (CBMSO,CSIC-UAM) Cantoblanco, 28049Madrid, Spain, 2Biomol-Informatics SL. C/ Faraday, 7. ParqueCientıfico de Madrid. Cantoblanco,28049 Madrid, Spain

Trypsin is one of the best characterized serine proteases.Enzyme acylation process is required for substrate degra-dation. There is a lot of information about how this processundergoes. However, in order to obtain a more detaileddescription of the catalytic triad mechanism, a QM/MMapproach was used. We used the hybrid QM/MM potentialimplemented in AMBER11 package. In the QM calculations aDFT Hamiltonian was used. We develop an approach basedon the adaptively biased MD in order to obtain the freeenergy surface of the conformational space defined by thereaction coordinates. This approach presents some charac-teristics of steered MD and umbrella sampling procedures.Our results offer information about the lowest energy trajec-tory, the barrier profile of the reaction, and the geometry ofthe transition state. This method also provides a furtherinsight into the role of specific residues in the reaction.Substituting Asp102, member of the catalytic triad, for Ala wewere able to detect an increase of the barrier profile. Thiswas due to the loss of the interaction of carbonyl group ofAsp102 with Nd of His57, which make Ne of this residue aworst proton acceptor. This results show our approach as avaluable method in the study of enzymatic mechanisms.

P-284Macromolecular crowding effects studied by THzspectroscopy and molecular modelingMaria Mernea1, Octavian Calborean1,Diana Zatreanu1, Oana Sandu2, Traian Dascalu2,Dan Mihailescu11University of Bucharest, Faculty of Biology, BucharestRomania, 2National Institute for Laser, Plasma and RadiationPhysics, Bucharest, Romania

The intracellular media comprise a great variety of macromo-lecular species that are not individually concentrated,but being preset in the same compartment they excludeeach other’s volume and produce crowding. Crowding has aprofound impact on protein structure and determines confor-mational transitions and macromolecular association. We

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investigated macromolecular association on a 50% w/wbovine serum albumin (BSA) solution by time-domain tera-hertz (THz) spectroscopy and molecular modeling. Molecularcrowdingwas simulated by including twoBSAmolecules in thesame water box. We generated *300 such dimeric models,computed their THz spectra by normal modes analysis andcompared them with the experimental data. The best BSAdimer model was selected based on the agreement with theexperiment in the lowest frequencies domain of up to 1 THz.

P-285Symmetry constraints improve accuracy of ion channelsmodels. Application to two-pore-domain channelsAdina L. Milac1,2, Andriy Anishkin3, Sarosh N. Fatakia4,Carson C. Chow4, Sergei Sukharev3, H. Robert Guy21Institute of Biochemistry of the Romanian Academy, SplaiulIndependentei 296, 060031, Bucharest 17, Romania,2National Cancer Institute, National Institutes of Health, 12South Drive, Bethesda, Maryland 20892, USA, 3Universityof Maryland, College Park, MD 20742, USA, 4NationalInstitute of Diabetes and Digestive and Kidney Diseases,National Institutes of Health, Bethesda, Maryland 20892, USA

Ion channels are important drug targets. Structural informa-tion required for structure-based drug design is often filled byhomology models (HM). Making accurate HM is challengingbecause few templates are available and these often havesubstantial structural differences. Besides, in moleculardynamics (MD) simulations channels deviate from idealsymmetry and accumulate thermal defects, which complicateHM refinement using MD. We evaluate the ability of sym-metry-constrained MD simulations to improve HM accuracy,using an approach conceptually similar to CASP competition:build HM of channels with known structure and evaluate theefficiency of various symmetry-constrained MD methods inimproving HMs accuracy (measured as deviation from x-raystructure). Results indicate that unrestrained MD does notimprove accuracy, instantaneous symmetrization improvesaccuracy but not stability during subsequent MD, whilegradually imposing symmetry constraints improves bothaccuracy (by 5–50%) and stability. Moreover, accuracy andstability are strongly correlated, making stability a reliablecriterion in predicting HM accuracy. We further used thismethod to refine HM of TREK channels. We also propose agating mechanism for mechanosensitive channels that wasfurther experimentally confirmed.

O-286Nucleotide modifications and tRNA anticodon- mRNAcodon interactions on the ribosomeOlof Allner and Lennart NilssonKarolinska Institutet, Stockholm, Sweden

Molecular dynamics simulations of the tRNA anticodon andmRNA codon have been used to study the effect of thecommon tRNA modifications cmo5U34 and m6A37. IntRNAVal these modifications allow all four nucleotides to besuccessfully read at the wobble position in a codon. Previousdata suggest entropic effects are mainly responsible for theextended reading capabilities but detailed mechanisms have

remained unknown. The aim of this paper is to elucidate thedetails of these mechanisms on an atomic level and quantifytheir effects. We have applied: extensive free energy per-turbation coupled with umbrella sampling, entropiccalculations of tRNA (free and bound to the ribosome) andthorough structural analysis of the ribosomal decoding cen-ter. The results show that there are three mechanismsresponsible for the expanded decoding capability by themodifications: 1. Prestructuring of the ASL decreasesentropy. A decrease of stem loop entropy when free insolution favors the tRNA-ribosome complex formation. 2.Alternate binding conformations. The further reach of thecmo5U34 allows an alternative outer conformation to beformed for the non cognate base pairs. 3. Increased contactsbetween tRNA and mRNA with the ribosome enhances the‘‘catalyzing’’ effect of the ribosome.

P-287Functional and dysfunctional conformers of humanneuroserpin: a comparative MD studyR. Noto1, M. Manno1, V. Martorana1

Institute of Biophysics at Palermo, Italian National ResearchCouncil, Via U. La Malfa 153, 90146 Palermo, Italy

Human neuroserpin (hNS) is a serine protease inhibitor(serpin) of a tissue-type plasminogen activator (tPA). Theconformational flexibility and the metastable state of thisproteins underlies to misfolding and to dysfunctional muta-tions causing a class of rare genetic diseases which sharethe same molecular basis. The conformational transition ofthe native form, triggered upon the cleavage at reactivecenter loop (RLC), releases a complex of the cleaved formbound to the inactivated target protease. Without RLCcleavage a stable inactive latent form can be obtained byintra/inter molecular loop insertion leading to polymerization.This work concerns the study of the three above mentionedforms of hNS by MD simulations to investigate the relationbetween their conformational stability and. The startingnative and cleaved configurations are based on the x-raystructure, while the latent form is here modelled. The resultsof the simulation reveal a striking conformational stabilityalong with the intrinsic flexibility of selected regions of thefold.The analysis of the essential collective modes of thenative hNS shows that the initial opening of the b-sheet Acoincides with several changes in the local pattern of saltbridges and of hydrogen bonds.

P-288Regulation of ubiquitin-conjugating enzymes: a commonmechanism based on a pattern of hydrophobicand acidic residuesElena Papaleo1, Alberto Arrigoni1, Nicola Casiraghi1, LucaBertini1, Roberto Pagliarin2, Marco Vanoni1, Paola Coccetti1,Luca De Gioia11University of Milano-Bicocca, Milan, Italy, 2Universityof Milano, Milan, Italy

E2 ubiquitin-conjugating enzymes play a central role in theubiquitin (Ub) pathway, influencing the fate of the targetsubstrates [Ye and Rape, Nat Rev Mol Cell Biol 2009,

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10:755-64]. Several E2s are characterized by an extendedloop (L7), which regulates Ub-charging activity [Papaleoet al, PLoS Comput Biol, in press].We studied L7 interactions, in representative E2s, integrat-ing phylogenetic and residue co-evolution analyses, normalmode and multi-replica molecular dynamics simulations. Apattern of hydrophobic residues emerged as the maindeterminant of the stabilization of E2 inactive conforma-tions. L7 can have a pivotal role in downstream eventsrelated to interactions with Ub and E3 enzymes. In fact,once the E2-Ub complex is formed, L7 open conformation,induced by phosphorylation, allows its acidic residues toreduce conformational freedom of Ub in the E2 catalyticcleft. Indeed, hydrophobic residues interact with E2 distalregions rich of hydrophobic residues determining a L7conformation competent for interactions with E3s. In light ofthe above scenario, L7 emerges as a suitable target fordrug design, prompting us to develop virtual screening anddocking simulations to identify compounds, which should beable to act as molecular hinges and to modify proteindynamics.

P-289Protein dynamics and temperature adaptationin extremophilic enzymesMatteo Tiberti1, Giulia Renzetti1, Gaetano Invernizzi1,Bjarni Asgeirsson2, Stefania Brocca1, Marina Lotti1,Luca De Gioia1, Elena Papaleo11University of Milano-Bicocca, Milan, Italy, 2Universityof Iceland, Reykjavik, Iceland

Enzyme temperature adaptation generally involves a mod-ulation of intramolecular interactions, affecting proteinsdynamics, stability and activity [1-2]. In this contribution, wediscuss studies of different classes of extremophilicenzymes, focusing on cold-adapted variants, as well astheir mesophilic-like mutants, performed by all-atommolecular dynamics simulations with particular attention tostructural communication among residues within the three-dimensional architecture [3-4]. Common adaptation strate-gies turned out to be based on improved local flexibility inthe proximity of the functional sites, decrease in intercon-nected electrostatic interactions, and modulation ofcorrelated motions and networks of communicating resi-dues. Specific differences related to the diverse proteinfolds can also be detected.References

1. Feller G, J Phys Cond Matter, 2010, 22:323101.2. Heidarsson PO, Sigurdsson ST, Asgeirsson B, FEBS J,

2009, 276:2725-35.3. Pasi M, Riccardi L, Fantucci P, De Gioia L, Papaleo E, J

Phys Chem B, 2009, 113:13585-95.4. Tiberti M, Papaleo E, J Struct Biol, 2011, 174:69-83.

AcknowledgementsThis research was supported by CASPUR Standard HPCGrant 2010. We also thank Dr. Marco Pasi and Alberto Ar-rigoni for fruitful discussion and comments.

P-292Molecular dynamics study of bNeurexin-NeuroligininteractionsLukasz Peplowski, Rafal Jakubowski, Adrian Jasinski,Wieslaw NowakTheoretical Molecular Biophysics Group, Institute of Physics,N. Copernicus University, Torun, Poland

bNeurexins and Neuroligins are cell adhesion molecules andplay important role in synapse junction formation, maturationand signal transduction between neurons. Mutations ingenes coding these proteins occurs in patients with cognitivediseases like autism disorders, Asperger syndrome andmental retardation [1]. It has been found that the complexbNeurexin-Neuroligin has also an important role in angio-genesis [2]. Herein we will present molecular foundations ofbNeurexin-Neuroligin interactions obtained from all-atommolecular dynamics simulations of bNeurexin, Neuroliginand their complex (3B3Q) [3]. 50 ns MD trajectories(CHARMM force field) were analyzed and roles of Ca2+ andN-actetyl-D-glucosamine posttranslational modifications inintermolecular interactions were scrutinized.Supported byPolish Ministry of Education and Science, grant no. N202262038.

[1] Lise M-F., El-Husseini A. Cell. Mol. Life Sci. 63, 1833(2006)

[2] Bottos A. et al. PNAS 106(49), 20782 (2009)[3] Chen X. et al. Nat. Struc. Mol. Biol. 15(1), 50 (2008)

O-293The quest for the perfect force fieldStefano Piana1, Kresten Lindorff-Larsen1, andDavid E. Shaw1,2

1D. E. Shaw Research, New York, NY 10036, USA, 2Centerfor Computational Biology and Bioinformatics, ColumbiaUniversity, New York, NY 10032, USA

Advances in hardware and software have enabled increas-ingly long atomistic molecular dynamics simulations ofbiomolecules, allowing the exploration of processes occur-ring on timescales of hundreds of microseconds to a fewmilliseconds. Increasing the length of simulations beyondthe microsecond time scale has exposed a number of limi-tations in the accuracy of commonly employed force fields.Such limitations become more severe as the size of thesystems investigated and the length of the simulationsincrease. Here I will describe the force field problems thatwe have encountered in our studies, how we identified andaddressed them, and what we have learned in the processabout the biophysics of the systems we are investigating.While the quest for a ‘‘perfect’’ force field is not over (andmay never be), our work has greatly improved the accuracyand range of applicability of simple physics-basedforce fields, to the point that reliable predictions can nowbe obtained from millisecond-timescale simulations ofbiomolecules.

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P-294Concentration effects on the encapsulation of prilocainein liposomes by computer simulationsM. Pickholz1 and G. Giupponi21FFyB, Universidad de Buenos Aires, Argentina,2Departament de Fisica Fonamental, Universitat deBarcelona, Carrer Marti i Franques, 08028 Barcelona, SpainE-mail: mpickholz@ffyb.uba.ar

Local anesthetics (LA) are pain-relief drugs, widely used inmedicine and dentistry. The relatively short duration ofanalgesia still restricts their clinical use for the treatment ofchronic pain. Nowadays, intensive research is focused onanesthetics entrapped into liposomes to enhance theiractivity and pharmacokinetic properties [1].In this work, we investigated the encapsulation of prilocaine(PLC), an aminoamide local anesthetic, into a small unila-mellar liposome. On the line of our previous work [2], wehave carried out Molecular Dynamics (MD) simulations usinga Coarse Grain model up the microsecond time scale. In thisway, we compare the effects of the concentration of LA atfisiological pH. We were able to capture important features ofthe PLC-vesicle interactions. The behavior of PLCs at fisio-logical pH is essentially a combination of high and low pH: wefound that all neutral PLC molecules rapidly diffuse into thehydrophobic region of the vesicle adopting an asymmetricbimodal density distribution. Protonated PLC molecules(pPLC) initially placed in water were instead only found onthe external monolayer, with a high rate of exchange with thewater phase and no access to the inner part of the liposomein a concentration dependent way.

[1] E. de Paula, C.M.S. Cereda, G.R. Tofolia, M. Franz-Montana, L.F. Fraceto, D.R. de Araujo; Recent Patentson Drug Delivery & Formulation, 1 (2010) 23.

[2] M. Pickholz and G. Giupponi; J. Phys. Chem. B 114(2010) 7009.

P-295Monte Carlo simulation study of diffusion controlledreactions in three dimensionalcrowded mediaLaura Pitulice1,2*, Eudald Vilaseca3, Adriana Isvoran1, 2,Josep Lluıs Garces4 and Francesc Mas31Department of Chemistry, West University of Timisoara,Timisoara, Romania, 2Nicholas Georgescu-RoegenInterdisciplinary Research and Formation Platform,Laboratory of Advanced Researches in EnvironmentalProtection, Timisoara, Romania, 3Physical ChemistryDepartment and Research Institute of Teoreticaland Computational Chemistry of Barcelona University(IQTCUB), Barcelona, Catalonia, Spain, 4ChemistryDepartment, Lleida University (UdL), Lleida, Catalonia, SpainE-mail: pituliceii@yahoo.com

Published data reveal that the rate coefficients of diffusioncontrolled reactions taking place in crowded media are timedependent. Within this study we have performed on lattice3D Monte Carlo simulations concerning Michaelis-Mentenenzymatic reactions in crowded media, such as the cyto-plasmatic region of the cells. We have considered the samesize and mobility of the reactant particles and differentcrowding conditions using distinct concentrations and sizes of

the immobile obstacles. The results we have obtained indicatea fractal like kinetics with the degree of fractality that changeswith the concentration and dimension of the obstacles. Thesimulation data also reflect that, depending on the temporalscale, molecular crowding can bring positive or negativeeffects on reaction kinetics. Comparing the cases studied, forshort periods of time the value of the initial rate constantgenerally increases with the degree of crowding. For longerperiods of time and larger distances, molecular crowdingslows down the reaction kinetics in every case, due to smallerdiffusion coefficients of the reactants, having a more intenseeffect for the cases with higher degree of crowding.

P-296Multiscale modeling of adhesion and traffickingJin Liu, Neeraj J. Agrawal, Vyas Ramanan,Ravi RadhakrishnanUniversity of Pennsylvania, Philadelphia, PA, USA

We focus on applications of molecular and mesoscale simu-lation methodologies to the cellular transport process ofendocytosis, i.e., active transport mechanisms characterizedby vesicle nucleation and budding of the cell membraneorchestrated by protein-interaction networks, and functional-ized carrier adhesion to cell surfaces. We discuss theoreticaland computationalmethodologies for quantitatively describinghow cell-membrane topologies are actively mediated andmanipulated by intracellular protein assemblies. We also dis-cussmethods for computing absolute binding free energies forcarrier adhesion.We present rigorous validation of our modelsby comparing to diverse range of experiments.

References:

N.J. Agrawal, R. Radhakrishnan, Calculation of Free ener-gies Calculation of free energies in fluid membranes subjectto heterogeneous curvature fields, 2009, Phys Rev E, 80,011925.N.J. Agrawal, J. Nukpezah, R. Radhakrishnan, MinimalMesoscale Model for Protein-Mediated Vesiculation inClathrin-Dependent Endocytosis, 2010, Plos: ComputationalBiology,, 6(9) e1000926, 2010.J. Liu, G. E. R. Weller, B. Zern, P. S. Ayyaswamy, D.M. Eckmann, V. Muzykantov, R. Radhakrishnan, A Compu-tational Model for Nanocarrier Binding to EndotheliumValidated Using In Vivo, In Vitro, and Atomic Force Micros-copy Experiments, 2010, Proceedings of the NationalAcademy of Sciences, 107(38), 16530-16535.

P-297Mg2+ is a temporary electron storage during the GTPHydrolysis MechanismTill Rudack1, Fei Xia2, Carsten Kotting1,Jurgen Schlitter1 and Klaus Gerwert1,21Ruhr University Bochum, Department of Biophysics,Bochum Germany, 2Shanghai Institutes for BiologicalScience, CAS-Max-Planck Partner Institutefor Computational Biology, Shanghai, P. R. China

Mg2+ is ubiquitous and often coordinated by the triphosphateof GTP or ATP in signal transduction and motor proteins. It

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plays an important role in catalytic hydrolysis. Here, weanalysed the GTPase Ras which is a crucial switch in cellularsignal transduction. It cleaves the substrate GTP hydrolyti-cally to diphosphate and Pi. The GAP protein acceleratesGTP hydrolysis by a factor of 105. FTIR spectra of GTP showremarkable changes when it binds to the enzyme in com-parison to water [1]. The main goal is to understand how thisacceleration is accomplished.Spectral features are computed by QM/MM. The simulationsprovide charge shifts and small deformations of bond lengthsand angles that are not accessible by experiments directly.With GROMACS we calculated 50ns MM trajectories of GTPand GTP bound Mg2+ in water, Ras�GTP, Ras�GTP�RasGAP,Ras�GDP�Pi�RasGAP and Ras�GDP. For each system wetook six snapshots from the last 25ns as starting structuresfor 5ps QM/MM simulations, calculated the FTIR spectra andcompared them with experiments for validation.The results give new insights into the role of the Mg2+ duringhydrolysis: It drags the electrons from the triphosphate,stores them and after the Pi release returns them to thediphosphate.

[1] Kotting et al., PNAS (2006) 13911-13916.

P-298Pathways to exit a receptor: a dynamical viewof agonists - delta-opioids interactionFrancesca Collu1,2, Matteo Ceccarelli2,Paolo Ruggerone21Department of Chemistry and Biochemistry, UniversitatBern, Freiestrasse 3, Bern, 3012, Switzerland, Italy, 2CNR-IOM, Unita SLACS, and Department of Physics, Universityof Cagliari, S.P. Monserrato-Sestu Km 0.700, Monserrato,CA, 09042, Italy

The importance of delta-opioid receptors as target of a largenumber of drugs is well recognized, but the molecular detailsof interaction and action of the compounds are largelyunknown. In an effort to shade some light on this importantissue we performed an extensive computational study on theinteraction of two compounds, clozapine and desmetilcloza-pine, with a delta-opioid receptor. According to experiments,the lacking of a single methyl group in desmetilclozapine withrespect of clozapine makes the former more active than thelatter, providing a system well suited for a comparative study.We investigated stable configurations of the two drugs insidethe receptor by simulating their escape routes by metady-namics, an algorithm that allows the simulation of events thatare otherwise out of range for standard molecular dynamicssimulations. Our results point out that the action of the com-pound is related to the spatial distribution of the affinity sites itvisits during its permanency. Desmetilclozapine has a largerdistribution of residues, which is interacting with, than cloza-pine. However, large conformational changes of the receptorwere not observed in the presence of both compounds. Thus,a more dynamical picture of ligand-receptor affinity is pro-posed on the basis of the results obtained, involving thecompetition among different stable states as well as theinteraction with the solvents. Such information might be usefulto provide hints and insights that can be exploited in morestructure-and-dynamics-oriented drug design.

P-299Functional modes and residue flexibility controlthe anisotropic response of Guanylate Kinaseto mechanical stressSophie Sacquin-Mora1, Olivier Delalande2 andMarc Baaden11Institut de Biologie Physico-Chimique, Laboratoire deBiochimie Theorique, CNRS UPR9080,13, rue Pierre etMarie Curie, 75005 Paris, France, 2Centre de BiophysiqueMoleculaire, CNRS UPR4301, Orleans, FranceE-mail: sacquin@ibpc.fr

Keywords: Protein mechanics, Brownian dynamics, PrincipalComponent Analysis, flexibility, mechanoenzymatics,mechanical anisotropy

The coupling between the mechanical properties of enzymesand their biological activity is a well-established feature thathas been the object of numerous experimental and theoret-ical works. In particular, recent experiments show thatenzymatic function can be modulated anisotropically bymechanical stress. We study such phenomena using amethod or investigating local flexibility on the residue scale,which combines a reduced protein representation withBrownian dynamics simulations. We performed calculationson the enzyme Guanylate Kinase in order to study itsmechanical response when submitted to anisotropic defor-mations. The resulting modifications of the protein’s rigidityprofile can be related to the changes in substrate bindingaffinities that were observed experimentally. Further analysisof the principal components of motion of the trajectoriesshows how the application of a mechanical constraint on theprotein can disrupt its dynamics, thus leading to a decreaseof the enzyme’s catalytic rate. Eventually, a systematic probeof the protein surface led to the prediction of potential hot-spots where the application of an external constraint wouldproduce a large functional response both from the mechan-ical and dynamical points of view. Such enzyme engineeringapproaches open the possibility to tune catalytic function byvarying selected external forces. (Sacquin-Mora et al., 2010,Biophys. J. 99, pp3412-3419)

P-300The initial maturation mechanism of HIV-1 proteaseS. Kashif Sadiq and Gianni De FabritiisUniversitat Pompeu Fabra, Barcelona Biomedical ResearchPark (PRBB), C/ Dr. Aiguader 88, 08003, Barcelona, Spain

HIV-1 protease autocatalyses its own release from Gag andGagPol precursor polyproteins into mature functional pro-teins. As it is functional in the dimeric form, whilst initially,only a single monomer is embedded within each GagPolchain, the question arises as to what cut’s the cutter. Twoindividual monomers in different GagPol chains are known tocome together to form an embedded-dimer precursor prote-ase. Mature-like protease activity is concomitant withN-terminal intramolecular cleavage of this transient embed-ded-dimer precursor, but how this crucial maturation-initiatingstep is physically achieved, has remained unknown.Here, we show via 400 all-atom explicit solvent moleculardynamics simulation runs of 400 ns each that the N-terminal

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of an immature-like protease, with the N-terminal initiallyunbound as in the GagPol polyprotein, can self-associate tothe active site and therefore be cleaved under conditions ofthermodynamic equilibrium, identifying possible bindingpathways at atomic resolution, in agreement with previousindirect experimental evidence [1]. The binding pathwaypredominantly makes use of the open conformation of thebeta-hairpin flaps characterised by us previously [2], and theN-terminal binds across the entire active site in goodagreement with crystal structures of a cleavage-site peptide-bound protease.The N-terminus serves two roles, firstly in the maturation ofthe protease itself by self-associating to the protein and thenas a stabilizing component of the dimer interface in a matureprotease. Targeting the prior mechanism could be the focusof a novel therapeutic strategy involving immature proteaseinhibition.

[1] Visualizing transient events in amino-terminal autopro-cessing of HIV-1 protease. Tang C, Louis JM, Aniana A,Suh JY, Clore GM. Nature. 2008; 455(7213):693-6.

[2] Explicit solvent dynamics and energetics of HIV-1protease flap opening and closing. Sadiq SK, DeFabritiis G. Proteins. 2010;78(14):2873-85.

P-301Force distribution analysis of allosteric mechanismsChristian Seifert, Frauke GraeterHeidelberg Institute for Theoretical Studies, Heidelberg,Germany

Revealing the pathways of signal transfer in allosteric pro-teins has remained a challenge for today’s biophysicalmethods. Previous approaches are primarily based on thecomparison of active and inactive structures and thermody-namic concepts. However, stiff regions of the protein mightmask signal propagation, even though they are able to carrysignals in form of high internal stresses.We here present a new method, force distribution analysis(FDA Stacklies et al 2011, accepted), that detects the distri-bution of stress upon external perturbations inmacromolecules like proteins, other (bio-)polymers or evensolids with high sensitivity. For tracing signal transfer througha protein structure, FDA calculates the changes, here causedby ligand binding, in the inter-atomic forces of the protein assampled in Molecular Dynamics simulations.The analyzed proteins are two homologues of the chaperoneHsp90 and the catabolite activator protein CAP.We propose a new model for the signal transduction in theE.Coli (HtpG) and Yeast (Hsp82) homologues of Hsp90. Theforce differences between the apo, ADP and ATP boundstates obtained by FDA based on all-atom trajectories total-ing 540 ns revealed a cross-talk between the binding site andthe middle domain via distinct paths.The catabolite activator protein (CAP) is a major player in thelac operon. CAP features a negative cooperativity for thebinding of cAMP, which is interestingly based not on achange in structure but in flexibility (Popovych et al. 2006).FDA of the apo, single- and double-bound state revealed asignaling network in CAP, which transfers a signal (first cAMPis bound) to the second binding niche without obvious

structural changes, thereby explaining the observedcooperativity.This work describes the effectiveness of FDA for resolvingallosteric communication pathways, which are directly test-able by experiments. As such, it has broad implications forour view on protein internal strain and function.

O-302The role of non-native interactions in knotted proteinsTatjana Skrbic1,2, Pietro Faccioli1,3,4 andCristian Micheletti51Interdisciplinary Laboratory for Computational Science(LISC), Trento, Italy, 2European Center for Theoreticalstudies in nuclear physics and related areas (ECT*), Trento,Italy, 3Universita degli Studi di Trento, Trento, Italy, 4IstitutoNazionale di Fisica Nucleare (INFN), Italy, 5ScuolaInternazionale Superiore di Studi Avanzati (SISSA), Trieste,Italy

Knotted proteins are the object of an increasing number ofexperimental and theoretical studies, because of their abilityto fold reversibly in the same topologically entangled con-formation. The topological constraint significantly affects theirfolding landscape, thus providing new insight and challengesinto the funnel folding theory [1]. Recently developedexperimental methods to trap and detect knots have sug-gested that denaturated ensembles of the knotted proteinsmay be knotted [2].We present numerical simulations of the early stage of fold-ing of the knotted proteins belonging to protein familiesMTase (methyltransferase) and SOTCase (succinyl-ornithinetranscarbamylase), and of their unknotted homologues [3].Our results show that native interactions are not sufficient togenerate the knot in the denaturated configurations. How-ever, when non-native interactions are included we observeformation of knots only in the chains whose native state isknotted. In addition, we find that the knots are formed througha universal mechanism. Such a knot formation mechanismcorrectly predicts the fraction of the knotted proteins found innature and can be used to make qualitative prediction ontheir folding times.

[1] L. Mallam, FEBS Journal 276, 365 (2008).[2] L. Mallam, J. M. Rogers and S. E. Jackson, PNAS 107,

8189 (2010).[3] R. Potestio, C. Micheletti and H. Orland, PLoS Comp.

Biol. 6, 1 (2010).

P-304Modeling cellular adhesion with Monte Carlo SimulationsEva Sunnick, Andreas Janshoff and Burkhard GeilUniversity of Goettingen, Institute of Physical Chemistry,Goettingen, GermanyE-mail: esunnic@gwdg.de

Cellular adhesion is an important process in cell develop-ment and survival, it plays a key role in maintaining cell

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shape and motility and also for numerous signaling pro-cesses. Adhesion is based on non-covalent interactionsbetween transmembrane proteins and the extracellularmatrix. Cells are able to create two-dimensional assembliesof integrins, so called focal adhesions, which they use to stickto the substrate and collect information about the environ-mental properties. The goal of this work is a deeperunderstanding of the formation and the stability of theseadhesion clusters.Bond cluster formation and disintegration is dynamicallymodeled with the aid of Monte Carlo Simulations. In themodel, a membrane is attached to a flat surface via a vari-able number of adhesion bonds. The spacial configuration ofthese adhesion points subjected to an inhomogeneousstress field maps into a distribution of local membrane/ sur-face distances. We introduce a model which explicitlyaccounts for the membrane elasticity and demonstrate thatsuch models are able to explain the spontaneous formationof adhesion bond clusters.

P-305Characterizeing the activation of protein kinase throughan hybrid all-atom structure-based modelL. Sutto1, I. Mereu1, F.L. Gervasio11Structural Biology and Biocomputing Programme, SpanishNational Cancer Research Center (CNIO), c/ MelchorFernandez Almagro, 3. E-28029 Madrid, Spain

Structure based models are successful at conjugating theessence of the energy landscape theory of protein folding[1]with an easy and efficient implementation. Recently theirrealm expanded beyond single protein structure, been usedprofitably to widely study large conformational transitions[2-3]. Still, when dealing with conformational transition betweentwo well-defined structures an unbiased and realisticdescription of the local backbone and sidechain interactionsis necessary. The proposed model merges a precisedescription of these interactions with a structure-based longrange potential that takes into account different conformers.We present the results of the activation of the catalyticdomain of human cSRC tyrosine kinase for which wereconstructed the transition free energy and the descriptionof the activation loop flexibility. The excellent model perfor-mances in terms of speed and the satisfactory accuracy ofthe description of the system and its flexibility are promisingfor a more systematic study of the tyrosine kinase familyactivation mechanisms.

[1] Luthey-Schulten, Z.; Wolynes, P. Annu. Rev. Phys.Chem 1997, 48, 545-600.

[2] Yang, S.; Roux, B. PLoS Computational Biology 2008,4, e1000047.

[3] Camilloni, C.; Sutto, L. J. Chem. Phys. 2009, 131,245105.

P-306Molecular dynamics simulations on the functionof the transmembrane Cav1.2 channel in dependenceof the content of cholesterol in the membraneChonticha Suwattanasophon1, Roland Faller2,Peter Wolschann1, Anna Stary-Weinzinger31Institute for Theoretical Chemistry, Universityof Vienna,Vienna, Austria, 2Department of ChemicalEngineering and Materials Science, University of California,Davis, California, USA, 3Department of Pharmacologyand Toxicology, University of Vienna,Vienna, Austria

Increased cholesterol levels are associated with multiplepathological conditions. Here, Molecular Dynamics simula-tions are applied to explain the influence of membranecholesterol levels on voltage-gated calcium channels. Weused different lipid compositions to obtain information aboutthe possible effects by which cholesterol influences Cav1.2channels. Cholesterol is not directly interacting with theprotein in the open or the closed conformation, but the Ca2+

ion mobility is decreased in POPC/CHOL systems comparedto pure POPC bilayers. Cholesterol increases lipid packingimplying that it plays a crucial rule in restricting lipid move-ment in the region around 1 nm of POPC. This leads to anindirect modulation of Cav1.2 channel function.

P-307Higher order numerical simulation of stochasticchemical reaction systemsTamas Szekely Jr.1, Konstantinos C. Zygalakis2,Kevin Burrage1 and Radek Erban21Oxford University Computing Laboratory, Oxford, OX1 3QD,UK, 2University of Oxford, Mathematical Institute, Oxford,OX1 3LB, UK

We introduce a previously undescribed technique for mod-elling the kinetics of stochastic chemical systems. We applyRichardson extrapolation, a sequence acceleration methodfor ordinary differential equations, to a fixed-step tau-leapingalgorithm, to produce an extrapolated tau-leaping methodwhich has weak order of accuracy two. We prove thismathematically for the case of linear propensity functions.We use four numerical examples, two linear and two non-linear, to show the higher accuracy of our technique inpractice. We illustrate this by using plots of absolute error fora fixed-step tau-leap and the extrapolated tau-leap. In allcases, the errors for our method are lower than for a fixed-step tau-leap; in most cases they are second order ofaccuracy.

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O-308Segment swapping between domains is an evolutionarymechanism that generates new protein foldsAndras Szilagyi1, Yang Zhang2, Peter Zavodszky11Institute of Enzymology, Budapest, Hungary, 2Universityof Michigan, Ann Arbor, MI, USA

How new protein folds arise during evolution is a long-standing, puzzling question. We present evidence suggest-ing that new protein structures can arise by a swap ofsegments between domains within a single-chain monomer.By a comprehensive structural search of the current PDB, weidentified 32 well-defined segment-swapped proteins (SSPs)belonging to 18 structural families. Nearly 13% of all multi-domain proteins in the PDB may have a segment-swappedevolutionary precursor as estimated by more permissivesearching criteria. The formation of SSPs can be explainedby two principal evolutionary mechanisms: (i) domainswapping and fusion (DSF), (ii) circular permutation (CP). Bylarge-scale comparative analyses using structural alignmentand HMM methods, it was found that the majority of SSPshave arisen by the DSF mechanism, and a much smallerfraction by CP. Functional analyses revealed that segmentswapping, which results in two linkers connecting thedomains, may impart directed flexibility to multidomain pro-teins. Inter-domain segment swapping represents a novelgeneral mechanism by which new protein folds and multi-domain architectures arise in evolution, and segment-swapped proteins have structural and functional propertiesthat make them worth defining as a separate group.

O-310Molecular dynamics simulations of ribosomaltranslocation based on cryo-EM dataAndrea C. Vaiana, Lars V. Bock, Christian Blau,Niels Fischer, Holger Stark, Wolfgang Wintermeyer,Marina V. Rodnina, Helmut GrubmullerMax-Planck-Institute for BiophysicalChemistttingen, Germany

High-resolution structural studies of the ribosome haverevealed many details of how the ribosomal machineryachieves efficient protein synthesis at the atomic level. Thesestudies provide atomic resolution structures of the ribosome inwell- defined states along the protein assembly cycle. Unfor-tunately, these structures shed little light on intermediatestates, transition pathways, and equilibrium states at physio-logical temperatures. Molecular dynamics simulations allowaddressing these dynamical features of the ribosome whilemaintaining a faithful all-atom, explicit solvent representation ofthe system. We combine crystallographic and cryo-EM datawith molecular dynamics simulations to target the elongationphase of protein synthesis. Using fitted models as startingpoints we performed molecular dynamics simulations to obtaina picture of the ribosome in different states of transfer RNAtranslocation. Analyzing the main modes of motion along thesimulation trajectories provided insight into the concertedmotion of different parts of the ribosome during elongation.

P-311Implicit solvation in different dielectric environmentswith the generalized Born model FACTSIoan Vancea, Birgit StrodelICS-6: Structural Biochemistry, Forschungszentrum Juelich,Germany

The generalized Born model was introduced for the efficientevaluation of continuum electrostatic energies. FACTS (fastanalytical continuum treatment of solvation) is an efficientimplicit solvent model based on the generalized Born modelfor calculating the free energy of solvation of a moleculeembedded in an aqueous continuum solvent.We discuss a modification to the standard FACTS implicitsolvent model for modelling heterogeneous dielectric envi-ronments with applications to biological membranes. Themembrane-water system in the modified model is repre-sented in the form of multiple layered dielectric regions withdielectric constants different from the solute cavity. Themodified FACTS model for membranes is implemented in theCHARMM molecular dynamics simulations package andresults are shown for a range of different proteins.

P-312Simulating bacterial efflux: how molecular featuresaffect functional rotationA.V. Vargiu1, R. Schulz 2, F. Collu3, U. Kleinekathofer2,P. Ruggerone11CNR-IOM, Unita SLACS, and Department of Physics,University of Cagliari, S.P. Monserrato-Sestu Km 0.700,Monserrato (CA), 09042, Italy, 2School of Engineeringand Science, Jacobs University Bremen, Campus Ring 1,Bremen, Bremen, 28759, Germany, 3Departmentof Chemistry and Biochemistry, Universitat Bern,Freiestrasse 3, Bern, 3012, Switzerland

The major tripartite efflux pump AcrAB-TolC is responsible forthe intrinsic and acquired multidrug resistance in Escherichiacoli. At heart of the extrusion machinery there is the homo-trimeric transporter AcrB, which is in charge of the selectivebinding of structurally and chemically different substrates andenergy transduction. The effects of conformational changes,which have been proposed as the key features of the extru-sion of drugs, are investigated at molecular level usingdifferent computational methods like targeted moleculardynamics. Simulations, including almost half a million atoms,have been used to assess several hypotheses concerning thestructure-dynamics-function relationship of the AcrB protein.The results indicate that, upon induction of conformationalchanges, the substrate detaches from the binding pocket andapproaches the gate to the central funnel. In addition, weprovide evidence for the proposed peristaltic transportinvolving a zipper-like closure of the binding pocket, respon-sible for the displacement of the drug. Using these atomisticsimulations the role of specific amino acids during the tran-sitions can be identified, providing an interpretation of site-directed mutagenesis experiments. Additionally, we discuss apossible role of water molecules in the extrusion process.

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O-313Trafficking of lipids between high density lipoproteinand cholesteryl ester transfer proteinArtturi Koivuniemi1, Timo Vuorela1, Petri T. Kovanen2, MarjaT. Hyvonen3 and Ilpo Vattulainen1,4,*1Department of Physics, Tampere University of Technology,Finland, 2Wihuri Research Institute, Helsinki, Finland,3Department of Physics, University of Oulu, Finland,4Department of Applied Physics, Aalto University Schoolof Science and Technology, FinlandE-mail: Ilpo.Vattulainen@tut.fi

Cardiovascular disease (CVD) is the main cause of death inWestern countries. The risk for being exposed to CVD largelydepends on the relative levels of low density lipoprotein(LDL) and high density lipoprotein (HDL) particles that arecarriers of cholesterol and its esters in human bloodstream.Meanwhile, cholesteryl ester transfer protein (CETP) trans-ports cholesteryl esters, triglycerides, and phospholipidsbetween the different lipoprotein fractions, such as HDL andLDL. Intriguingly, the inhibition of CETP has been shown toprevent and treat the development of CVD. In this work, weemployed atomistic and coarse-grained molecular dynamicssimulations to unravel the mechanisms associated with theCETP-mediated lipid exchange. We found CETP to bind tothe surface of HDL-sized lipid droplets through its chargedand tryptophan residues. We further found CETP to inducethe formation of a channel from the core of the lipid droplet tothe binding pocket of CETP. We also clarified the role of helixX in the opening of CETP binding pocket and the mechanismassociated with diffusion of lipids between CETP and thelipid droplet. The findings presented here can be exploited inthe design of molecular agents to inhibit the activity of CETP.

P-314An optimized MM/PBSA virtual screening approachapplied on an HIV-1 gp41 fusion peptide inhibitorTom Venken1, Daniela Krnavek2, Jan Munch2,Frank Kirchhoff2, Petra Henklein3, Marc De Maeyer1

and Arnout Voet11Laboratory for Biomolecular Modelling, KULeuven,Heverlee, Belgium, 2Institute of Molecular Virology,University Hospital Ulm, Ulm, Germany, 3Charite-Universitatsmedizin Berlin, Institute of Biochemistry, Berlin,GermanyE-mail: tom.venken@chem.kuleuven.be

VIRus Inhibitory Peptide (VIRIP), a 20 amino acid peptide,binds to the fusion peptide (FP) of human immunodeficiencyvirus type 1 (HIV-1) gp41 and blocks viral entry. Moleculardynamics (MD) and molecular mechanics/Poisson-Boltz-mann surface area (MM/PBSA) free energy calculationswere executed to explore the binding interaction betweenseveral VIRIP derivatives and gp41 FP. A promising corre-lation between antiviral activity and simulated binding freeenergy was established thanks to restriction of the flexibilityof the peptides, inclusion of configurational entropy calcula-tions and the use of multiple internal dielectric constants for

the MM/PBSA calculations depending on the amino acidssequence. Bases on these results, a virtual screeningexperiment was carried out to design enhanced VIRIP ana-logues. A selection of peptides was tested for inhibitoryactivity and several improved VIRIP derivatives were identi-fied. These results demonstrate that computational modellingstrategies using an improved MM/PBSA methodology can beused for the simulation of peptide complexes. As such, wewere able to obtain enhanced HIV-1 entry inhibitor peptidesvia an MM/PBSA based virtual screening approach.

P-317Structure based discovery and optimization of first-in-class inhibitors of the HIV-1 IN–LEDGF/p75 interactionArnout R.D. Voet1, Frauke Christ2, Arnaud Marchand3,Patrick Chaltin3, Stefan Nicolet4, Sergei Strelkov4, ZegerDebyser2, Marc De Maeyer11The Laboratory for Biomolecular Modeling, KULeuven,B-3001 Leuven, Belgium, 2Division of Molecular Medicine,Katholieke Universiteit Leuven, B-3000 Leuven and IRCKatholieke Universiteit Leuven Campus Kortrijk, Flanders,Belgium, 3CD3-CISTIM, KULeuven, B-3000 Leuven,Belgium, 4Deparment of Pharmacology, KULeuven, B-3000Leuven, Belgium

An essential step during the HIV life cycle is the integration ofthe viral cDNA into the human genome. HIV-1 integrasemediates integration in a tight complex with the cellularcofactor: LEDGF/p75 [1]. Disruption of the interaction inter-feres with HIV replication and therefore provides aninteresting new drug target for antiretroviral therapy [2,3].Here we present the structure based discovery and optimi-zation of a series of small molecule inhibitors that bind to HIV-1 integrase and block the interaction with LEDGF/p75. Thework flow was set up according to a funnel principle in which aseries of virtual screening tools were applied in such way todiscard at each step molecules unlikely to be active againstthe desired target (including 2D filtering, pharmacophoremodelling and molecular docking) The activity and selectivityof the selected molecules were confirmed in an ALPHAscreen based assay, that measure protein-protein interactionin vitro, and furthermore by in vivo experiments. Active com-pounds proceeded towards crystallographic soaking into thereceptor protein crystals. These crystal structures not onlyvalidated the binding mode and activity of the hit compounds,but furthermore were used as a platform for structure baseddrug design which resulted in the rational discovery of new hitcompounds and optimized lead compounds. In vitro and invivo experiments validated the mechanism of action of thesecompounds and show that they are a novel class of antiret-roviral compounds with in vivo inhibitory activity by targetingthe interaction between LEDGF/p75 and HIV-1 integrase.Cross resistance profiling indicate that these compounds areactive against current resistant viral strains.[4] Currently themost potent inhibitors show an in vivo IC50 of 55nM. Thesecompounds are promising for future pharmaceutical optimi-zations to be used in the clinic as new antiretroviral agents.

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Figure: Crystallography was used to validate the bindingmode of the discovered inhibitors. Insights in the interactionof the ligand-protein complex allowed for rational design ofoptimized inhibitors.

References:

[1] Cherepanov P, et al. HIV-1 integrase forms stabletetramers and associates with LEDGF/p75 protein inhuman cells. J Biol Chem. 2003 Jan 3;278(1):372-81.

[2] Vandekerckhove L, et al. Transient and stable knock-down of the integrase cofactor LEDGF/p75 reveals itsrole in the replication cycle of human immunodeficiencyvirus. J Virol. 2006 Feb;80(4):1886-96.

[3] De Rijck J, et al. Overexpression of the lens epithelium-derived growth factor/p75 integrase binding domaininhibits human immunodeficiency virus replication. JVirol. 2006 Dec;80(23):11498-509.

[4] Christ F, Voet A, Marchand A, et al. Rational design ofsmall-molecule inhibitors of the LEDGF/p75-integraseinteraction and HIV replication. Nat Chem Biol. 2010Jun;6(6):442-8.

P-318Modelling allosteric signalling in protein homodimersThomas Rodgers1, David Burnell1, Mark R. Wilson1, EhmkePohl1, Martin Cann1, Philip D. Townsend1,Tom C. B. McLeish1, Hedvika Toncrova21Biophysical Sciences Institute, Durham University, Durham,UK, 2Physics and Astronomy, University of Leeds, Leeds, UK

Allostery in protein systems is a thermodynamic phenom-enon. Allosteric response is driven by the free energydifferences obtained in different binding events, which, inprinciple, contain contributions from both enthalpic andentropic changes. While traditional views of allostery haveconcentrated on structural changes induced by the bindingof ligands (i.e. ‘‘enthalpically dominated’’), it is nowincreasingly recognised that fluctuations in structure cancontribute to allosteric regulation. In some cases, where

ligand-induced structural changes are small, thermal fluc-tuations can play a dominant role in determining allostericsignalling. In thermodynamic terms, the entropy change forsubsequent binding is influenced by global vibrationalmodes being either damped or activated by an initial bind-ing event. One advantage of such a mechanism is thepossibility for long range allosteric signalling. Here, chan-ges to slow internal motion can be harnessed to providesignalling across long distances. This paper considershomotropic allostery in homodimeric proteins, and presentsresults from a theoretical approach designed to understandthe mechanisms responsible for both cooperativity and anti-cooperativity. Theoretical results are presented for thebinding of cAMP to the catabolite activator protein (CAP)[1], where it is shown that coupling strength within a dimeris of key importance in determining the nature of the allo-steric response. Results from theory are presented alongside both atomistic simulations and simple coarse-grainedmodels, designed to show how fluctuations can play a keyrole in allosteric signalling in homodimeric proteins.

[1] Substrate-Modulated Thermal Fluctuations Affect Long-Range Allosteric Signaling in Protein Homodimers:Exemplified in CAP. H. Toncrova and T. C.B. McLeish.Biophysical J., (2010), 98, 2317-2326.

P-319A forward-backward mathematical model of bloodpressure and blood flow in human upper limbDejan Zikic1, Dragoslav Zikich21Biophysics Institute, Medical School, Belgrade University,Serbia, 2Ella Institute for Melanoma Treatmentand Research, Sheba Medical Center, Tel Hashomer, IsraelE-mail: dzikic@med.bg.ac.rs; dzikich@gmail.com

Keywords: Pulse wave reflection, blood pressure waveforms,arterial hemodynamics

A forward-backward mathematical model of the blood flowand the blood pressure of the large arteries in human upperlimb have been developed. The model is based on Wo-mersley solution for one-dimensional equations derived fromthe axisymmetric Navier-Stokes equations for flow in avessel. Blood flow was measured at brachial and radialartery using IR sensor, positioned at 3 cm distance fromarterial branching point, where the pulse was easily detectedby hand. The arterial pulse wave velocity, reflected coeffi-cient and terminal impedance were calculated from themeasured data reflected coefficient and used in simulation.Blood flow and blood pressure simulations were comparedwith the measured signals as well as with the publishedclinical data. The model predicts the blood pressure wave-form composed of a forward propagation waveform and anumber of reflected waveforms from many branching sites.The model predictions are in a good correlation of with theage-related characteristics of blood vessels. The model isconsistent with clinically observed blood pressure and canbe useful for proper biophysical understanding of arterialhemodynamics.

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Imaging and optical microscopy

P-320A new class of reversibly switchable fluorescentproteinsM. Andresen, T. Brakemann, A.C. Stiel, I. Testa, C. Eggeling,S. Hell, S. JakobsMax-Planck-Institute for Biophysical Chemistry, Gottingen,Germany

Reversibly switchable fluorescent proteins (RSFPs) can beswitched between a fluorescent (on) and a nonfluorescent(off) state which is accompanied by a cis-trans isomerizationof the chromophore on the molecular level 1,2. This uniqueproperty has already provided new aspects to variousmicroscopy techniques, such as high resolution microscopy,FCS or monochromatic multicolor microscopy 3-5. Despite oftheir established potential, RSFPs still have a major draw-back: the wavelength for fluorescence excitation is alwaysone of the two switching wavelengths. The imaging processthus inevitably results in the switching of a small fraction ofthe RSFPs, which might hinder or complicate some experi-ments. We developed a new reversibly switchablefluorescent protein which eliminates the problem of thecoupling between switching and fluorescence excitation. Thisfluorescent protein follows an unusual and currently unknownmechanism of switching between a fluorescent and a non-fluorescent state. It is brightly fluorescent and exhibits anexcellent signal to noise ratio.

1. Andresen, M. et al. Proc Natl Acad Sci U S A,13005-13009 (2007).

2. Andresen, M. et al. Proc Natl Acad Sci U S A,13070-13074 (2005).

3. Andresen, M. et al. Nat Biotechnol, 1035-1040 (2008).4. Eggeling, C. et al. Microsc Res Tech, 1003-1009 (2007).5. Egner, A. et al. Biophys J, 3285-3290 (2007).

O-321Programmable array microscope imaging of living cellsand spin-off to tumor diagnosticsDonna J. Arndt-Jovin1, Michelle G. Botelho1,Sven Kantelhardt2, Anthony H.B. de Vries1,Pieter A.A. De Beule1,3, Thomas M. Jovin1Laboratory of Cellular Dynamics, Max Planck Institutefor Biophysical Chemistry, 37077 Gottingen, Germany,2Department of Neurosurgery, University of Mainz, 55101Mainz, Germany, 3INL Nanotechnolgy Laboratory, Braga,Portugal

EGFR exhibits diffusion but undergoes retrograde transportto the cell body after ligand association, dimerization, andtransautophosphorylation. Filopodial transport allows remotesensing and was discovered by sensitive imaging of QD-EGF [1]. Recently we identified Shc1 as an essential com-ponent for coupling of activated EGFR to the actin filaments[Gralle Botelho et al.]In parallel studies [2], QD-based ligands (EGF, Mabs) weretargeted to EGFR in gliomas. Cell-cultures, animal models andex vivo biopsies of human high-grade as well as low-grade

gliomas showed high probe specificity.. The aim is to definemore precisely the tumor boundaries at the time of resection.We used the Programmable Array Microscope designed forsensitive, high-speed optical sectioning, particularly of livingcells. The PAM is based on structured illumination and con-jugate detection using a digital micromirror device (DMD) [3]located in a primary image plane. The unique feature is therapid, (re)programmable adjustment of local excitationintensity, Dynamic, on-the-fly optimization is thus achieved,e.g. multipoint FRAP [4], light exposure minimization andobject tracking [5], or super-resolution strategies. The fea-tures and operation of the 3rd generation PAM will bepresented.

[1] D.S. Lidke et al., J. Cell Biol. 170 (2005) 619.[2] S.R. Kantelhardt et al. PLoS One (2010) 5: doi:10.1371/

journal.pone.0011323[3] P.A.A. De Beule et al. Proc. SPIE 7932, 79320G (2011);

doi:10.1117/12.879611[4] G.M. Hagen et al., Microsc. Res. Tech. 72 (2009)

431-440[5] W. Caarls et al., J. Microsc. (2010) 1365-2818.doi:2010.

03413.x

O-322Three-dimensional video-rate nonlinear microscopyof contracting myocytesMasood Samim, Nicole Prent, Richard Cisek, Daaf Sandkuijl,Sergey Musikhin and Virginijus BarzdaDepartment of Chemical and Physical Sciences, Departmentof Physics and Institute for Optical Sciences, Universityof Toronto, 3359 Mississauga Road N., Mississauga, ON,L5L 1C6 Canada

Contraction of muscle cells, motility of microorganisms,neuronal activity, and other fast cellular processes requiremicroscopic imaging of a three-dimensional (3D) volume witha video-rate scanning. We present 3D video-rate investiga-tions of structural dynamics in biological samples with themulticontrast third- and second-harmonic generation as wellas fluorescence microscope. The multidepth scanning isachieved by two combined laser beams with staggeredfemtosecond pulses. Each of the beams is equipped with apair of deformable mirrors for dynamic wavefront manipula-tion enabling multidepth refocusing with simultaneouscorrections for optical aberrations. Combined, more than 250frames per second lateral scanning with fast refocusingenables the 3D video-rate imaging of dynamically movingstructures. In addition, combination of two laser beams isaccomplished at two perpendicular polarizations enablinglive imaging of sample anisotropy, which is important forstructural studies particularly with the second harmonicgeneration microscopy. Investigations of beating chickembryo hearts with the 3D video-rate scanning microscoperevealed multidirectional cardiomyocyte contraction dynam-ics in myocardial tissue. Intricate synchronization ofcontractions between different layers of myocytes in the tis-sue will be presented. The video-rate 3D microscopy opensnew possibilities of imaging fast biological processes in livingorganisms.

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P-323Spinning-disk confocal fluorescencemicrospectroscopy: instrumental setup and featuresDaniele Biglino1,2, Zoran Arsov1,2, Iztok Urbancic1,Tilen Koklic1,2, Janez Strancar1,21Jozef Stefan Institute, Condensed Matter Physics,Laboratory of Biophysics, Ljubljana, Slovenia, 2Centreof excellence CO-NAMASTE, Ljubljana, Slovenia

Confocal fluorescence microscopy is an invaluable tool tostudy biological systems at cellular level also thanks to thesynthesis of always new specific fluorescent probes, e.g.multiprobe labelling enables complex system characteriza-tion. However, only the recent employment of narrowbandtunable filters overcomes the problems due to the use of thebroadband ones. The possibility of acquiring the emissionspectra in a spatially resolved way extends simple imageintensity studies into characterization of complex probe-environment relationship through the sensitivity of fluores-cence spectra to the local molecular environmentdifferences. Consequently, fluorescence microspectroscopy(FMS) is able to provide the spectral information in a welldefined spatial region allowing the researcher to simulta-neously obtain spatial and spectroscopic information. Ourinstrument has been specially built to study live cells andtheir interaction with nanomaterials, drug carriers and modi-fied cell environment. Other main characteristics arereducing the bleaching effect and employing a white lightsource that does not limit the use to specific probes.Graphical tools, such as colour coded images, have alsobeen introduced to provide explicit and straightforward visualinformation.

P-325High speed FPGA based multi-tau correlation for single-photon avalanche diode arraysJan Buchholz1,2, Jan Krieger1,2, Agata Pernus1,2,Edoardo Charbon4, Udo Kebschull3, Jorg Langowski21Equally contributing authors, 2German Cancer ResearchCenter, Biophysics of Macromolecules (B040), ImNeuenheimer Feld 580, 69120 Heidelberg, Germany,3Goethe-Universitat Frankfurt, Senckenberganlage 31,60325 Frankfurt, Germany, 4Technische Universiteit Delft,Mekelweg 4, 2628 CD Delft, The Netherlands

With the evolving technology in CMOS integration, high-density arrays of single-photon avalanche diodes (SPAD)have become available. We use a 32x32 SPAD pixel array asimage sensor in a custom built Single Plane IlluminationMicroscope for Fluorescence Correlation Spectroscopy(SPIM-FCS). Our array can be read out at rates above100kHz for full frames, thus enabling observation of fastbiophysical and -chemical processes at the microsecondtimescale. The time-dependent fluorescence fluctuations areevaluated by means of an autocorrelation analysis for everysingle pixel. To cover a wide range of lag-times, the multiple-tau correlation algorithm is used. We implemented thisalgorithm in a Field Programmable Gate Arrays (FPGA), areconfigurable integrated circuit. Due to their parallelismthese devices are ideally suited for such algorithms.Our current hardware design is capable of calculating full

autocorrelation of all 1024 pixels in real-time for a givenframe-time of 10ls. The same design can also be used forcross-correlation. This enables us to observe diffusionprocesses of the samples in real-time.

P-326FRET-Imaging to reveal cross-bridge dynamics in singleskeletal muscle fibresV. Caorsi, D. Ushakov, N. Setta-Kaffetzi, M.A. FerencziMolecular Medicine Section, NHLI, Imperial College London,London SW7 2AZ, UK

We demonstrate the use of FRET-imaging (Forster Reso-nance Energy Transfer) as an assay to directly monitor thedynamics of cross-bridge conformational changes in singlefibres of skeletal muscle. We measured nm-distances ofseveral FRET pairs located at strategic positions to sensemyosin head conformational changes: we focused ourattention on the essential light chain, ELC (specificallylabelling a modified ELC and exchanging it with the naturalELC of the fibre) and we investigated its interaction with theSH1 helix, with the nucleotide binding pocket and with actin.We characterized FRET in single rigor muscle fibres, deter-mining distances in agreement with those from thecrystallographic data. The results demonstrate the viability ofthe approach in sensing different FRET efficiencies over afew nanometres, an essential requirement to follow theexpected small FRET variations in contracting muscle fibres.We are now performing dynamic experiments on rigor andactive fibres by applying small stretch/release cycles to alterthe interaction distances (estimated time resolution of nearly20ms/frame). In this configuration, it will be possible tomeasure functional changes, shedding light on the myosinhead dynamics during contraction.

P-327Direct visualization of CFTR conformation by atomicforce microscopy imagingCarlotta Marasini1,2 Massimo Vassalli11CNR, Istituto di Biofisica, Genova, Italy, 2Fondazione per laRicerca sulla Fibrosi Cistica onlus, Verona, ItalyE-mail: carlottamarasini@gmail.com

Cystic Fibrosis (CF) is one of the recessive Mendeliangenetic diseases more popular in the world. There is nodecisive treatment for this disease and, only recently, aneffort to find pharmacological therapies for CF has beencarried out. This disease is caused by a mutation in thegene encoding for the CF trans-membrane regulator(CFTR) chloride channel present in epithelial cells. A fullknowledge of the CFTR structure and conformationalchanges associated with the activation process would allowa better understanding of the disease, possibly leading tothe identification of suitable drugs. Up to now a detailedmolecular structure of the first nucleotide binding domain(NBD1) was determined by x-ray diffraction and an homol-ogy model of the NBD1-NBD2 dimer was build andsuccessively extended to the complete CFTR. However, the

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quaternary organization of the protein is still controversial:X-ray experiments suggest that isolated CFTR is organizedas monomer, whereas a dimeric organization is proposedon the basis of electron microscopy reconstructions. Inaddition, Schillers et al. (2004) used Atomic Force Micros-copy (AFM) to image the cytoplasmic side of CFTR ontransfected Oocytes, showing the presence of peculiarannular structures, probably associated to CFTR oligomers,but never observing features comparable with the proposedstructures for monomers and dimers. The aim of our work isto study CFTR in its natural environment, the plasmamembrane, to gather definite information about the nativeorganization of the channel and to open for future studieson the putative functional role of the supramolecular orga-nization of the channel in the plasma membrane that hasnot yet been fully addressed. Atomic Force Microscopy(AFM) imaging was performed on the cytoplasmic side ofcellular membranes extracted from fetal rat thyroid (FRT)cells permanently transfected with CFTR. The protocol toobtain flat and clean membranes, crucial to reach thehighest resolution in AFM images, was carefully assessedby means of AFM and confocal microscopy. CFTR channelswere identified in the plasma membrane using an immu-nogold labeling approach and the organization of theirquaternary structure was studied. Our preliminary resultsindicate that several CFTR conformations co-exist in theplasma membrane: monomers, dimers and annular rings.

P-328Focal delivery of BDNF to cultured neurons with opticaltweezersElisa D’Este1, Gabriele Baj2, Giulietta Pinato1,Enrico Tongiorgi2, Dan Cojoc11Istituto Officina dei Materiali IOM-CNR, Laboratorio TASC,Trieste, Italy, 2Department of Life Sciences, Universityof Trieste, Trieste, Italy

Focal stimulation of cultured neurons is crucial since itmimics physiological molecules release. Indeed, the nervoussystem finely tunes the activity of each synapse by regulatingthe secretion of molecules spatially and temporally. Currentlyused techniques have some drawbacks such as a poorspatial resolution or a low flexibility. We propose a novelapproach based on Optical Tweezers (OT) [1] to overcomethese limitations.OT allow an ease manipulation with sub-micrometric preci-sion of silica beads, which can be functionalized with anyprotein. For a proof-of-principle study we coated 1,5lm largebeads with Brain-Derived Neurotrophic Factor (BDNF) orBovine Serum Album (BSA) as control. We showed that asingle bead was able to activate the BDNF receptor TrkB,inducing its phosphorylation. Moreover BDNF beads but notcontrol beads were able to induce c-Fos translocation intothe nucleus [2], indicating that the whole pathway was acti-vated. Finally, we positioned vectors in proximity to thegrowth cones of cultured hippocampal neurons [3]. Controlbeads didn’t affect the normal development of these struc-tures while BDNF beads significantly did. These findingssupport the use of the OT technology for long-term, localizedstimulation of specific subcellular neuronal compartments.

[1] K. C. Neumann and S. M. Block (2004). ‘‘Opticaltrapping.’’ Rev. Sci. Instrum. 75, 2787-2809.

[2] Reichardt, L.F. (2006). ‘‘Neurotrophin-regulated signal-ling pathways.’’ Philos Trans R Soc Lond B Biol Sci.361(1473):1545-64

[3] K. Shen and C. W. Cowan, ‘‘Guidance Molecules inSynapse Formation and Plasticity’’ Cold Spring HarbPerspect Biol. 2010 Apr 1;2(4)

O-329Optical nanoscopy and individual moleculelocalization with focused light under linearand non linear regimesAlberto Diaspro, Paolo Bianchini, Francesca Cella Zanacchi,Benjamin Harke, Silvia Galiani, Zeno Lavagnino, JenuChacko, Emiliano RonzittiIIT – Istituto Italiano di Tecnologia, Genova, Italy, LAMBS-MicroScobio and Department of Physics, Universityof Genoa, Italy

A recognized advantage of optical microscopy lies in the factthat allows non-invasive three-dimensional (3D) imaging oflive cells at the submicron scale with high specificity. Theadvent of the visible fluorescent proteins and of a myriad offluorescent tags pushed fluorescence microscopy to becomethe most popular imaging tool in cell biology. The confocaland multiphoton versions of fluorescence microscopy rein-force this condition. In general, is a well-known paradigm thegiven inability of a lens-based optical microscope to discerndetails that are closer together than half of the wavelength oflight. Recently, the viewpoint for improving resolution movedfrom optical solutions to the side of the fluorescent moleculeto be detected. Today, for the most popular imaging mode inoptical microscopy, i.e. fluorescence, the diffraction barrier iscrumbling and the term ‘‘optical nanoscopy’’, coined earlier,comes to be a real far field optical microscope available forthe scientific community as the ones allowing individualmolecule localization at high precision. Here we discussabout architectures, calibrations and applications of targetedand stochastic readout methods using both single and mul-tiphoton excitation.Work supported by IIT, IFOM and Italian PRIN 2008JZ4MLB.

P-330Fluorescence resonance energy transfer analysisof nuclear proteins by laser scanning cytometryDoan Xuan Quang Minh, Nikoletta Szaloki,Gyorgy Vamosi and Zsolt BacsoUniversity of Debrecen, Medical and Health Science CenterDepartment of Biophysics and Cell Biology, Debrecen,Hungary

Defining location and nature of the interactions betweenmolecular species in living cells is hampered by the limitedresolution of the employed instruments. We present here aslide–based cytometric method of fluorescence resonanceenergy transfer analysis that utilizes spectral properties ofcyan and yellow fluorescent proteins as markers to investi-gate the interaction of Fos and Jun transcription factors in

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substratum attached cells. FRET was measured by a laserscanning imaging cytometer which combines the advantagesof flow cytometry with those of the image analysis system.Large number cells was automatically identified, their fluo-rescence data and digital images were determined on cell bycell basis. FRET efficiencies of CFP and YFP tagged Fos-Jun heterocluster pairs was 8.35±3.00% representing6.98nm proximity of Fos-Jun protein terminals. The back-ground FRET efficiency due to spectral crosstalk in theabsence of molecular interaction was 0.42±0.64%. Onpopulation level the applied transfection method providesFRET events with low efficiency, FRET signals from Fos-Junprotein pairs, however, were clearly located in the nuclei ofthe cells.

P-331Chromosome imaging: fluorescence in situhybridisation (FISH) in human peripheral bloodlymphocytesA. Durgaryan1, S. Sommer21H.Buniatian Institute of Biochemistry, Yerevan, Armenia,2German Research Centre for Heavy Ion Physics (GSI),Darmstadt, Germany

Fluorescence in situ hybridisation (FISH) relies on thehybridisation of labelled DNA probes in a complementaryway and allows direct visualization of chromosome region ofinterest. Study aimed to describe FISH technique applicationin human peripheral blood lymphocytes for further use inradiobiological investigations. The whole chromosome 2XCPprobe for chromosome 2 (MetaSystems, Germany), 6q21/Alphasatellite Cocktail probe for chromosome 21 telomericand centromer region (Q-biogene) and Telomere PNA Probe/Cy3 (Dako) for telomeric regions were applied on lympho-cytes slides. Samples were counterstained with DAPI +Antifade solution. Localisations of above mentioned specificprobes were analyzed with Olympus fluorescent microscopeand ISIS software (MetaSystems, Germany) on 100 metha-phases for each samples using DAPI and FITC filters. Cellswith good signals for each of applied probes were scored. Itwas showed that 2XCP probe for chromosome 2 and Telo-mere PNA Probe/Cy3 for telomeric regions worked veryefficiently and could be recommended for further analysis.Most of the cells hybridised with 6q21/Alphasatellite Cocktailprobe had splited signal, especially in case of green signal(centromer region) and eventual modification of protocols forthis type of cells may be proposed.

P-332Visualization of subcellular localization of pro/anti -apoptotic factors after Hypericin induced photodynamictreatmentL. Dzurova1, V. Huntosova1, D. Petrovajova1,F. Sureau2, P. Miskovsky1, Z. Nadova11Department of Biophysics P. J. Safarik University, Kosice,Slovakia, 2ANbioPhy CNRS UMR 7033, University Pierreand Marie Curie, Paris, France

Hypericin (Hyp) is a highly hydrophobic plant pigment withphotosensitizing properties. Hyp localization inside cell plays

a key role of its photoactivity, due to the singlet oxygenproduction, which has a very short lifetime (ns-ls, dependingof Hyp environment). Hyp sub-cellular localization dependson its concentration in the medium, incubation time and useddelivery system.Variations in activity of Protein Kinase C, (anti-apoptoticPKCa and pro-apoptotic PKCd) in correlation with the activityof Bcl-2 protein, cytochrome c release from mitochondria anddecreasing of mitochondrial membrane potential after pho-todynamic action were monitored. The study was performedfor two different delivery modes of Hyp to U- 87MG gliomacells- Hyp alone (membrane diffusion) vs. Hyp loaded in lowdensity lipoprotein (LDL) (endocytosis). Confocal fluores-cence microscopy, flow-cytometry and specific fluorescencelabeling were used as main experimental techniques. Ourresults show that Hyp photoaction strongly affects apoptoticresponse of the cells and that the dynamics of this actionsignificantly depends on used delivery system. Correlationanalysis between the monitored parameters (see above)determined for both delivery system is presented and criti-cally discussed.

AcknowledgementsThis work was supported by the Slovak Research andDevelopment Agency under contract num. APVV-LPP-0072-07, by the Scientific Grant Agency of the Ministry of Educa-tion of Slovak VEGA 1/0241/09, by the projects ’’Center forexcellent research of atherosclerosis’’ (034/2009/2.1/OPR&D) (20%) and ’’Network of excellent laboratories inoncology’’ (26220120024) (20%) Operation ProgrammeResearch and Development funded by European RegionalDevelopment Fund.

P-333Correlative dynamic optical microscopy(TL, FRAP, FCS, FLIM) to decipher medicalbiofilm reactivitySamia Daddi Oubekka1,2, Arnaud Bridier3, RomainBriandet3, Karine Steenkeste1,2, Marie-Pierre Fontaine-Aupart1,21Institut des Sciences Moleculaires d’Orsay (ISMO), CNRSUMR 8214, Univ Paris-Sud, Bat.210, F-91405 Orsay, France,2Centre de Photonique Biomedicale, Federation LUMAT, FR2764, Univ Paris-Sud, Bat. 106, F-91405 Orsay c-, France,3INRA, UMR1319 MICALIS, F-91744 Massy cedex, France

Surface contamination by bacteria is a natural and sponta-neous process occurring in any natural environment onbiotic (mucosa, tissues…) and abiotic surfaces (medicalequipments, food surfaces…). Whatever the bacterial nature(Gram-positive or -negative), the environmental fluid (air,water, blood…) and the receptor surface (implants, medicalequipments, food surfaces…), the surface contaminationinitiated by the first adherent bacteria can evolve to a threedimensional structure named biofilm (cohesive bacteriaassembly ensured by an autoproduced extracellular organicmatrix). The mechanisms by which these biofilms offer pro-tective environment to viral particles or hypertolerance toantimicrobial action are not yet elucidated. To reach a betterunderstanding of biofilm reactivity, we reported for the firsttime successful applications of correlative time-resolvedoptical microscopy approach by time-lapse (TL), FRAP, FCS,

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FLIM, for real-time analysis of molecular mobility and reac-tion inside biofilms. By means of non-biological or biological(virus, biocides and antibiotics) reactive compounds, signifi-cant advances to understand the roles of the extracellularmatrix and bacteria physiological properties were obtained,an important step to improve pathogenic biofilm inactivation.

P-334Towards skin cancer diagnostics by two-photonmicroscopyAlbrecht Haase1, Francesco Tessarlo1, Claudia Caldini1,Paolo Bauer2, Guiseppe Zumiani2, Sebastiana Boi3, RenzoAntolini11BIOtech Research Centre and Physics Department,University of Trento, Mattarello, Italy, 2Departmentof Dermatology, APSS, S. Chiara Hospital, Trento, Italy,3Department of Histopathology, APSS, S. Chiara Hospital,Trento, Italy

Here we present a feasibility study to develop two-photonmicroscopy (2PM) into a standard diagnostic tool for non-invasive, skin cancer diagnosis. The goal is defining experi-mental parameters to maximize image quality of opticalbiopsies in human skin, while avoiding tissue damage.Possible diagnostic indicators will be compared for healthytissue, benign, and malignant melanocytic lesions.We report on preliminary results of a study on 2PM imagingof ‘‘ex-vivo’’ biopsy samples, were autofluorescence inten-sity and contrast between lesion and surrounding tissueswere optimised varying excitation wavelength, detectionband, and dispersion pre-compensation. Moreover, wedetermined modulation functions for laser power anddetector gain to compensate losses in deep tissue imaging.As the main process of photo-damage, thermo-mechanicalmodifications were quantified and damage threshold powerswere determined.In order to image structural changes in ordered tissue likecollagen fibres, second-harmonic generation signals wererecorded and optimised.

P-335In-vivo two-photon imaging of the honeybee antennallobeAlbrecht Haase1, Elisa Rigosi2,3, Gianfranco Anfora3, ClaudioVinegoni4, Giorgio Vallortigara2, Renzo Antolini11BIOtech Research Centre and Physics Department,University of Trento, Mattarello, Italy, 2CIMeC, Centrefor Mind/Brain Sciences, University of Trento, Rovereto (TN),Italy, 3IASMA Research and Innovation Center, FondazioneE. Mach, San Michele all’Adige, Italy, 4Center for SystemsBiology and Center for Molecular Imaging Research,Massachusetts General Hospital, Harvard Medical School,Boston, USA

We adapted a two-photon microscope for in-vivo imaging ofthe honeybee olfactory system, focusing on its primarycentres, the antennal lobes. The setup allowed obtainingboth 3D-tomographic measurements of the antennal lobe

morphology and time-resolved in-vivo calcium imaging of itsneuronal activity.The morphological data were used to precisely measure theglomerular volume in both sides of the brain, investigating thequestion of volumetric lateralization. Functional calcium imag-ing allowed to record the characteristic glomerular responsemaps to external odour stimuli applied to the bees’ antennae.Compared to previous neural imaging experiments in thehoneybee, this work enhanced spatial and temporal resolution,penetration depth, and it minimized photo-damage. Final goalof this study is the extension of the existing functional atlases ofthe antennal lobe to 3D and into the temporal dimension byinvestigating the time-resolved activity pattern.

P-336Fluorescent analysis of apoptosis inducedby Pseudomonas aeruginosa in endothelial cellsF. Iordache1, C. Iordache2, M. Lixandru, 2A. Holban3,C. Bleotu3, V. Lazar3, C. Chifiriuc3, H Maniu11Institute of Cellular Biology and Pathology ‘‘NicolaeSimionescu’’, Bucharest, Romania, 2National Institutefor Research and Development in Microbiologyand Immunology Cantacuzino, Romania, 3Faculty of Biology,University of Bucharest, Romania

A consequence of invasion by an intracellular pathogen isapoptosis of the host cell, usually observed in phagocyticcells. The aim of this study was to evaluate if Pseudomonas(P.) aeruginosa, although not considered a classic intracel-lular pathogen, could induce apoptosis in endothelial cells(EC). Strains were evaluated for virulence factors expression:pore forming toxins (haemolysins, lecithinases, lipases),proteases (caseinases, gelatinasse), DNA-ses, mucinases.The adherence and invasion capacity of P. aeruginosa to ECwas done using Cravioto’s method. For detection of apoptosiswas used acridine orange/ethidium bromide staining. Thequalitative assay of bacterial adherence to cellular substratereveled that all tested strains adhered to EC, exhibiting adiffuse, aggregative or mixed diffuse-aggregative pattern and20-70% adherence rates. Cell free culture of P. aeruginosadid not determine a cell death response, but in case of wholebacterial culture, acridine orange/ethidium bromide stainingshowed the capacity to induce apoptosis of EC, fluorescentmicroscopy revealed the chromatin condensation, frag-mented nuclei, membrane blebbing, and apoptotic bodies. P.aeruginosa have the ability to adhere and invaded EC and toinduce morphological changes, including apoptosis.

P-337Photo-toxicity of near-infrared voltage sensitivefluorescent dyesGiedrius Kanaporis1, Jonas Jurevicius1, Irma Martisiene1,Ruta Vosyliute1, Antanas Navalinskas1, Rimantas Treinys1,Arvydas Matiukas2, Arkady M. Pertsov21Lithuanian University of Health Sciences, Kaunas,Lithuania, 2SUNY Upstate Medical University, Syracuse,NY, USA

The use of voltage sensitive fluorescent dyes (VSD) fornoninvasive measurement of the action potential (AP) in

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blood perfused heart have been hindered by low interroga-tion depth, high absorption and auto-fluorescence of cardiactissue. These limitations are diminished using new nearinfrared (NIR) VSD (di-4-ANBDQBS). Here we validatedtoxicity and photo-toxicity of these dyes in guinea pig andhuman cardiac muscle slabs.Application of NIR VSD showed no effect on cardiac musclecontraction force or relaxation. Optical action potentialsclosely tracked kinetics of microelectrode recorded APs inboth field and electrode stimulated preparations. For photo-toxicity assessment dye (50 lM) preloaded cardiac slabswere exposed to prolonged laser radiation of various power.Microelectrode AP recordings show that exposure to pro-longed laser radiation (10min.; 2mW/mm2) of dye loadedtissue had no statistically significant effect on APD50 orconduction velocity, thus indicating no or weak photo-tox-icity on the NIR VSD. In contrast, exposure to 5 min. laserradiation of phototoxic dyes (mitotracker deep-red) pre-loaded tissue caused significant reduction in APD50 (by13%) and conduction velocity (30%). Thus, due to the lowphoto-toxicity, NIR VSD are well suited for in vivo cardiacimaging.

P-338Morphological analysis of spores with variousmicroscopic techniques in Streptomyces coelicolorK. Kovacs1,2, G. Madar1, L. Matyus1, A. Jenei1, A. Penyige21University of Debrecen, Department of Biophysics and CellBiology, Hungary, 2University of Debrecen, Departmentof Human Genetics, 4032 Debrecen, Egyetem ter 1, Hungary

Streptomycesetes are filamentous Gram-positive soil bacte-ria well known for their complex morphological developmentand secondary metabolite production. During their life cyclespores germinate to form a network of hyphae, which laterdevelops into aerial mycelium when cross-walls are gener-ated and spores are formed. We have examined andcompared the last stage of the differentiation process in awild-type S. coelicolor (M145) and its DcabBmutant lacking acalmodulin-like calcium binding protein.The strains were grown on four kinds of media: SMMS,SMMS with 10 % saccharose, R5 and R5 with reduced cal-cium in order to study the effect of environment and osmoticstress on the sporulation of the two strains to assess thefunction of CabB protein.From the cultures pictures were taken at 48 hours and after 7days using phase contrast, atomic force and confocal laserscanning microscope and the sizes of spores weremeasured.Our results showed that the DcabB mutant made smallerspores, its differentiation and stress response were slower.We could conclude from it that the aberrant protein slows themetabolism, the signal transduction and has effects onsporulation, septation and air-mycelium formation. Based onit we can tell that the CabB has a significant role in normaldevelopment.

P-339Implementation of dual auto- and cross correlationfunction calculation for FCS/FCCS measurementsby FPGABalazs Kreith, Gabor Mocsar, Sandor Damjanovich,Gyorgy VamosiBiology and Signaling Research Group of the Hung. Acad.Sci. and Dept. of Biophysics and Cell Biology, Medicaland Health Science Center, Univ. of Debrecen, Hungary

Fluorescence correlation and crosscorrelation spectroscopyhave become widely used techniques for studying the diffu-sion and interactions of molecules. From a combination ofautocorrelation (ACF) with dual color crosscorrelation func-tions (XCF) a more complex picture can be gained about thediffusing species and their interactions. Most hardware cor-relator cards lack the capacity to calculate auto- andcrosscorrelation functions and store raw data simulta-neously. Here we present a new approach to calculate ACFsand XCFs online simultaneously by using the multiple tauand multiple sampling time algorithm implemented on a fieldprogrammable gate array (FPGA, National Instruments).Although the FPGA has strict source restriction, the numberof available correlation functions can be multiplied by usingthis new approach. Thus the dual ACF and dual XCF can becalculated for continuous illumination. For calculation ofthe desired correlation functions a series of interconnectedlinear correlator blocks were implemented. The first 16 cor-relator blocks had a minimal lag time of 50 ns, followed by 23blocks of 8 correlators with stepwise doubled sampling times.The new approach is based on the interleaved scheduling ofthe execution of linear correlator blocks.

P-340Single plane illumination fluorescence correlationspectroscopy using high speed image sensorsJan Krieger1,2, Jan Buchholz1,2, Agata Pernus1,2, LucioCarrara4, Edoardo Charbon3, Jorg Langowski11Equally contributing authors, 2German Cancer ResearchCenter, Biophysics of Macromolecules (B040), ImNeuenheimer Feld 580, D-69120 Heidelberg, Germany,3Technische Universiteit Delft, Mekelweg 4, 2628 CD Delft,The Netherlands, 4ESPROS, Zug, Switzerland

The mobility and reaction parameters of molecules insideliving cells can be conveniently measured using fluorescentprobes. Typically fluorescence correlation spectroscopy(FCS) based on confocal microscopy is used for such mea-surements. This implies high time-resolution but only for asingle spot at a time. In order to achieve a high time-reso-lution at multiple spots, we built a single plane illuminationmicroscope (SPIM) equipped with high-speed image acqui-sition devices and a high-NA detection optics. This allows usto do parallel FCS measurements in a thin plane (width *2-3 lm) of the sample. Our setup is equipped with a fastEMCCD camera (full frame time resolution 2000ls) and a32932 pixel array of SPADs. The SPAD array has a fullframe time resolution of 3-10ls, which is even fast enough to

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resolve the typical motion time-scale of small molecules (likeeGFP) inside living cells. The performance of the system ischaracterized by diffusion measurements of water-solublefluorescent beads, as well as FCS measurements in livingcells. Our data acquisition system uses programmablehardware for some tasks and is fast enough to allow real-timecorrelation of 1024 pixels, as well as saving the completedataset for later evaluation.

O-341Cryo-EM of membranes and membrane proteinsWerner KuhlbrandtMax-Planck-Institute of Biophysics, Max-von-Laue-Str.3,60438 Frankfurt am Main, Germany

Electron cryo-microscopy (cryo-EM) covers a larger size rangethan any other technique in structural biology, from atomicresolution structures of membrane proteins, to large non-crystalline single molecules, entire organelles or even cells.Electron crystallography of two-dimensional (2D) crystalsmakes it possible to examine membrane proteins in thequasi-native environment of a lipid bilayer at high to moder-ately high resolution (1.8-8A). Recently, we have usedelectron crystallography to investigate functionally importantconformational changes in membrane transport proteinssuch as the sodium/proton antiporters NhaA and NhaP, orthe structure of channelrhodopsin.‘‘Single particle’’ cryo-EM is well suited to study the structureof large macromolecular assemblies in the 3.3 to 20A reso-lution range. A recent example is our 19A map of amitochondrial respiratory chain supercomplex consisting ofone copy of complex 1, two copies of complex III, and one ofcomplex IV. The fit of the x-ray structures to our map indi-cates short pathways for efficient electron shuttling betweencomplex I and III by ubiquinol, and between complex III andIV by cytochrome c.Electron cryo-tomography can visualize large protein com-plexes in their cellular context at 30-50A resolution, and thusbridges the gap between protein crystallography and lightmicroscopy. Cryo-ET is particularly suitable for studying bio-logical membranes and large membrane protein complexesin situ. We found that long rows of ATP synthase dimers alongridges of inner membrane cristae are an ubiquitous feature ofmitochondria from all 6 species we investigated (2 mammals,3 fungi, 1 plant). The proton pumps of the respiratory chainappear to be confined to the flat membrane regions on eitherside of the ridges. This highly conserved pattern suggests afundamental role of the mitochondrial cristae as proton trapsfor efficient ATP synthesis.

References:

Single-particle analysis:Gipson P, Mills D, Wouts R, Grininger M, Vonck J, KuhlbrandtW (2010): Direct structural insight into the substrate-shuttlingmechanism of yeast fatty acid synthase by electron cryomi-croscopy. Proc Natl Acad Sci USA 107, 9164-9169Electron crystallography:Kuhlbrandt W, Wang DN, Fujiyoshi Y (1994): Atomic modelof plant light-harvesting complex by electron crystallography.Nature 367, 614-621.

Appel M, Hizlan D, Vinothkumar KR, Ziegler C and Kuhl-brandt W (2009): Conformations of NhaA, the Na+/H+

exchanger from Escherichia coli, in the pH-activated and ion-translocating states. J Mol Biol 388, 659-672.Electron cryo-tomography:Strauss M, Hofhaus G, Schroder RR, Kuhlbrandt W (2008):Dimer ribbons of ATP synthase shape the inner mitochon-drial membrane. EMBO J 27, 1154-1160.Daum B, Nicastro D, Austin II J, McIntosh JR, Kuhlbrandt W(2010): Arrangement of photosystem-II and ATP synthase inchloroplast membranes of spinach and pea. Plant Cell 22,1299-1312.

P-342Modeling the red blood cell shapes for holographicimagingMona Mihailescu1,2, Eugen Scarlat2, Mihai Kusko11National Institute for Research and Developmentin Microtechnologies (IMT-Bucharest), Bucharest, Romania,2Politehnica University of Bucharest, Bucharest, RomaniaE-mail: mona_m@physics.pub.ro

Digital holographic microscopy is a label free, noncontact,nonintrusive and nondestructive technique which givesquantitative information about phase shift introduced by thetransparent samples. From one single hologram, withoutmechanical scanning in the experimental setup, we recon-struct the amplitude and the phase, which allow 3D imagingof the sample with nanometer accuracy along propagationaxis. On the CCD camera, we record the diffraction pattern ofthe sample, superposed with the reference beam. The studyis focused on investigating the iso-intensity curves in thediffraction pattern coming from the phase-only transmissionfunctions which contain oblate spheroid models for the redblood cell (RBC) shapes. In simulations, the values intervalfor dimensions and refractive indices are in concordance withthe real ones, in different situations. The experimentally dif-fraction patterns in given conditions are obtained usingspatial light modulator. Results obtained in Fresnel approxi-mation will be used as preprocessing observations in ourmicrofluidic applications. Results obtained in Fraunhoferapproximation can be used in ektacytometry.This research is financed by the contract POSDRU/89/1.5/S/63700 and equipments is acquired by romanian contract4/CP/I/2007-2009 Capacities.

O-343Rational design and applications of photoconvertibleand bi-photochromic fluorescent proteinsBenjamien Moeyaert1, Virgile Adam1, Charlotte C. David1,Hideaki Mizuno1, Mickael Lelimousin2, Peter Dedecker1,Ryoko Ando3, Atsushi Miyawaki3, Jan Michiels1, YvesEngelborghs1 and Johan Hofkens11Katholieke Universiteit Leuven, Leuven, Belgium,2University of Oxford, Oxford, UK, 3RIKEN Brain ScienceInstitute, Wako-city, Japan

Advanced fluorescence imaging, including subdiffractionmicroscopy, relies on fluorophores with controllable emissionproperties. Chief among these fluorophores are the on/offphotoswitchable and green-to-red photoconvertible

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fluorescent proteins. IrisFP was recently reported as the firstfluorescent protein to combine irreversible photoconversionfrom a green-emitting to a red-emitting state with reversibleon/off photoswitching in both the green and red states. Theintroduction of this protein resulted in new applications suchas super-resolution pulse-chase imaging, but the propertiesof IrisFP are far from being optimal from a spectroscopic pointof view and its tetrameric organization complicates its use asa fusion tag. We have demonstrated how four-state opticalhighlighting can be rationally introduced into photoconvertiblefluorescent proteins by developing and characterizing a newset of such enhanced optical highlighters derived from mEo-sFP and Dendra2. One of these, which we called NijiFP, wasidentified as a promising new multi-photoactivatable fluores-cent protein with optical properties that make it ideal foradvanced fluorescence-based imaging applications.

P-344Synthesis and optical properties of ultrasmall inorganicoptical markers based on lanthanides emission for bio-medical applicationsA. Podhorodecki, M. Banski, J. MisiewiczInstitute of Physics, Wroclaw University of Technology,Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland

Introducing tomedicine and biology concept of opticalmarkersin tremendousway has changed the recent status of these twoimportant disciplines. This was mainly due to strong develop-ment in imaging techniques which recently allow us toinvestigate both static aswell dynamic properties of living cells,their components and their interactions with external factors.Recently used molecular markers including organic dyes,fluorescent proteins or chelates containing lanthanide ionshave several significant limitations. One of the alternativesfor molecular markers are inorganic quantum dots (ie. CdSe,CdS) which are recently commonly used in many academicworks. However, even if they are much better from physico-chemical point of view, from the application point of view atthis moment they are rather useless mainly because of theirhigh risk of toxicity. One of the solution combining advanta-ges of both concepts is to make nontoxic inorganicnanocrystals doped by lanthanide ions.In this work, we will present optical results obtained for NaYF4

nanocrystals doped by different lanthanide ions. The aim ofthisworkwas to design and to synthesize thesemarkers and tounderstand physical processes responsible for their emissionand to optimize these processes to the physical limits.

P-345Natural killer cells fate at the draining lymph nodes:a physical portrait of the biological contestL. Sironi1, M. Caccia1,2, N. Gritti1, T. Gorletta2, I. Zanoni2,C. Salvetti1, S. Pozzi1, S. Freddi1, S. Daglio1, C. E. Villa1,M. Collini1, L. D’Alfonso1, F. Granucci2, G. Chirico11Dep. Physics, Universita degli Studi di Milano-Bicocca, Italy,2Dept. Biotechnology and Biosciences, Universita degli Studidi Milano-Bicocca, Italy

Intravital microscopy has fostered a full blown of publicationregarding the behavior of cells in different tissues andphysiological conditions. However, a few papers describe

how the motility parameters can be used to understandwhether an interaction is occurring and, on balance, thedistinction between interacting and non interacting cells isperformed visually on the image time stack.Here we describe a multi-parameter approach that allows todiscern among different cell behaviors on an objective groundandwedemonstrate its effectiveness valuating themutual fateof Natural Killer (NK) and Dendritic (DC) cells at the draininglymph-nodes in inflammatory and stationary conditions.The method is time saving and allows a wide scale charac-terization of the lymphocyte tracks and to build up statistics ofthe cell-cell interaction duration. This has allowed thedevelopment of a numerical model of the NK-DC interaction,based on a molecular-stochastic dynamic approach, whoseoutput can be directly compared to the data. The Smolu-chowsky equation is resolved for two superimposed radialpotentials irradiating from the DC cells and describing theshort and long range DC-NK interaction mediated by differenttypes of interleukins.

P-346Time-resolved Raman imaging of malarial hemozoinNicholas Isaac Smith1, Katsumasa Fujita2, Kota Nishibe2,Satoshi Kawata2, Cevayir Coban31Biophotonics Laboratory, Immunology Frontier ResearchCenter (IFReC), Osaka University, Japan, 2Departmentof Applied Physics, Grad. School of Engineering, OsakaUniversity, Japan, 3Malaria Immunology Laboratory,Immunology Frontier Research Center (IFReC), OsakaUniversity, Japan

Hemozoin is formed during malaria infection of red bloodcells, the malaria parasite cleaves hemoglobin, leaving freeheme which is then toxic to the parasite. The free heme isthen bio-crystalized to form hemozoin which allows the par-asite to remain viable. The hemozoin is released during thebreakdown of the red blood cells, is small and can be difficultto resolve spatially. Since it contains an abundance of hemeprotein, which has a strong absorbance at 532 nm, it can bereadily detected and tracked by using resonant Ramanscattering spectroscopy. Here we use slit-scanning confocalRaman imaging to detect the hemozoin and resolve it againstthe background molecules. Inside a red blood cell, hemo-globin is the strongest background signal since it alsocontains large amounts of heme. Nevertheless, the discrim-ination is possible, and the time-resolved observation ofhemozoin is an important tool to understand effects ofmalaria since the hemozoin can trigger the immune responseand cause inflammation in tissue.

P-347Superresolution optical microscopy of isolated cardiacmitochondrial proteinsStefani Enrico, Singh Harpreet, Gardeazabal RodrıguezPedro F., Lu Rong, Bopassa Jean C., Wu Yong, andToro LigiaUniversity of California, Los Angeles, Los Angeles, CA90095-7115, USA

To study the structural organization of mitochondrial proteins,we applied Stimulated Emission Depletion (STED)

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microscopy in isolated mitochondria. In STED microscopy,two laser beams are used: one for excitation of fluorophoresand the other, with doughnut shape, to deplete them in orderto allow fluorescence emission only from the excited volumelocated at the doughnut’s center. Using a custom-madeSTED, we achieved a lateral resolution of *20 nm in imagesof purified mitochondria, where we investigated the locali-zation pattern and distribution of Cox 4 and VDAC1 proteins,and confirmed the existence of Na/Ca2+ exchanger. Resultsshow that Cox 4 proteins are distributed in clusters of *20and *28 nm; whereas VDAC1 displays four size distribu-tions of *22, *33, *55 and *83 nm. We havedemonstrated that protein clusters in the mitochondria can beresolved with a separation power of *20 nm, and that it ispossible to retrieve quantitative information about their clus-ter sizes. This approach can be extended to other proteins inmitochondria and subcellular organelles and to investigatedisease-induce changes in mitochondrial protein distribution.Supported by NIH and AHA.

O-349Integrin-dependent activation of the JNK signalingpathway in live cells by mechanical stressAndrea Maria Pereira1,3, Cicerone Tudor2,3, Johannes S.Kanger2, Enrique Martin-Blanco1 andVinod Subramaniam2

1Instituto de Biologıa Molecular de Barcelona (CSIC), ParcCientific de Barcelona, Spain, 2Nanobiophysics, MESA+Institute for Nanotechnology & MIRA Institute for BiomedicalTechnology and Technical Medicine, University of Twente,Enschede, The Netherlands, 3These authors contributedequally to the reported work

Mechanical force is known to modulate the activity of the JunN-terminal kinase (JNK) signaling cascade. However, it is notknown how this activation is regulated in vivo. We assessedin real-time the activity of the JNK pathway in DrosophilaS2R+ cells by Fluorescence Lifetime Imaging Microscopy(FLIM) using an intramolecular phosphorylation-dependentdJun-FRET (Fluorescence Resonance Energy Transfer)biosensor. We found that Drosophila S2R+ cells plated ondifferent substrates showed distinct levels of JNK signalingthat associate with differences in cell morphology, stiffness,and integrin and focal adhesion organization. QuantitativeFRET-FLIM analysis also revealed the sustained activationof the JNK pathway in response to multidirectional staticstretch. Importantly, this was contingent on the presence offunctional integrins in focal adhesions but not on Talin. Incontrast, at rest, both integrins and Talin suppressed JNKactivation. Our results showing sustained JNK activation andstable cell morphology changes in response to mechanicalstretch suggest that alterations in the cytoskeleton andmatrix attachments may act as regulators of JNK signalingand that this property itself might feed back to modulate celladhesion and the cytoskeleton. This dynamic system ishighly plastic; at rest focal adhesions and Talin are keyfactors in modulating JNK activity, while mechanical stretchleads to a Talin-independent JNK activation. The level ofJNK activity seems to be a major element, but not the onlyone, in the regulation of the cytoskeleton and cell shaperemodeling associated with stretch. Finally, in combination

with RNA silencing experiments we have been able to dis-sect the signal transduction pathway for mechanical stressin this system.

O-350Direct mapping of nanoscale compositional connectivityon intact cell membranesThomas S. van Zanten1, Jordi Gomez2, Carlo Manzo1,Alessandra Cambi3, Javier Buceta4, Ramon Reigada2, MariaF. Garcia-Parajo11BioNanoPhotonics group, IBEC-Institute for Bioengineeringof Catalonia, Barcelona, Spain, 2Departament de Quımica-Fısica, Universitat de Barcelona, Barcelona, Spain,3Department of Tumor Immunology, NCMLS, RadboudUniversity Nijmegen Medical Center, Nijmegen, TheNetherlands, 4Computer Simulation and Modeling(Co.S.Mo.) Lab, Barcelona Science Park, Barcelona, Spain

Lateral segregation of cell membranes is accepted as a mainmechanism for cells to regulate a diversity of cellular func-tions. In this context, lipid rafts have been conceptualized asorganizing principle of biological membranes where under-lying cholesterol-mediated selective connectivity must existat the resting state. However, such a level of nanoscalecompositional connectivity has been challenging to prove.We used single-molecule near-field scanning opticalmicroscopy (NSOM) to visualize the nanolandscape of raftganglioside GM1 after tightening by its ligand cholera toxin(CTxB) on intact cell membranes. We show that CTxBtightening of GM1 is sufficient to initiate a minimal raft coa-lescence unit, resulting in the formation of cholesterol-dependent GM1 nanodomains \120nm in size. Simulta-neous dual color high-resolution images revealed that GPIanchored proteins were recruited to regions proximal(\150nm) to CTxB-GM1 nanodomains without physicalintermixing. Together with in-silico experiments, our high-resolution data conclusively demonstrate the existence ofraft-based interconnectivity at the nanoscale. Such a linkedstate on resting cell membranes constitutes thus an obliga-tory step towards the hierarchical evolution of large-scale raftcoalescence upon cell activation.

P-351X-ray spectro-microscopy at ID21: mapping of elementsand chemical states at subcellular levelG. Veronesi1, M. Salome1, B. Fayard1,2, H. Castillo-Michel1,E. Koudouna3, A. Quantock3, M. Cotte1,41European Synchrotron Radiation Facility, B.P.220, 38043Grenoble Cedex, France, 2Laboratoire de Physique desSolides, UMR 8502- Bat 510, Universite Paris Sud, 91405Orsay cedex, France, 3Structural Biophysics Group, Schoolof Optometry and Vision Sciences, Cardiff University, CardiffCF24 4LU, Wales, UK, 4C2RMF, CNRS UMR 171, Palais duLouvre, 14, Quai F. Mitterrand, F-75001 Paris, France

Microscopy techniques at sub-micron resolution are key toolsfor research in life sciences, and one of their most desirablefeatures is elemental sensitivity. The beamline ID21 of the

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European Synchrotron Radiation Facility [1] hosts a Scan-ning X-ray Microscope (SXM) for X-ray fluorescence spectro-microscopy: its relatively wide energy range (2-9.2 keV) andthe small X-ray beam size (0.3x0.7 lm2, achieved by meansof Zone Plate or Kirk-Patrick Baez mirror focusing) allow toimage elemental distributions at cellular and sub-cellularresolution, with sensitivity to trace elements down to a fewppm. Furthermore, the chemical state of a selected elementcan be determined by means of micro-X-ray-AbsorptionSpectroscopy (lXAS).Molecular mapping with *5lm lateral resolution can aswell be achieved on ID21, thanks to a Synchrotron-Radi-ation (SR) FTIR-microscope: the combination of SXM andSR-FTIR provides a set of complementary informationnecessary to investigate the physics and chemistry ofbiosystems.The experimental capabilities of the beamline and its ver-satility (wide variety of sample environments and detectionsystems) will be presented, together with examples ofapplications, notably to stem cells research.

[1] M.Salome et al (2009), J. Phys.: Conf. Ser. 186, 012014

P-352Inside the polyribosome by super-resolutionmicroscopiesG. Viero1, L. Lunelli2, P. Bianchini3, N. Arseni1,A. Diaspro3, R. Gilbert4, C. Pederzolli2, A. Quattrone11Laboratory of Translational Genomics, Centre for IntegrativeBiology, University of Trento Via delle Regole 101 38123Mattarello, Italy, 2Center for Materials and Microsystems,Fondazione Bruno Kessler, Povo (Trento), Italy,3Nanophysics, IIT–Italian Institute of Technology, 16163Genova, Italy, 4Division of Structural Biology, HenryWellcome Building for Genomic Medicine, Oxford, OX3 7BN,UK

During translation initiation, the 5’ cap and the 3’ poly(A) tailof a eukaryotic mRNA are recognized by eIF4E and PABP(poly(A)-binding protein), respectively. Both yeast and humaneIF4E interact with eIF4G, eIF4A and eIF4B forming theeIF4F complex. This 5’ cap complex cooperates with thepoly(A) tail to promote a functional circularization of mRNA,which is necessary for efficient translation by bridging of its 5’and 3’ termini by the protein-protein interaction. Despitebiochemical and genetic evidences demonstrate the inter-action of eIF4G and Pab1, up to now the imaging informationon these proteins and their relationship with the mRNA, thenative closed-loop and the organization of the UTRs in thepolysome are completely lacking. Here, we focus on thesefundamental topics by integrating three different high reso-lution imaging approaches by comparing images of nativehuman polysomes obtained by Atomic Force Microscopy,cryo-EM and STED microscopy.

P-353Optical signal interrogation depth in Tyrode and blood-perfused myocardial tissueGiedrius Kanaporis1, Jonas Jurevicius1, AntanasNavalinskas1, Ruta Vosyliute1, Arvydas Matiukas2,Bogdan G. Mitrea2, Arkady M. Pertsov21Lithuanian University of Health Sciences, Kaunas,Lithuania, 2SUNY Upstate Medical University, Syracuse,NY, USA

Photon attenuation length (d) in biological tissues determinesinterrogation depth, spatial resolution, and amplitude offluorescence signal in various types of optical imaging,including imaging of cardiac excitation using voltage-sensi-tive dyes. We assessed d in human and pig myocardium atexcitation/emission wavelengths of commonly used andrecently developed near-infrared voltage-sensitive dyes(NIR). We also compare d in Tyrode vs blood-perfused tis-sues and simulate respective fluorescent signals in thecontext of potential clinical applications.Experiments were conducted in isolated slabs of ventricularmyocardium. Light decay inside the tissue was measured at520, 650, and 715nm. d in blood-perfused porcine andhuman myocardium were similar (at 520nm 0.46±0.01 mmand 0.48±0.02 mm; at 650nm 1.83 ± 0.02 mm and 1.98 ±0.07 mm, at 715nm 2.31 ± 0.05 mm and 2.74 ± 0.13 mm inporcine and human respectively), which makes porcinemyocardium a good model for development of clinicalimaging applications. Blood- vs Tyrode perfusion signifi-canctly reduces d, particularly at 520nm. However, oursimulations show that, the resulting reduction of opticalaction potential is\16% (for NIR dyes), which would not be amajor impediment for in vivo imaging of cardiac excitation.

Molecular motors

O-354Myosin on an optical leash: load dependenceof the working stroke in a single myosin headMarco Capitanio, Francesco Pavone, Roberto Bottinelli,Monica Canepari, Manuela Maffei, Diego BenventiLENS-European Laboratory for Non-Linear Spectroscopy,Florence, Italy

Muscle performance at the molecular level is determined bythe elementary displacement (working stroke) produced bythe motor protein myosin II, and its dependence on load. Wedeveloped a laser trap assay (the optical leash) capable ofapplying controlled loads to a single myosin head before theworking stroke is initiated and probing actin-myosin interac-tion on the microsecond time scale. We found that theworking stroke size depends both on load and on thedetachment pathway followed by myosin. In a first pathway,myosin detaches very rapidly from actin (\1ms) withoutproducing any movement. In a second pathway, myosinsteps and remains bound to actin for a time inversely

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proportional to ATP concentration; the working strokeremains constant (*5 nm) as the load is increased, until itsuddenly vanishes as the isometric force is reached(5.7±0.6pN). A third dissociation pathway becomes morepopulated as the force is increased, due to prematureunbinding of myosin from actin, resulting in a working strokethat decreases with load. Taken together, these results give anew insight into the molecular mechanism of load depen-dence of the myosin working stroke, which is a primarydeterminant of skeletal muscle performance and efficiency.

P-355Mechanism of DNA sequence recognition by the SpoIIIEtranslocaseDiego Cattoni and Marcelo NollmannCentre de Biochimie Structurale, INSERM U554, Universitede Montpellier, CNRS UMR 5048, 29 rue de Navacelles,34090 Montpellier Cedex, France

During cell division, chromosomal DNA must be segregatedrapidly and accurately. In bacteria, chromosomal segregation isconductedbyDNA translocases,e.g.FtsK inEscherichia coliorSpoIIIE in Bacillus subtilis. Sporulating B. subtilis dividesasymmetrically. To assure the genetic material integrity aftersporulation, SpoIIIE assembles at the division septum andtranslocate the 2/3 of the chromosome (*3 Mbp) remaining inthe mother cell towards the forespore by using the energy ofATP hydrolysis converted into mechanical force by their AAA+motor domains. Several mechanisms have been proposed inorder to understand the determinants of translocation direc-tionality of DNA translocases. It is accepted that direction oftranslocation issignedbyspecificandasymmetrical 8 basepairchromosomal sequences, but how these enzymes recognizethis specific sequences and what is their role in activity anddirectionality regulation still generates controversy. Here, wecombine information from SpoIIIE/DNA equilibrium and pre-equilibriumbindingassayswith continuous triplexdisplacementmeasurements to further elucidate the mechanism of SpoIIIEsequence recognition and its effect on transport directionality.

O-356Rotation of F1-ATPase with a protrusion-less gammarotorRyohei Chiwata1, Ayako Kohori1, Tomoya Kawakami1,Mohammad Delawar Hossain1,2, Shou Furuike1, KatsuyukiShiroguchi1, Kazuo Sutoh1, Masasuke Yoshida3,4, KazuhikoKinosita Jr. 1

1Department of Physics, Faculty of Science and Engineering,Waseda University, Tokyo, Japan, 2Department of Physics,School of Physical Sciences, Shahjalal University of Scienceand Technology, Sylhet, Bangladesh, 3ATP SynthesisRegulation Project, ICORP, Japan Science and TechnologyAgency (JST), Tokyo, Japan, 4Department of MolecularBioscience, Kyoto Sangyo University, Kyoto, Japan,Dr.ShouFuruike’spresentaddress isFaculty ofPhysics,OsakaMedical College, Osaka, Japan, Dr. Katsuyuki Shiroguchi’spresent address is Department of Chemistry and ChemicalBiology, Harvard University, Cambridge, MA, USA

F1-ATPase is an ATP-driven rotary motor in which the centralc subunit rotates inside a stator made of an a3b3 ring. ATP

hydrolysis in the b subunits generates torque for c rotationby a mechanism not fully understood. The c rotor consists ofa globular domain that protrudes from the stator and aslender shaft that penetrates the a3b3 ring. The shaft is ananti-parallel coiled coil of the N- and C- terminal helices of c.Previously we have deleted either or both of these terminalhelices genetically. Surprisingly, all mutants rotated in thecorrect direction, showing that the shaft portion is dispens-able. Here we inquire if the rest of the c rotor, the globularprotrusion that accounts for *70 % of the c residues, is alsodispensable. Keeping the N- and C-terminal helices thatconstitute the shaft, we have replaced the middle *200residues with a short helix-turn-helix motif borrowed from adifferent protein. The protrusion-less mutant thus maderetained a high ATPase activity and rotated fast in the cor-rect direction. This may not be unexpected because, incrystal structures, most of the removed residues do notcontact with the a3b3 ring. Combined with the previousresults, however, the present results indicate that none ofthe c residues are needed for rotation.

P-357The rotary mechanism of a molecular engine,the vacuolar proton-ATPase, workingin a biomembraneCsilla Ferencz1, Pal Petrovszki1, Zoltan Kota1, ElfriedaFodor1, Andras Der1, Lajos Haracska2, Ferhan Ayaydin3,Zoltan Varga4, Attila Bota4 and Tibor Pali11Institute of Biophysics, Biological Research Centre, Szeged,Hungary, 2Institute of Genetics, Biological Research Centre,Szeged, Hungary, 3Institute of Cellular imaging laboratory,Biological Research Centre, Szeged, Hungary;4ChemicalResearch Centre, Budapest, Hungary

The rotary mechanism of vacuolar proton-ATPase (V-ATP-ase) couples ATP hydrolysis and trans-membrane protontranslocation. We tested the effect of oscillating electric (AC)field on V-ATPase activity in yeast vacuoles. The AC tech-nique has several advantages over direct observations: itcan be applied on native membranes, there are no labels andattachments involved, and the target protein is in its naturalenvironment. This was/is the first of its kind of experiment onV-ATPase, and we got strikingly different results from previ-ous studies on other proteins: Both low and high frequencyAC field reduces ATPase activity in a wide frequency range.A sharp resonance is seen at 88.3 Hz, where the ATPaseactivity reaches or exceeds the control (no AC) level. Wethink that the resonance happens at that of the 60 degreesrotor steps, meaning that the rotation rate of the rotor isaround 15 Hz, under the given conditions. Synchronisation ofindividual ATPases by slow or matching, but not fast AC islikely via a hold-and-release mechanism. We can explain theabove observations by assuming that the AC field interactswith the proton movements, and if we consider the estimatedgeometry of the hydrophilic proton channels and the protonbinding cites on the rotor.

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P-358The effect of stretch on phosphate release in skeletalmuscle fibresM.A. Ferenczi, D.C Bickham, T.G. West, R.C. Woledge,N.A. CurtinMolecular Medicine Section, NHLI, Imperial College London,London SW7 2AZ, UK

We applied ramp stretches to contracting permeabilizedfibres of mammalian skeletal muscle at 12 and 20�C, with aview to better understand the relationship between force andthe elementary steps of the actomyosin ATPase. Using afluorescent, genetically engineered phosphate binding pro-tein to report on phosphate (Pi) release resulting from ATPhydrolysis, we determined the time course of Pi releaseduring 1) development of isometric force, 2) a ramp stretchapplied when the isometric force plateau has been reached,3) an isometric phase after stretch and 4) reversal of thestretch. The results show that the rate of Pi release rapidlydecreases upon the application of stretch, with observablechanges in Pi occurring as quickly as those observed in theforce signal. At the end of the stretch, force decreases whilstthe rate of Pi release accelerates. During ramp shortening,force decreases to the level expected from the force-velocityrelation, and the rate of Pi release accelerates as ATPhydrolysis fuels the exertion of power by the muscle fibre.The rapid decrease in the rate of Pi release upon stretch isexplained by a redistribution of cross-bridge states toenhance Pi-containing intermediate states at the expense ofintermediate states containing ADP only.

P-359Stepping rotation in H+-ATPase/synthase has beenrevealed with an essentially drag-free probeShou Furuike1,2, Masahiro Nakano3, Kengo Adachi1, HiroyukiNoji3, Kazuhiko Kinosita Jr. 1 and Ken Yokoyama41Waseda Univ., Tokyo, Japan, 2Osaka Medical College,Osaka, Japan, 3Osaka Univ. Osaka, Japan, 4Kyoto SangyoUniv., Kyoto, Japan

Vacuole-type ATPases (VoV1) and FoF1 ATP synthasesconstitute a superfamily of rotary molecular machines thatcouple ATP hydrolysis/synthesis in the soluble V1/F1 portionwith proton flow in the membrane-embedded Vo/Fo portion,by sharing one joint rotary shaft. Here we have compared atsubmillisecond resolutions the ATP-driven rotation of isolatedV1 and the whole VoV1 derived from Thermus thermophilus,using a 40-nm gold bead for which viscous drag is almostnegligible. At saturating ATP of 4 mM, V1 rotated at *60revolutions per second, with *5 ms dwells every 120�. Dwelltime analyses indicated that at least two events other thanATP binding, one likely ATP hydrolysis, occur in each dwell,as in F1. Unlike F1, however, the dwells were at ATP-waitingpositions that were resolved at lM ATP. VoV1 rotated anorder of magnitude slower, and exhibited dwells separated by*30�. The twelve positions, though not always fully popu-lated, match the twelve-fold symmetry of the Vo rotor in T.thermophilus, indicating that the ATP-driven rotation must gothrough stator-rotor interactions in Vo, possibly coupled withproton translocation. We plan to examine the pH depen-dence of the dwells to see if they are indeed coupled toprotonation/deprotonation.

O-360How myosin traps and how actin triggers the releaseof phosphate at the beginning of force generationTatiana Isabet1, Paola Llinas1, Lin Song2, Allan Zong2,Olena Pylypenko1, Julie Menetrey1, H. Lee Sweeney2

and Anne Houdusse11Institut Curie/CNRS, France, 2University of PennsylvaniaSchool of Medicine, USA

Coupling structural and kinetic studies has elucidated detailsof a subset of the transitions of the actin-myosin catalyticcycle, including the ATP-induced dissociation of myosin fromactin and for the ATP hydrolysis step that allows priming ofthe lever arm. Not yet visualized however are the structuralrearrangements triggered by actin binding that are coupled toforce generation and product release. While phosphate isproduced in the pre-powerstroke (ADP.Pi) state, it is alsotrapped in this state until an actin-activated transition opens aback-door to promote its release from the active site. It isunclear, however, which of the three elements of the activesite (Switch I, Switch II and the P-loop) play a role in thistransition and whether the major cleft within the motor par-tially closes to allow this release while keeping the lever armprimed. New structures of the myosin VI motor in the pre-powerstroke state with Pi trapped reveal the post-hydrolysisstate that traps the motor. We have also crystallized a newstructural state for myosin VI in the presence of MgADP andphosphate that has an open back-door due to a Switch IIrearrangement and a lever arm primed. Thus it has theexpected features of the previously unseen Pi release stateof myosin. Kinetic and structural studies on a number ofmutants from different myosins are currently being performedto evaluate this possibility. The structure is consistent withcleft closure occurring subsequent to Pi release, which isalso being tested. An interesting finding in this new structureis that the SH1 helix kinks to allow the converter to stay in theprimed position, the signature of a state at the beginning offorce production.

O-361Assembly and function of the type 3 secretion systemneedle from Salmonella typhimuriumM. KolbeMax-Planck-Institute for Infection Biology, CellularMicrobiology, Chariteplatz 1 10117 Berlin, Germany

Pathogenic Gram-negative bacteria including Salmonellatyphimurium, Shigella flexneri and Yersinia pestis use a TypeThree Secretion System (TTSS) to deliver virulence factorsinto host cells[1] [2]. The TTSS constitutes a continuous pro-tein transport channel of constant length through thebacterial envelope[3]. The needle of the Type Three SecretionSystem is made of a single small protein (protomer). Weanalyzed the assembly and the structure of the TTSS needleusing different biophysical methods including Fourier Trans-form-Infrared Spectroscopy, nmR spectroscopy and X-raycrystallography. We show that the TTSS needle protomerrefolds spontaneously to extend the needle from the distalend. The protomer refolds from a-helix into b-strand confor-mation to form the TTSS needle[4]. Regulated secretion ofvirulence factors requires the presence of additional protein

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at the TTSS needle tip. X-ray crystal structure analysis of thetip complex revealed major conformational changes in boththe needle and the tip proteins during assembly of the S.typhimurium TTSS. Our structural analysis provides the firstdetailed insight into both the open state of the TTSS needletip and the conformational changes occurring at the patho-gen-host interface[5].

[1] J. E. Galan, H. Wolf-Watz, Protein delivery into eukary-otic cells by type III secretion machines. Nature 444,567-573 (2006).

[2] M. Lunelli, R. K. Lokareddy, A. Zychlinsky, M. Kolbe,IpaB–IpgC interaction defines binding motif for type IIIsecretion translocator. Proc. Natl. Acad. Sci. 106,9661-9666 (2009).

[3] T. C. Marlovits et al., Structural Insights into theAssembly of the Type III Secretion Needle Complex.Science 306, 1040-1042 (2004).

[4] O. Poyraz et al., Nat. Stuct. & Mol. Biol. 17, 788-792(2010).

[5] M. Lunelli et al., submitted.

P-362Binding change mechanism of membrane-bound Fo

rotary motor driven by proton motive forceJung-Hsin Lin 1,2,3

1Division of Mechanics, Research Center for AppliedSciences, Academia Sinica, Taiwan, 2Institute of BiomedicalSciences, Academia Sinica, Taiwan, 3School of Pharmacy,National Taiwan University, Taipei, TaiwanE-mail: jhlin@gate.sinica.edu.tw, jlin@ntu.edu.tw

The membrane-bound component Fo of the ATP synthaseworks as a rotary motor and plays the central role of drivingthe F1 component to transform chemi-osmotic energy intoATP synthesis. We have conducted molecular dynamicssimulations of the membrane-bound Fo sector with explicitlipid bilayer, in which the particular interest was to observethe onset of helix motion in the c ring upon the change of theprotonation state of Asp61 of the c1 subunit, which is theessential element of the Boyer’s binding-change mecha-nism. To investigate the influence of transmembranepotential and pH gradient, i.e., the proton motive force, onthe structure and dynamics of the a-c complex, differentelectric fields have been applied along the membrane nor-mal. Correlation map analysis indicated that the correlatedmotions of residues in the interface of the a-c complex weresignificantly reduced by external electric fields. The deute-rium order parameter (SCD) profile calculated by averagingall the lipids in the Fo-bound bilayer was not very differentfrom that of the pure bilayer system, which agrees withrecent 2H solid-state nmR experiments. However, by delin-eating the lipid properties according to their vicinity to Fo, wefound that the SCD profiles of different lipid shells areprominently different. Lipids close to Fo formed a moreordered structure. Similarly, the lateral diffusion of lipids onthe membrane surface also followed a shell-dependentbehavior. The lipids in the proximity of Fo exhibited verysignificantly reduced diffusional motion. The numerical valueof SCD was anti-correlated with that of the diffusion

coefficient, i.e., the more ordered lipid structures led toslower lipid diffusion. Our findings will not only help forelucidating the dynamics of Fo depending on the protonationstates and electric fields, but may also shed some light tothe interactions between the motor Fo and its surroundinglipids under physiological condition, which could help torationalize its extraordinary energy conversion efficiency.This work has been published1 in March 2010, and it wasselected as one of the two Featured Articles of that issue.

1. Lin, Y.-S.; Lin, J.-H.; Chang, C.-C., Molecular DynamicsSimulations of the Rotary Motor F0 under ExternalElectric Fields across the Membrane. Biophys. J. 2010,98, 1009-1017.

P-363Neck linker docking coordinates the kineticsof kinesin’s headsImre Derenyi, Andras Czovek, and Gergely J. Szoll}osiDepartment of Biological Physics, Eotvos University,Budapest, Hungary

Conventional kinesin is a two-headed motor protein, which isable to walk along microtubules processively by hydrolyzingATP. Its neck linkers, which connect the twomotor domains andcan undergo a docking/undocking transition, are widelybelieved to play the key role in the coordination of the chemicalcycles of the two motor domains and, consequently, in forceproduction and directional stepping. Although many experi-ments, often complemented with partial kinetic modeling ofspecific pathways, support this idea, the ultimate test of theviability of this hypothesis requires the construction of a com-plete kineticmodel.Considering the twoneck linkersasentropicsprings that are allowed to dock to their head domains andincorporating only the few most relevant kinetic and structuralproperties of the individual heads, we have developed the firstdetailed, thermodynamically consistent model of kinesin thatcan (i) explain the cooperation of the heads (including theirgating mechanisms) during walking and (ii) reproduce much ofthe available experimental data (speed, dwell time distribution,randomness, processivity, hydrolysis rate, etc.) under a widerange of conditions (nucleotide concentrations, loading force,neck linker length and composition, etc.). Besides revealing themechanism by which kinesin operates, our model alsomakes itpossible to look into the experimentally inaccessible details ofthemechanochemical cycle and predict howcertain changes inthe protein affect its motion.

P-364Toward the construction of a myosin based machinePasquale Bianco1, Dan Cojoc2, Luca Melli1, ManuelaMaffei3, Andrea Causero2, Giulietta Pinato2, Mario Dolfi1,Roberto Bottinelli3, Vincenzo Lombardi11Laboratory of Physiology, DBE, University of Florence,Firenze, Italy, 2Istituto Officina dei Materiali, CNR, Trieste,Italy, 3University of Pavia, Pavia, Italy

The research project is directed toward the construction ofa synthetic bio-inorganic machine that consists in a single

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actin filament that interacts with a linear array of myosin IImotors regularly disposed on a nano-structured device. Themotor array is intended to simulate the unique properties ofthe ensemble of motor proteins in the half-sarcomere of themuscle by providing the condition for developing steadyforce and shortening by cyclic interactions with the actinfilament. The mechanical outputs in the range 0.5-200 pNforce and 1-10,000 nm shortening will be measured andcontrolled with millisecond time resolution by means of adouble laser optical tweezers apparatus (DLOT, Bonginiet al., Biophys J, Supplement 1, 98:752a/3908-Pos, 2010).The objectives pursued by the project are: a. Production ofnative and artificial myosins; b. Interfacing motor proteinsand inorganic devices; c. Preparation of bead tailed actinwith correct polarity; d. Design and fabrication of nano-structures to control the geometry of the motor proteinarray by nano-patterning of the silica/quartz support; e.Synthetic machine imaging by optical and electronmicroscopies. The advancement of the research in each ofthese objectives is reported.

Supported by IIT-SEED MYOMAC.

O-366Mechanism of the force generation in the bacterialflagellar motor: a structural biology perspectiveCyril F. Reboul1, Jenna O’Neill2, Ashley M. Buckle1,Anna Roujeinikova11Department of Microbiology and Departmentof Biochemistry and Molecular Biology, Monash University,Clayton, Vic. 3800, Australia, 2Manchester InterdisciplinaryBiocentre, Faculty of Life Sciences, Universityof Manchester, 131 Princess Street, Manchester, M1 7DN,UK

The bacterial flagellar motor is a membrane-embeddedmolecular machine that rotates filaments, providing a pro-pulsive force for bacteria to swim. The molecular mechanismof torque (turning force) generation is being investigatedthrough the study of the properties and three-dimensionalstructure of the motor’s stator unit. We are taking both top-down and bottom-up approaches, combining data from themolecular genetics studies, cross-linking, X-ray proteincrystallography and molecular dynamics simulations. Wehave recently determined the first crystal structure of theprotein domain that anchors the proton-motive-force-gen-erating mechanism of the flagellar motor to the cell wall, andformulated a model of how the stator attaches to peptido-glycan. The work presented at the meeting will inform theaudience on our latest work that establishes the relationshipbetween the structure, dynamics and function of a keycomponent of the bacterial flagellar motor, the motility proteinB (MotB). This work will be put in the perspective of themechanism of rotation, stator assembly, anchoring to pepti-doglycan and interaction with the rotor, and discussed in thelight of the elementary events composing the cycle of elec-trochemical-to-mechanical energy conversion that drivesflagellar rotation.

P-367Directionality of the yeast Kinesin-5 Cin8 is controlledby molecular structure and salt conditionsChristina Thiede1, Adina Gerson-Gurwitz2,Natalia Movshovich2, Vladimir Fridman2, Maria Podolskaya2,Tsafi Danieli3, Stefan Lakamper1, Dieter Kopfenstein1,Christoph F. Schmidt1 and Larisa Gheber21Georg-August-Universitat Gottingen, Gottingen, Germany,2Ben-Gurion University of the Negev, Beer-Sheva, Israel,3Hebrew University of Jerusalem, Jerusalem, Israel

Members of the conserved Kinesin-5 family fulfill essentialroles in mitotic spindle morphogenesis and dynamics andwere thought to be slow, processive microtubule (MT)-plus-end directed motors. The mechanisms that regulate Kinesin-5 function are still not well understood. We have examined invitro and in vivo functions of the Saccharomyces cerevisiaeKinesin-5 Cin8 using single-molecule motility assays andsingle-molecule fluorescence microscopy and found thatCin8 motility is exceptional in the Kinesin-5 family.In vitro, individual Cin8 motors could be switched by ionicconditions from rapid (up to 50 lm/min) and processiveminus-end, to bidirectional, to slow plus-end motion. Deletionof the uniquely large insert of 99 amino acids in loop 8 of Cin8induced bias towards minus-end motility and strongly affec-ted the directional switching of Cin8 both in vivo and in vitro.We further found that deletion of the functionally overlappingKinesin-5 Kip1 and of the spindle-organizing protein Ase1affected Cin8 velocity and processivity, but directionality wasnot affected.The entirely unexpected finding of switching of Cin8 direc-tionality in vivo and in vitro demonstrates that the ‘‘gear box’’of kinesins is much more complex and versatile than thought.

O-368Longitudinal and rotational motion of microtubulesdriven by the kinesin-14 motor NcdAndrej Vilfan1, Bert Nitzsche2, El _zbieta Kocik3,Andrzej A. Kasprzak3 and Stefan Diez21J. Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia,2Technische Universitat Dresden and Max Planck Instituteof Molecular Cell Biology and Genetics, Dresden, Germany,3Nencki Institute of Experimental Biology, Warsaw,Poland

In gliding motility assays, various motor proteins have beenshown to drive not only translational, but also rotationalmotion of filaments around their longitudinal axis. Using FLICmicroscopy [1], we measured the longitudinal and rotationalmotion of quantum-dot labeled microtubules driven by Ncd (akinesin-14 motor protein) as a function of the ATP concen-tration. The data show two striking features: (i) Thelongitudinal velocity shows a strong deviation from Michaelis-Menten shape. (ii) The rotational pitch depends strongly onthe ATP concentration. We propose a simple mechanicalmodel, which explains both findings. The underlying mech-anism of the non-processive Ncd motor assumes that thepower stroke comprises a longitudinal, as well as a lateral

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(off-axis) component and that the waiting (apo) state takesplace before the power stroke. Our model is distinct from themodels for filament rotation by myosin kinesin-1 motors,which is a consequence of the filament structure [2, 1]. Ourresults support the finding that the Ncd power stroke is trig-gered by ATP binding [3].

[1] B. Nitzsche, F. Ruhnow and S. Diez, Nature Nanotech-nology 3:552-556 (2008)

[2] A. Vilfan, Biophys. J. 97:1130-1137 (2009)[3] N.F. Endres, C. Yoshioka, R.A. Milligan and R.D. Vale,

Nature 439:875-878 (2006)

O-369Switch between large hand-over-hand and smallinchworm-like steps in myosin VIToshio YanagidaGraduate School of Frontier Bioscience, Osaka University,Riken QBiC (Quantitative Biological Center) and NICT CiNet(Center for Neural and Information Networks), Osaka Japan

Many biological motor molecules move within cells usingstepsizes predictable from their structures. Myosin-VI, how-ever, has much larger and more broadly-distributed stepsizesthan those predicted from its short lever arms. We explain thediscrepancy by monitoring Qdots and gold nano-particlesattached to the myosin VI motor domains using high-sensi-tivity nano-imaging. The large stepsizes were attributed to anextended and relatively rigid lever arm; their variability to twostepsizes, one large (72 nm) and one small (44 nm). Theseresults suggest there exist two tilt-angles during myosin-VIstepping, which correspond to the pre- and post-power-strokes states and regulate the leading head. The large stepsare consistent with the previously reported hand-over-handmechanism, while the small steps follow an inchworm-likemechanism and increase in frequency with ADP. Switchingbetween these two mechanisms in a strain sensitive, ADP-dependent manner allows myosin-VI to fulfill its multiple cel-lular tasks including vesicle transport and membraneanchoring. http://www.fbs.osaka-u.ac.jp/labs/yanagida/, http://www.qbic.riken.jp/.

Structure of proteins and protein complexes

P-370Mossbauer spectroscopy of chicken liver and spleen,human liver ferritin and pharmaceutical modelsI.V. Alenkina1,2, M.I. Oshtrakh1, Yu.V. Klepova3,N.V. Sadovnikov3, V.A. Semionkin1,21Faculty of Physical Techniques and Devices for QualityControl, Ural Federal University, 620002, Ekaterinburg,Russia, 2Faculty of Experimental Physics, Ural FederalUniversity, 620002, Ekaterinburg, Russia, 3Facultyof Physiology and Biotechnology, Ural State AgriculturalAcademy, K. Liebknecht str., 42, Ekaterinburg, 620075, Russia

Ferritin deposits iron in oxyhydroxide iron core surrounded byprotein shell. The iron core structure may vary in differentferritins in both normal and pathological cases. To study iron

core variations the Mossbauer spectroscopy with a highvelocity resolution was applied for comparative analysis ofnormal and leukemia chicken liver and spleen tissues,human liver ferritin and commercial pharmaceutical productsImferon, Maltofer� and Ferrum Lek as ferritin models.Mossbauer spectra of these samples measured with a highvelocity resolution at room temperature were fitted using twomodels: homogenous iron core (one quadrupole doublet)and heterogeneous iron core (several quadrupole doublets).The results of both fits demonstrated small variations ofMossbauer hyperfine parameters related to structural varia-tions of the iron cores. These small structural variations maybe a result of different degree of crystallinity, the way of ironpackage, nonequivalent iron position, etc. Obtained smalldifferences for normal and leukemia tissues may be usefulfor distinguishing ferritins from normal and pathologicalcases. This work was supported in part by the RussianFoundation for Basic Research (grant # 09-02-00055-a).

P-371Structural study of the interaction of SopB, a TTSSeffector of Salmonella, and its chaperone SigEFrederique Dewitte1, Prakash Rucktooa2, Pierre Lebrun3,Vincent Villeret1 and Coralie Bompard11Interdisciplinary Research Institute CNRS, Lille, France,2Netherland Cancer Institute, Amsterdam, The Netherlands,3CIIL, Institut Pasteur de Lille, Lille, France

Invasion of epithelial cells by Salmonella enterica is mediatedby bacterial ‘‘effector’’ proteins that are delivered into the hostcell by a type III secretion system (TTSS). The collaborativeaction of these translocated effectors modulates a variety ofcellular processes leading to bacterial uptake into mammaliancells. Type III effectors require the presence in the bacterialcytosol of specific TTS chaperones. Effectors are known tointeract with their chaperone via a Chaperone Binding Domain(CBD) situated at their N-terminus. This work focus on SopB,an effector with phosphoinositide phosphatase activity andparticularly its interaction with the specific chaperone SigE bybiochemical, biophysical and structural approaches.We have co-expressed SopB with its specific chaperoneSigE and purified the complex, determined the limits of theCBD and purified the SopBCBD/SigE complex. The structureof SigE has been solved previously but no crystals could beobtained for structure determination of both complexes. Weused SAXS experiment combined with biophysical approachto analyse the interaction between SopB and its chaperoneas well as the quaternary structure on the complex that willbe described in this presentation.

P-372A spectroscopic study of GMP kinase secondarystructure in interaction with ligandsC. Chilom1, I. Matei2, G. Baranga1, D. M. Gazdaru1,A. Popescu11Faculty of Physics, University of Bucharest, Magurele,Romania, 2Faculty of Chemistry, University of Bucharest,Bucharest, Romania

Guanylate monophosphate kinase (GMPK) is a cytosolicenzyme involved in nucleotide metabolic pathways. One of

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the physiological roles of GMPKs is the reversible phos-phoryl group transfer from ATP to GMP (its specific ligand),yielding ADP and GDP. The GMPK from Haemophilus in-fluenzae is a small protein, with a number of 208 amino acidsin the primary structure.In order to determine the secondary structure changes of thisenzyme, as well as some physical characteristics of itscomplexes with GMP and ATP ligands, circular dichroism(CD) and ATR - FTIR studies were performed. The enzymeand its ligands were dissolved in Tris - HCl buffer, at pH 7.4and 25 �C.From the CD spectra the content of the secondary structureelements of GMPK and GMPK/GMP, GMPK/ATP (with andwithout Mg2+) were determined. The major secondarystructure elements of GMPK from Haemophilus influenzaewere a-helix (* 37 %) and b-sheet (* 36 %). ATR - FTIRexperiments show that the amide I and amide II bands of theGMPK are typical for a protein with great a-helix content.From the second derivative spectra, the content of the sec-ondary structure elements were estimated. These data werein agreement with those obtained by CD.

P-374Helical peptides derived from the dimerization helixof the capsid protein of HIV-1bind protein wtRosa Domenech1, Rebeca Bocanegra2, Rosario Gonzalez-Muniz3, Javier Gomez1, Mauricio G. Mateu2 and Jose L.Neira11Instituto de Biologıa Molecular y Celular, Universidad MiguelHernandez, Elche, Alicante, Spain, 2Centro de BiologıaMolecular Severo Ochoa, CSIC-UAM, UniversidadAutonoma de Madrid, Cantoblanco, Madrid, Spain, 3Institutode Quımica-Medica, CSIC, Madrid; Instituto deBiocomputacion y Fısica de Sistemas Complejos,Zaragoza, Spain

Assembly of the mature human immunodeficiency virus type1 capsid involves the oligomerization of the capsid protein,CA. The C-terminal domain of CA, CTD, participates both inthe formation of CA hexamers, and in the joining of hexamersthrough homodimerization. Intact CA and the isolated CTDare able to homodimerize in solution with similar affinity(dissociation constant in the order of 10 lM); CTD homodi-merization involves mainly an a-helical region. In this work,we show that peptides derived from the dimerization helix(which keep residues energetically important for dimerizationand with higher helical propensities than the wild-typesequence) are able to self-associate with affinities similar tothat of the whole CTD.Moreover, the peptides have a higher helicity than that of thewild-type sequence, although is not as high as the theoreti-cally predicted. Interesting enough, the peptides bind to CTD,but binding in all peptides, but one, does not occur at thedimerization interface of CTD (helix 9). Rather, bindingoccurs at the last helical region of CTD, even for the wild-typepeptide, as shown by HSQC-NMR. As a consequence, allpeptides, but one, are unable to inhibit capsid assembly ofthe whole CA in vitro. The peptide whose binding occurs atthe CTD dimerization helix has an Val?Arg mutation inposition 191, which has been involved in dimer-dimer con-tacts. These findings suggest that event keeping the

energetically important residues to attain CTD dimerizationwithin a more largely populated helical structure is notenough to hamper dimerization of CTD.

P-375Structure and Function of the Sodium/ProlineTransporter PutP studied by EPR SpectroscopySabrina Dunkel1, Michael Raba2, Johann P. Klare1, HeinrichJung2 and Heinz-Jurgen Steinhoff11Physics Department, University of Osnabruck,Barabarastr.7, 49072 Osnabruck, Germany, 2LMU Munich,Biocentre, Microbiology, Grosshaderner Str. 2-4-, 82152Planegg-Martinsried, Munich, Germany

PutP is an integral membrane protein located in the cyto-plasmic membrane of Escherichia coli, being responsiblefor the coupled transport of Na+ and proline. It belongs tothe family of sodium solute symporters (SSS). Structuraldata for PutP is not available, but secondary structurepredictions together with biochemical and biophysicalanalyses suggest a 13 transmembrane motif. From a recenthomology model based on the X-ray structure of the relatedNa+/galactose symporter vSGLT, previously publishedelectron paramagnetic resonance (EPR) studies, andrecent crystallographic and EPR studies on the cognatebacterial homolog of a neurotransmitter:Na+ symporter,LeuT, it has been proposed that helices VIII and IX as wellas the interconnecting ‘‘loop 9’’ region determine theaccessibility of the periplasmic cavities which bind sodiumand proline.We performed site-directed spin labeling of ‘‘loop 9’’ incombination with EPR spectroscopy to investigate thestructural features of this region and possible conformationalchanges induced by sodium and proline. Analyses of spinlabel mobility and polarity as well as accessibility to para-magnetic quenchers allow us to refine this region in thepresent homology model. Furthermore, our data suggestconformational changes in this region upon substrate bindingincluding an overall motion of a helical segment.

P-376Interactions of Brain Fatty Acid Binding Proteinas monitored by site directed spin labelingF.H. Dyszy1, A.P.A. Pinto1, A.P.U. Araujo1,F. Batista1, I. Polikarpov1, A.J. Costa-Filho1,21Instituto de Fısica de Sao Carlos, Universidade de SaoPaulo, Sao Carlos, SP, Brazil, 2Faculdade de Filosofia,Ciencias e Letras de Ribeirao Preto, Universidade de SaoPaulo, Ribeirao Preto, SP, Brazil

Fatty acid-binding proteins (FABP) are a family of lowmolecular weight proteins that share structural homologyand the ability to bind fatty acids. The common structuralfeature is a b-barrel of 10 antiparallel b-strands forming alarge inner cavity that accommodates nonpolar ligands,capped by a portal region, comprising two short a-helices.B-FABP exhibits high affinity for the docosahexaenoic(DHA) and oleic acid (OA). It is also postulated that B-FABPmay interact with nuclear receptors from PPAR family. In thepresent work, we used molecular biology and spectroscopic

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techniques to correlate structure, dynamics and function.Site-directed mutagenesis was used to produce 5 mutantsof B-FABP with a nitroxide spin probe (MTSL) selectivelyattached to residues located at the portal region. ESRspectra of the labeled B-FABP mutants were sensitive tothe location of the mutation and were able to monitorinteractions in three cases: (1) FABP in the presence ofDHA and OA ligands; (2) FABP in the presence of POPCand POPG lipid model membranes, where we evidencedthe importance of electrostatic interactions and a possiblecollisional mechanism for the delivery of FA; (3) FABP in thepresence of PPAR nuclear receptor, in which case we wereable to detect the complex formation.

P-377Effect of DNA binding on the self-associationof the human recombinase protein Rad51M. Fekete1, G. Schay2, J. Kardos3, Cs. Pongor2,M. S. Kellermayer2, J. Fidy1,21Biomembrane Research Group, Hungarian Academyof Sciences, Budapest, Hungary, 2Department of Biophysicsand Radiation Biology, Semmelweis University, Budapest,Hungary, 3Department of Biochemistry, Eotvos LorandUniversity, Budapest, Hungary

HsRad51 is a protein that plays key role in the homologousrecombination repair (HRR) of double strand DNA breaks(DSB) in human cells. It has been found that the presence ofRad51 homologues is essential for cell viability in highereukaryotes. It is supposed that in vivo, the recombinationprocess is initiated by the formation of 3’-ssDNA overhangswhich are then recognized by Rad51 with the help of otherinteracting proteins. Rad51 then forms a helical nucleopro-tein filament around ssDNA. In concert with other key playersthe nucleoprotein filament then binds the dsDNA substrate,recognizes homology and exchanges the DNA strands in anATP dependent manner. It was shown that in highereukaryotes HRR requires the presence of the breast cancerassociated protein BRCA2. Based on co-crystallization andstructure determination of the complex of BRCA2 and theATP-binding domain of HsRad51 it was suggested that theself-association of Rad51 is regulated by the repeats ofBRCA2 intruding into the interface of polymer formation(Pellegrini et al. Nature, (2002) 420, 287-293). Indeed, theinterface of homo-polymerization may be an important site ofthe regulation of filament formation.The present study was based on the reported finding that inthe absence of DNA, Rad51 forms ring-like self-aggregatesin solution. In the experiments, HsRad51 protein and itsmutants were produced by bacterial expression and purifiedin our laboratory. The ring-like associates were characterizedby dynamic light scattering and AFM. Binding to ssDNA anddsDNA was verified by fluorescence spectroscopy and singlemolecule techniques. Fluorescence label studies combinedwith high pressure technique were used to determine thedissociation constant of the interface (Schay et al., JBC(2006) 281, 25972-25983) and its dependence on the solu-tion components. The fluorescence high pressure techniquewas applied to study the role of the protein interface inter-action in the nucleoprotein formation.

AcknowledgementsThe authors acknowledge support by OTKA (Hung. ScientificFoundation Agency) grant no. K 84271.

P-378New insight in the assembly and function of humansmall heat shock proteins (sHSP)F. Skouri-Panet1, M. Michiel2, C. Ferard1, E. Duprat1,S. Finet11Institut de Mineralogie et de Physique des MilieuxCondenses, CNRS, Univ. Pierre et Marie Curie, Paris,France, 2CEA, DSV, iBiTec-S, Laboratoire Stress Oxydantset Cancer, Gif-sur-Yvette, France

sHSP are ubiquitous proteins involved in cellular resistance tovarious stress (oxidative, heat, osmotic…). They are able toprevent aggregation of non-native proteins through the abilityof forming large soluble complexes and preventing their non-specific and insoluble aggregation. In consequence to thismolecular chaperone function, they can regulate many pro-cesses (resistance to chemotherapy, modulation of the cellularadhesion and invasion, inflammatory response in skin), andthe modulation of their expression has been found to be amolecular marker in cancers, spermatogenesis, or cartilagedegeneration. Furthermore, they are involved in severalpathologies: myopathies, neuropathies, cancers, cataracts.Among the 10 human members (HspB1-10), this studyfocused on HspB1 (Hsp27 involved in some cancers), HspB4(lens specific), HspB5 (lens, muscle, heart, lung) and HspB5-R120G (responsible for a desmin-related myopathy and acataract). As sHSP form large, soluble (but polydisperse inmammals) hetero-oligomers, molecular biology, biochemistry,biophysics and bioinformatics were successfully combined tocompare the functional/dysfunctional assemblies in order tounderstand the critical parameters between sHSP membersdepending upon their tissue and cellular localization.

P-379Molecular damage caused by radiation exposureFelipe Folgosa1,21REQUIMTE - Departamento de Quımica, CQFB, Faculdadede Ciencias e Tecnologia, Universidade Nova de Lisboa,2829-516 Caparica, Portugal, 2CEFITEC - Departamento deFısica, Faculdade de Ciencias e Tecnologia, UniversidadeNova de Lisboa, 2829-516 Caparica, PortugalE-mail: filipe.folgosa@dq.fct.unl.pt

Ionizing radiation is a type of radiation that contains enoughenergy to displace electrons and break chemical bonds. Thiscanpromote the removalof at least oneelectron fromanatomormolecule, creating radical species, namely, Reactive OxygenSpecies (ROS) [1, 2]. Theseare oftenassociatedwith damagesat cellular level, such as, DNA mutations, cell cycle modifica-tions and, in animal cells, cancer. To overcome this problem,organisms developed different protection/repair mechanismsthat enable them to survive to these threats. DNA glycosylasesare enzymes that are part of baseexcision repair (BER) system,mainly responsible for DNA repair. They can recognize a DNAlesionand, insomecases, areable to remove themutatedbase.Herewepropose tostudyoneof thoseenzymes, endonucleaseIII, which contains a [4Fe-4S] cluster [3, 4]. Samples were

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exposed to different doses of UV-C radiation and the effectswere studied by electrophoretic and spectroscopic methods.

[1] J. A. Imlay, Ann Rev Biochem, 77, 755-776, 2008[2] M. Valko, et al., Chem Biol Interact, 160, 1-40, 2006[3] J.C. Fromme, et al., Curr Opin Struct Biol, 14, 43-49, 2004[4] S. Maynard, et al., Carcinogenesis, 30, 2–10, 2009

Acknowledgement:FF would like to acknowledge to FCT for the grant SFRH/BPD/48430/2008

P-380Cholesterol affects Na,K-ATPase thermostabilityand catalytic activityCarolina Fortes Rigos1, Juliana Sakamoto Yoneda1,Guillermo Montich2, Bruno Maggio2 andPietro Ciancaglini11Faculdade de Filosofia Ciencias e Letras de Ribeirao Preto,FFCLRP-USP; Depto. Quımica, 14040-901. Ribeirao Preto,SP, Brasil, 2Facultad de Ciencias Quımicas, Depto. QuımicaBiologica, Universidad Nacional de Cordoba, CIQUIBIC,5000, Cordoba, Argentina

Na,K-ATPase is an integral protein present in the plasmamembrane of animal cells, and consists of two main subunits:the a and b. Cholesterol is an essential constituent of the animalmembrane cells. In order to study the interactionbetweenNa,K-ATPase andcholesterol, wehaveused theDSC technique, anda proteoliposome system composed by the enzyme andDPPC:DPPE, with different percentage in mol of cholesterol.The heat capacity of purified Na,K-ATPase profile exhibitsthree transitions with 31, 189 and 60 kcal/mol at 55, 62 and69�C. Multiple components in the unfolding transition couldbe attributed either to different steps in the pathway or toindependent unfolding of different domains. This denatur-ation of Na,K-ATPase is an irreversible process. For theproteoliposome, we also observed three peaks, with 180,217 and 41 kcal/mol and 54, 64 and 72�C. This increase inDH indicates that the lipids stabilize the protein. When cho-lesterol was used (from 10 to 40 mol %), the first transitionwas shifted to a lower temperature value around 35�C. Theseresults confirm that cholesterol has an influence on thepacking and fluidity of lipid bilayer and changes in lipidmicroenvironment alter the thermostability as well as theactivity of Na,K-ATPase. Financial support: FAPESP.

P-381Apolipoprotein E3 mutants linked with developmentof Type III Hyperlipoproteinemia alter the protein’sthermodynamic propertiesDimitra Georgiadou1, Angeliki Chroni2, Alexander Vezeridis3,Vassilis I. Zannis3 and Efstratios Stratikos11Protein Chemistry Laboratory, IRRP, National Centrefor Scientific Research Demokritos, Agia Paraskevi, Athens15310, Greece, 2Institute of Biology, National Centrefor Scientific Research Demokritos, Agia Paraskevi, Athens15310, Greece, 3Molecular Genetics, Departmentsof Medicine and Biochemistry, Whitaker CardiovascularInstitute, Boston University School of Medicine, Boston,Massachusetts 02118, USA

Apolipoprotein E is a major lipid carrier protein in humans,plays important roles in lipid homeostasis and protectionfrom atherosclerosis. ApoE is characterized by structuralplasticity and thermodynamic instability and can undergosignificant structural rearrangements as part of its biologi-cal function. Mutations in the LDL binding region 136-150of apoE have been linked with lipoprotein disorders suchas Type III Hyperlipoproteinemia in humans and experi-mental animals. In this study we examined whether threesuch mutations, namely R136S, R145C and K146E affectthe thermodynamic stability and conformation of apoE3.Circular dichroism spectroscopy revealed that the muta-tions do not alter the secondary structure of the protein.However, all three mutants have altered thermodynamicstability compared to wild-type as evidenced by thermaland chemical denaturation experiments. Furthermore, allthree variants are able to clarify DMPC emulsions, but withsubtle changes in kinetics that suggest differences inequilibrium distribution of conformations. Overall, our find-ings suggest that single amino acid changes in thefunctionally important region 136-150 of apoE3 can affectthe molecule’s stability and structural lability in solution. Wepropose that these thermodynamic alterations be takeninto account when evaluating the pathogenic potential ofapoE3 variants.

O-382Structure-function relationships in different MFE-2proteins by various biophysical techniquesMaija Mehtala1, Tatu Haataja1, Clement Blanchet2,Marc Lensink3, Kalervo Hiltunen1, Dmitri Svergun2 andTuomo Glumoff11University of Oulu, Oulu, Finland; 2EMBL-Hamburg,Hamburg, Germany, 3IRI-CNRS Institut de RechercheInterdisciplinaire, Centre National de la RechercheScientifique, Lille, France

We have undertaken to study the structure and function ofperoxisomal multifunctional enzyme type 2 (MFE-2) fromdifferent organisms. MFE-2 is a key enzyme in long- andbranched-chain fatty acids breakdown in peroxisomes. Itcontains two enzymes in the same polypeptide and alsoconsists of differing amount of domains depending on thespecies. Crystal structure and enzyme kinetics of Dro-sophila melanogaster MFE-2 has revealed the domainassembly and raised a question about existence of a sub-strate channeling mechanism. Small-angle x-ray scatteringstudies have further resolved the assembly of domains inthe human MFE-2. Mutations in the MFE-2-coding gene inhumans may cause D-bifunctional protein deficiency - ametabolic disease characterized by accumulation of fattyacyl-CoA intermediates due to inactive or residually activeMFE-2 protein. We have also studied the structure, stabilityand dynamics of such mutant proteins both experimentallyand in silico. The latest results on all these studies will bepresented.

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P-383Self-organisation of FtsZ protein filamentson a substratePablo Gonzalez de Prado and Pedro TarazonaDepartamento de Fısica Teorica de la Materia Condensada,Universidad Autonoma de Madrid, Spain

FtsZ is a protein that plays a key role in bacterial division,forming a protein ring directly related to the constriction of themembrane. This process has been observed to occur withoutthe help of molecular motors. Nonetheless, the details of theself-assembly and subsequent force generation of the septalring are still obscure.AFM observations allows the study of the behaviour ofFtsZ solutions on a substrate with unprecedented resolu-tion, permitting the identification of individual proteinfilaments. The different resulting structures can be com-pared to Monte Carlo models for a 2D lattice accountingfor the essential interactions between monomers. Theseinclude a strong longitudinal bond that allows a limitedflexibility (i.e., curvature of the filaments) and a weakerlateral interaction.The work we present follows this approach, focussing on thelatest experiments with FtsZ mutants. By using thesemutants, it is possible to choose the specific region of themonomer that will anchor to the substrate, thus generatingnew structures that provide an insight into monomer-mono-mer interactions. In this way, we explore the anisotropy of thelateral bond in FtsZ, a factor that has not been taken intoaccount before but may prove to be of importance in FstZbehaviour in vivo.

P-384Modeling protein structures and their complexeswith limited experimental dataDominik GrontUniversity of Warsaw, Pasteura 1, 02-093 Warsaw, Poland

Conventional methods for protein structure determinationrequire collecting huge amounts of high-quality experi-mental data. In many cases the data (possibly fragmentaryand/or ambiguous) on itself cannot discriminate betweenalternative conformations and a unique structure cannot bedetermined. Small Angle Xray Scattering is an example ofsuch a ‘‘weak’’ experiment. The spectrum encodes onlyseveral independent degrees of freedom that provide aglobal description of a molecular geometry in a very syn-thetic way. In this contribution we utilized both localinformation obtained from nmR measurements and globaldescription of a macromolecule as given by SAXS profilecombined with a knowledge-based bimolecular force fieldto determine tertiary and quaternary structure of modelprotein systems. SAXS curve as well as various kinds oflocal nmR data such as isotropic Chemical Shifts and theirtensors, J-couplings, RDC, backbone NOE and REDORfrom nmR in solid phase are parsed with the ‘‘experimen-tal’’ module of BioShell toolkit and utilized by Rosettamodeling suite to generate plausible conformations.Obtained results show the new protocol is capable todeliver very accurate models.

P-385NOEnet—use of NOE networks for nmR resonanceassignment of proteins with known 3D structureDirk Stratmann, Carine van Heijenoort and Eric GuittetLaboratoire de Chimie et Biologie Structurales, ICSN-CNRS,Gif-sur-Yvette, France

Structural genomics programs today yield an increasingnumber of protein structures, obtained by X-Ray diffraction,whose functions remain to be elucidated. nmR plays here acrucial role through its ability to readily identify binding sites intheir complexes or to map dynamic features on the structure.An important nmR limiting step is the often fastidious assignmentof thenmRspectra.Forproteinswhose3Dstructuresarealreadyknown, the matching of experimental and back-calculated dataallows a straightforward assignment of the nmR spectra.We developed NOEnet, a structure-based assignmentapproach. It is based on a complete search algorithm, robustagainst assignment errors, even for sparse input data. Itallows functional studies, like modeling of protein-complexesor protein dynamics studies for proteins as large as 28 kD.Almost any type of additional restraints can be used as filtersto speed up the procedure or restrict the assignmentensemble. NOEnet being mainly based on nmR data (NOEs)orthogonal to those used in triple resonance experiments (J-couplings), its combination even with a low number ofambiguous J-coupling based sequential connectivities yieldsa high precision assignment ensemble.

P-386Temperature adaptation requires specific changesin the conformational flexibility pattern of IPMDHIstvan Hajdu, Zoltan Csala, Laszlo Barna,Andras Szilagyi and Peter ZavodszkyInstitute of Enzymology, Biological Research Centre,Hungarian Academy of Sciences H-1113 Karolina ut 29,Budapest, Hungary

We observed, that T. thermophilus isopropylmalate dehydro-genase (IPMDH) has higher rigidity and lower enzyme activityat room temperature than its mesophilic counterpart (E. coli),while the enzymes have nearly identical flexibilities under theirrespective physiological working conditions, suggesting thatevolutionary adaptation tends to maintain optimum activity byadjusting a ‘‘corresponding state’’ regarding conformationalflexibility. In order to reveal the nature of the conformationalflexibility change related to enzymatic activity, we designed aseries of mutations involving non conserved prolines specific tothermophilic IPMDH. Proline to glycine mutations substantiallyincreased conformational flexibility and decreased conforma-tional stability. The mutant enzyme variants did not showenhanced catalytic activity, but the non Arrhenius temperaturedependence of enzyme activity of the wild type was abolished.This phenomenon underlines the fact that the delicate balancebetween flexibility, stability and activity which is required for theenvironmental adaptation of enzymes can be easily disruptedwith mutations even distant from the active site, providing fur-ther evidence that optimization of proper functional motions arealso a selective force in the evolution of enzymes.

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P-388Structures of Trypanosoma brucei dUTPase provideinsight into the reaction mechanismGlyn Hemsworth1, Dolores Gonzalez-Pacanowska2 & KeithS. Wilson11Structural Biology Laboratory, Department of Chemistry,University of York, York YO10 5YW, UK, 2Instituto deParasitologıa y Biomedicina ‘‘Lopez-Neyra’’, ConsejoSuperior de Investigaciones Cientıficas, ParqueTecnologicode Ciencias de la Salud, 18100-Armilla, Granada, Spain

The kinetoplastids Trypanosoma brucei, T. cruzi and Leish-mania major are responsible for causing great morbidity andmortality in developing countries. The all a-helical dimericdUTPases from these organisms represent promising drugtargets due to their essential nature and markedly differentstructural and biochemical properties compared to the tri-meric human enzyme. To aid in the development of dUTPaseinhibitors we have been structurally characterizing theenzymes from these species. Here we present the structureof the T. brucei enzyme in open and closed conformations,completing the view of the enzymes from the kinetoplastids.Furthermore, we sought to probe the reaction mechanism forthis family of enzymes as a mechanism has been proposedbased on previous structural work but has not received anyfurther verification. The proposed scheme is similar to that ofthe trimeric enzyme but differs in detail. Using tryptophanfluorescence quenching in the presence of the transitionstate mimic AlF3 we have been able to identify which speciesis the likely transition state in the reaction. The crystalstructure of T. brucei in complex with this transition stateanalogue confirms the nature of the nucleophilic attackclearly showing how it differs from trimeric enzymes.

O-389The structure of factor H-C3d complex explainsregulation of immune complement alternative pathwayT. Kajander1, M.J. Lehtinen 2, S. Hyvarinen2,A. Bhattacharjee2, E. Leung3, D.E. Isenman3,S. Meri, A. Goldman1, T.S. Jokiranta2.1Institute of Biotechnology, University of Helsinki, Viikinkaari,FIN-00014, Helsinki, Finland, 2Haartman Institute, Universityof Helsinki, Haartmaninkatu, FIN-00014, Helsinki, Finland;3Department of Biochemistry, University of Toronto, Toronto,ON, Canada M5S 1A8

The alternative pathway of complement is important in innateimmunity, attacking not only microbes but all unprotected bio-logical surfaces through powerful amplification. It is unresolvedhow host and nonhost surfaces are distinguished at themolecular level, but key components are domains 19-20 of thecomplement regulator factor H (FH), which interact with host(i.e., nonactivator surface glycosaminoglycans or sialic acids)and the C3d part of C3b. Our structure of the FH19-20:C3dcomplex at 2.3 A resolution shows that FH19-20 has two dis-tinct binding sites, FH19 and FH20, for C3b. We showsimultaneous binding of FH19 to C3b and FH20 to nonactivatorsurface glycosaminoglycans, and we show that both of theseinteractions are necessary for full binding of FH to C3b onnonactivator surfaces (i.e., for target discrimination). We alsoshow that C3d could replace glycosaminoglycan binding to

FH20, thus providing a feedback control for preventing excessC3b deposition and complement amplification. This explainsthe molecular basis of atypical hemolytic uremic syndrome,where mutations on the binding interfaces between FH19-20and C3d or between FH20 and glycosaminoglycans lead tocomplement attack against host surfaces.

P-390Improved secondary structure determination of proteinswith SRCD: distinguishing parallel and antiparallelb-structureAndras Micsonai1, Frank Wien2 and Jozsef Kardos11Department of Biochemistry, Institute of Biology, EotvosLorand University, Budapest, Hungary, 2SynchrotronSOLEIL, DISCO beamline, Gif sur Yvette, France

Circular Dichroism (CD) spectroscopy is a widely usedtechnique for studying the secondary structure (SS) of pro-teins. Numerous algorithms have been developed for theestimation of the SS composition from the CD spectra.Although, these methods give more or less accurate esti-mation for proteins rich in a-helical structure, they often fail toprovide acceptable results on mixed or b-rich proteins. Theproblem arises from the diversity of b-structures, which isthought to be an intrinsic limitation of the technique. Theworst predictions are provided for proteins of unusualb-structures and for amyloid fibrils. Our aim was to develop anew algorithm for the more accurate estimation of SS con-tents for a broader range of protein folds with special interestto amyloid fibrils. Using synchrotron radiation CD (SRCD),we were able to collect high quality spectra of amyloid fibrilswith good S/N ratios down to 175nm. The novel referencedataset with spectra that significantly differ from presentreference sets, extends the information content for SSdetermination. Our algorithm takes into account the diversetwist of the b-sheets that has a great influence on the spectralfeatures. For the first time, we can reliably distinguish paralleland antiparallel b-structure using CD spectroscopy.

P-391Monitoring the assembly of the membrane proteininsertaseAlexej Kedrov1, Marko Sustarsic2, Arnold J.M. Driessen11Groningen Biomolecular Sciences and BioengineeringInstitute, University of Groningen, The Netherlands,2University of Oxford, UK

Molecular forces that governmembrane protein integration andfolding remainamajor question incurrentmolecular biologyandbiophysics. Each nascent polypeptide chain should acquire itsunique three-dimensional folded state within a complex envi-ronment formed by the anisotropic lipid membrane and themembrane-water interface.SecYEG translocase andmembersof a recently described YidC/Oxa1/Alb3 chaperone family arerecognized as primary players in the membrane protein gene-sis. These proteins, so called insertases, serve as membrane-embedded molecular pores where the newly synthesizedprotein is loaded prior its release into the bilayer. Here we applyfluorescence correlation spectroscopy to monitor the assembly

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of the insertase:ribosome:nascent polypeptide chain com-plexes in solution and reconstituted into nanodiscs and modelmembranes. Results provide insights on molecular mecha-nisms and dynamics of the insertase functioning.

O-392Conformational changes during GTPase activity inducedself-assembly of human guanylate binding protein 1revealed by EPR spectroscopyTobias Vopel1, Daniel Klose2, Carola-Sophie Hengstenberg1,Heinz-Jurgen Steinhoff2, Christian Herrmann2 andJohann P. Klare11Physical Chemistry I, Faculty of Chemistryand Biochemistry, Ruhr University Bochum, 44780 Bochum,Germany, 2Macromolecular Structure Group, Departmentof Physics, University of Osnabrueck, 49076 Osnabruck,GermanyEmail: jklare@uos.de

Human guanylate binding protein 1 (hGBP1) belongs to theDynamin Superfamily of large GTPases. hGBP1 is an elon-gated 67 kDa protein comprising three distinct domains, alarge GTPase (LG) domain, the middle domain - a five-helixbundle, and the GTPase effector domain (GED) consisting oftwo helices. The latter subdomain flanks the whole proteinand has contacts with the LG domain.During nucleotide hydrolysis the protein forms oligomerswhich lead to a self stimulation of its GTPase reaction. Forthis reason hGBP1 can be categorized as G protein activatedby nucleotide-dependent dimerization (GAD). Up to date, nostructural information about about the overall conformation ofthe full-length protein in the predicted dimeric or tetramericstate could be obtained.To the structural properties of hGBP1 upon oligomerization, weapplied site-directed spin labeling (SDSL) in combination withelectron paramagnetic resonance (EPR) spectroscopy. WeusedcontinuouswaveandpulseEPRmethods, namelyDoubleElectron Electron Resonance (DEER) spectroscopy, to deter-mine distance distributions between spin label side chains. Weused these distance constraints to model the arrangement ofhGBP1 in the dimeric state and conformational changes,especially of the GED, taking place upon dimerization.

O-393Structural insights into the control of type threesecretionP. Abrusci, S. Johnson, P. Roversi, M. McDowell,S.M. LeaSir William Dunn School of Pathology, South Parks Road,Oxford OX1 3RE, UK

Correct assembly and regulation of multi-component molec-ular machines is essential for many biological activities. TheType III Secretion System (T3SS) is a complex molecularmachine that is a key virulence determinant for importantGram-negative pathogens including Shigella, Yersinia andSalmonella species [1-2]. The T3SS consists of multiplecopies of *25 different proteins (totalling *7MDa), spansboth bacterial membranes and drives insertion of a contigu-ous pore into the host-cell membrane. Virulence factors aresecreted via this apparatus directly into the host cell.In all T3SS various levels of regulation occur with switchingbetween secretion off and on states overlaid on control ofwhich substrates are secreted. Genes involved in a variety ofthese switches have been identified but the molecularmechanisms underlying these functions is poorly under-stood. We are studying the T3SS of Shigella flexneri, thecausative agent of dysentery and will present the structure ofthe so-called ‘‘gatekeeper protein’’ MxiA.

1. J. Deane et al. (2010). Cell Mol Life Sci, 67, 1065-752. A.J. Blocker et al (2008). Proc. Natl. Acad. Sci. USA 105,

6507-13

P-394Circular dichroism and the secondary structureof the ROF2 protein from Arabidopsis thalianaLiliana Lighezan1, Adina Breiman2, Adrian Neagu31West University of Timisoara, Faculty of Physics, Blvd.Vasile Parvan No. 4, 300223 Timisoara, Romania, 2Tel AvivUniversity, Department of Plant Sciences, Tel Aviv, Israel69978, 3Victor Babes University of Medicine and PharmacyTimisoara, Department of Biophysics and Medical Informatics,Piata E. Murgu No. 2-4, 300041 Timisoara, RomaniaE-mail: llighezan@physics.uvt.ro; adina@post.tau.ac.il;neagu@umft.ro

Keywords: Thermal denaturation, thermotolerance, CDProsoftware.

The protein ROF2 from the plant Arabidopsis Thaliana actsas a heat stress modulator, being involved in the long-termacquired thermotolerance of the plant. Here we investigatethe relationship between the biological function and thestructure of ROF2 by circular dichroism (CD) spectroscopy inthe far and near UV domains. By analysing our CD spectruawith the CDPro software developed by Sreerama et al.(2000), we have obtained the percentages of a-helices,b-sheets, and turns in the secondary structure of ROF2. Thenear UV results suggest that ROF2 proteins can associate,forming super-secondary structures. CD experiments per-formed at temperatures between 5 �C and 97 �C indicate thatthe thermal denaturation of ROF2 caused by a raise in

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temperature up to 55 �C is followed by a partial recovery ofthe secondary structure as the temperature is raised further.This reversibility of the thermal denaturation of ROF2 mightplay an important role in the thermotolerance of ArabidopsisThaliana.

P-395Investigation of the bovine DGAT1 enzyme binding sitesspecificityJ.L.S. Lopes1; N.C. Noronha1; L.M. Beltramini1;E.M. Cilli2; A.P.U. Araujo11Instituto de Fısica de Sao Carlos, Universidade de SaoPaulo, Sao Carlos, Brazil, 2Instituto de Quımica,Universidade Estadual Paulista, Araraquara, Brazil

The diacylglycerolacyltransferase1 (DGAT1) is an integralprotein from the reticulum endoplasmic membrane that playsan essential role in triacylglyceride synthesis. In cattle, thisenzyme is associated to the fat content regulation on milkand meat. In this study, synthetic peptides corresponding toboth DGAT1 binding sites (Sit1 and Sit2) were designed,purified and employed to investigate the enzyme interactionwith substrates and membrane models. Different bindingspecificities in the interaction with phospholipid vesicles andmicelles were noted. Sit1 showed to bind more strongly innonpolar membrane models, while Sit2 was electrostaticallyattracted to negative phospholipid surfaces. The binding ofboth peptides was followed by significant conformationalchanges (like unordered to helix transition) in circulardichroism spectra and a 20nm blue shift in fluorescenceemission. The binding of Sit1 and Sit2 peptides to negativeliposome gave dissociation constants (KD) of 170 and0.44lM, respectively, and a leakage action 24-fold higher toSit2. The difference in specificity is related to the features ofthe putative substrates (acyl-CoAs and diacylglycerol) andcan be attributed to the distinct role of each DGAT1 bindingsite during lipid synthesis. Supported by FAPESP.

P-396Molecular dynamics simulation of short polymersof the cell division protein FtsZ suggests that GTPaseactivity is restricted to filament endsFernando Martın-Garcıa1,2, Jesus Ignacio Mendieta-Moreno1, Jesus Mendieta1,2 & Paulino Gomez-Puertas1.1Centro de Biologıa Molecular ‘‘Severo Ochoa’’ (CSIC-UAM),C/ Nicolas Cabrera, 1, Cantoblanco, 28049 Madrid, Spain,2Biomol-Informatics SL, Parque Cientıfico de Madrid,C/ Faraday, 7, Cantoblanco, 28049 Madrid, Spain

FtsZ, the bacterial homologue of tubulin, assembles intopolymers in the bacterial division ring. The interfacesbetween monomers include a GTP molecule, although therelationship between polymerization and GTPase activity isstill controversial. A set of short FtsZ polymers were mod-elled and the formation of active GTPase structures wasmonitored using molecular dynamics. Only the interfacesnearest the polymer ends exhibited geometry adequate forGTP hydrolysis. Conversion of a mid-polymer interface to aclose-to-end interface resulted in its spontaneous rear-rangement from an inactive to an active conformation.

P-397Reversible and irreversible photoactivation in mIrisFPvariantsSusan Gayda1, Karin Nienhaus1 and G. Ulrich Nienhaus1,21Institute of Applied Physics and Center for FunctionalNanostructures, Karlsruhe Institute of Technology (KIT),76131 Karlsruhe, Germany, 2Department of Physics,University of Illinois at Urbana-Champaign, Urbana, IL61801, USA, *Correspondence to Karin.Nienhaus@kit.edu

Fluorescent proteins (FPs) have become extremely valuabletools in the life sciences. Due to the latest advances in thelight microscopy, there is a steady need for FPs withimproved spectral properties. mIrisFP is a monomeric FPthat can be switched reversibly between a bright greenfluorescent and a dark state by illumination with light ofspecific wavelengths [1]. Structurally, this photo-switching isbased on a cis-trans isomerization of the chromophore. Uponillumination with violet light, mIrisFP can be irreversiblyphotoconverted from the green-emitting to a red-emittingform. The red form can again be switched reversibly betweena fluorescent and a dark state. To elucidate the mechanisticdetails of the photoinduced reactions, we have generatedmIrisGFP1. This variant can still undergo reversible photos-witching, but lacks the ability to photoconvert to the red stateso that the photoinduced transitions of the green form can bestudied without ‘artifacts’ due to green-to-red photoconver-sion. Using UV/visible spectroscopy, we have characterizedthe on- and off- switching processes in great detail. Severallight-activated reaction pathways have been identified. Theyare highly intertwined so that the net effect achieved with lightof a particular wavelength depends on the relative probabil-ities to photoinduce the various processes.

[1] J. Fuchs, S. Bohme, S., F. Oswald, P. N. Hedde, M.Krause, J. Wiedenmann, and G. U. Nienhaus, A Pho-toactivatable Marker Protein for Pulse-chase Imaging withSuperresolution, Nature Methods 7, 627-630 (2010).

P-398Biophysical study of the complex betweenthe 14-3-3 protein and phosducinM. Kacirova1,2, L. Rezabkova1,2, P. Herman3,J. Vecer3, M. Sulc4 and T. Obsil1,21Faculty of Science, Charles University in Prague,Prague, Czech Republic, 2Institute of Physiology,Academy of Sciences of the Czech Republic, Prague 4,Czech Republic, 3Faculty of Mathematics and Physics,Inst. Physics, Charles University in Prague; Prague,Czech Republic, 4Institute of Microbiology, Academyof Sciences of the Czech Republic; Prague,Czech Republic

Phosducin (Pd) is a Gtbc-binding protein that is highly expres-sed in photoreceptors. Pd is phosphorylated in dark-adaptedretina and is dephosphorylated in response to light. Dephos-phorylated Pd binds Gt protein bc-heterodimer with high affinityand inhibits its interaction with Gta or other effectors, whereasphosphorylated Pd does not. Therefore Pd down-regulates thelight response in photoreceptors. Phosphorylation of Pd at S54and S73 leads to the binding of the 14-3-3 protein. The 14-3-3

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proteins function as scaffolds modulating the activity of theirbinding partners and their role in Pd regulation is still unclear.The 14-3-3 protein binding may serve to sequester phosphor-ylated Pd from Gtbc or to decrease the rate of Pddephosphorylation and degradation. We performed severalbiophysical studies of the 14-3-3:Pd complex. Analytical ultra-centrifugation was used to determine the complexstoichiometry and dissociation constant. Conformationalchanges of Pd induced both by the phosphorylation itself andby 14-3-3 binding were studied using the time-resolved fluo-rescence spectroscopy techniques. This work was funded byGrant P305/11/0708 of the Czech Science Foundation and byResearch Project MSM0021620857 of the Ministry of Educa-tion, Youth, and Sports of the Czech Republic.

P-399BMH-dependent activation of yeast enzyme neutraltrehalaseV. Obsilova1, D. Veisova1, E. Macakova1, L. Rezabkova1,21Institute of Physiology Academy of Sciences of the CzechRepublic, 14220 Prague, Czech Republic, 2Departmentof Physical and Macromolecular Chemistry, Facultyof Science, Charles University, 12843 Prague, Czech Republic

Trehalase (EC 3.2.1.28) is a glycoside hydrolase enzyme thatcatalyzes the conversion of a, a-trehalose to two molecules ofglucose. Yeast BMH1 and BMH2 proteins (yeast 14-3-3 iso-forms) form a complex with neutral trehalase (NTH1) after itsphosphorylation by PKA. We showed that PKA phosphory-lates NTH1 in vitro on four Serine residues: 20, 21, 60 and 83.To find out which site or sites are essential for the BMH bindingwe produced NTH1 WT (both phosphorylated and non-phosphorylated), four NTH1 mutants containing single phos-phorylation site, one double phosphorylated NTH1 mutant (atSer20 and 21) and a mutant containing none of these studiedphosphorylation sites as well. The interaction between BMH1and BMH2 protein with enzyme NTH1 was monitored usingnative electrophoresis and sedimentation velocity measure-ments. The sedimentation equilibrium analysis was used todefine the stoichiometry of NTH1/BMH complexes. Finally, weused enzyme kinetic measurements to monitor the BMH-dependent activation of NTH1.Supported by Grant IAA501110801 of the Grant Agency ofthe Academy of Sciences of the Czech Republic, by Grant350111 of the Grant Agency of the Charles University and byResearch Project AV0Z50110509 of the Academy of Sci-ences of the Czech Republic.

P-400Mossbauer spectroscopy of various oxyhemoglobinsin relation to their structure and functionsM.I. Oshtrakh1, A.L. Berkovsky2, A. Kumar3,A.V. Vinogradov4, T.S. Konstantinova4,V.A. Semionkin1, S. Kundu31Ural Federal University, Ekaterinburg, Russia,2Hematological Research Center of the Russian Academyof Sciences, Moscow, Russia, 3University of Delhi SouthCampus, New Delhi, India, 4Ural State Medical Academy,Ekaterinburg, Russia

Mossbauer spectroscopy with a high velocity resolution wasused for comparative study of various oxyhemoglobins for

analysis of the heme iron electronic structure and proteinstructure-function relationship. Samples of pig, rabbit andnormal human oxyhemoglobins and oxyhemoglobins frompatients with chronic myeloleukemia and multiple myelomawere measured using Mossbauer spectrometric system witha high velocity resolution at 90 K. Mossbauer spectra werefitted using two models: one quadrupole doublet (model ofequivalent iron electronic structure in a– and b–subunits ofhemoglobins) and superposition of two quadrupole doublets(model of non-equivalent iron electronic structure in a– andb–subunits of hemoglobins). In both models small variationsof Mossbauer hyperfine parameters (quadrupole splitting andisomer shift) were observed for normal human, rabbit and pigoxyhemoglobins and related to different heme iron stereo-chemistry and oxygen affinity. Small variations of Mossbauerhyperfine parameters for oxyhemoglobins from patients wererelated to possible variations in the heme iron stereochem-istry and function.This work was supported in part by the RFBR (grant # 09-02-00055-a) for M.I.O. and Government of India (BT/PR11275/GBD/27/150/2008) for S.K.

P-401Structure-function relationships in spider silkMichel Pezolet and Thierry LefevreUniversite Laval, Quebec, Canada

The different types of silk produced by orb-weaving spidersdisplay variousmechanical properties to fulfill diverse functions.For example, the dragline silk produced by the major ampulateglandsexhibitshigh toughness that comes fromagood trade-offbetween stiffness and extensibility. On the other hand, flagelli-form silk of the capture spirals of the web is highly elastic due tothe presence of proline and glycine residues. These propertiesare completelydictatedby the structural organization of the fiber(crystallinity, degree of molecular orientation, secondary struc-ture, microstructure), which in turn results from the proteinprimary structure and the mechanism of spinning. Although thespinning process of dragline silk begins to be understood, themolecular events occurring in the secretory glands and leadingto the formation of other silk fibers are unknown,mainly due to alack of information regarding their initial and final structures.Taking advantage of the efficiency of Raman spectromicros-copy to investigate micrometer-sized biological samples, wehave determined the conformation of proteins in the completeset of glands of the orb-weaving spiderNephila clavipes as wellas in the fibers that are spun from these glands.

P-402Structural study of the 14-3-3/RGS3 protein complexL. Rezabkova1,2, P. Man2,3, P. Novak2,3, P. Herman4,J. Vecer4, T. Obsil1,21Faculty of Science, Charles University in Prague, Prague,Czech Republic; 2Institute of Physiology, Prague, CzechRepublic; 3Institute of Microbiology, Prague, Czech Republic;4Institute of Physics, Charles University in Prague, Prague,Czech Republic

In this study we were particularly interested in 14-3-3 f /RGS3 protein complex. The crystal structure of RGS

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domain of RGS3 at 2.3A resolution was solved. The stoi-chiometry of 14-3-3 f /RGS3 protein complex waselucidated using the analytical ultracentrifugation. To mapthe interaction between 14-3-3f and RGS3 protein weperformed a wide range of biophysical measurements: H/Dexchange and cross link experiments coupled to massspectrometry, FRET (Forster resonance energy transfer)time-resolved fluorescence experiments, time-resolvedtryptophan fluorescence spectroscopy and SAXS (smallangle X-ray scattering) measurements. Based on all theseresults we build 3D model of 14-3-3 f /RGS3 proteincomplex. Our model revealed new details on architecture ofcomplex formed by 14-3-3 proteins. To date all knownstructure of 14-3-3 proteins complexes suggests that theligand is docked in the central channel of 14-3-3 protein.Our results indicate that the RGS domain of RGS3 proteinis located outside the central channel of 14-3-3f proteininteracting with less-conserved residues of 14-3-3f.This work was funded by Grant IAA501110801 of the GrantAgency of the Academy of Sciences of the Czech Republicand by Grant 28510 of the Grant Agency of the CharlesUniversity.

P-403Solution structure and self assembly propertiesof soluble RAGEZsuzsa Sarkany1, Teemu Ikonen2, Frederico Ferreira-da-Silva1, Maria Joao Saraiva1,3, Dmitri Svergun2 and AnaMargarida Damas1,31IBMC-Institute for Molecular and Cell Biology, Universityof Porto, Portugal, 2European Molecular Biology Laboratory,Hamburg Outstation, Germany, 3ICBAS - Instituto deCiencias Biomedicas Abel Salazar, University of Porto,Portugal

The Receptor for Advanced Glycation End-products(RAGE) is a multiligand cell surface receptor involved invarious human diseases. The major alternative spliceproduct of RAGE comprises its extracellular region thatoccurs as a soluble protein (sRAGE). Although the struc-tures of sRAGE domains were available, their assembly intothe functional full length protein remained unknown. Herewe employed synchrotron small-angle X-ray scattering tocharacterize the solution structure of human sRAGE. Theprotein revealed concentration-dependent oligomerizationbehaviour, which was also mediated by the presence ofCa2+ ions. Rigid body models of monomeric and dimericsRAGE were obtained from the scattering data recorded atdifferent solvent conditions. The monomer displays a J-likeshape while the dimer is formed through the association ofthe two N-terminal domains and has an elongated structure.The results provided insight into the assembly of i) theheterodimer sRAGE:RAGE, which is responsible for block-age of the receptor signalling, and ii) RAGE homodimer,which is necessary for signal transduction, paving the wayfor the design of therapeutical strategies for a large numberof different pathologies.

O-406Three-dimensional structure of ClpB, a chaperonethat rescues aggregated proteinsMelisa Lazaro1, Urko del Castillo2,3, Gorka Lasso1, SergioPerez-Acebron2,3, Fernando Moro2,3, Xabier Agirrezabala1,David Gil1, Arturo Muga2,3, and Mikel Valle1,31Structural Biology Unit, Centre for Cooperative Researchin Biosciences, CICbioGUNE. Derio, Spain, 2Biophysics Unit(CSIC/UPV) and 3Department of Biochemistry and MolecularBiology, University of the Basque Country, Bilbao, Spain

ClpB is a hexameric AAA? ATPase that extracts unfoldedpolypeptides from aggregates by threading them through itscentral pore. The contribution of coiled-coil M domains isfundamental for the functional mechanism of this chaperone,and its location within the protein structure in previousstructural models is contradictory. We present cryo-electronmicroscopy structural analysis of ClpB from E. coli in severalnucleotide states. The study reveals a novel architecture forClpB and shows that M domains form an internal scaffoldlocated in the central chamber of ClpB hexamers. This innerstructure transmits local signals due to ATP binding andhydrolysis by AAA? domains. Surprisingly, coiled-coil Mdomains are seen to bend significantly around a hinge regionthat separates two structural motifs. Our results presenta new framework to understand ClpB-mediated proteindisaggregation.

P-407Streptomyces clavuligerus isoenzymes involvedin clavulanic acid biosynthesis: a structural approachD.F. Vieira1, L.S. Goto2, C.O. Hokka2,A.P.U. Araujo11Physics Institute of Sao Carlos (IFSC)/USP, Sao Carlos, SP,Brazil, 2Chemical Engineering Department/ UniversidadeFederal de Sao Carlos (UFSCar), Sao Carlos, SP, Brazil

Clavulanic acid (CA) is a potent b-lactamase inhibitor pro-duced by Streptomyces clavuligerus. N2-(2-carboxyethyl)arginine synthase (CEAS) and proclavaminate amidinohydrolase (PAH) catalyze the initial steps in the biosynthesisof CA. Recently ceas1and pah1genes (paralogous of ceasand pah) were related to the CA biosynthesis but theirproducts have not been studied yet. Here we present theinitial structural analysis of CEAS1 and PAH1 using bio-physical techniques. Pah1 and ceas1 genes were isolatedfrom the genomic DNA of S. clavuligerus and overexpressedin E. coli. The recombinant proteins were purified by affinitychromatography and analyzed by size exclusion chroma-tography, non-denaturing PAGE, dynamic light scattering,far-UV Circular Dichroism (CD) and fluorescence spectros-copy. Our results showed that PAH1 and CEAS1 wereobtained as hexamer and dimer respectively. Both proteinsshowed an a/b folding, being stable up to 35�C. Above thistemperature protein unfolding was observed but the com-plete unfolding was not observed, even at 100�C. MoreoverCEAS1 and PAH1 showed to be stable over a wide pH range(pH 5.5 – 9.5). We are currently working on improvingCEAS1 crystals which are a promising step towards theelucidation of the CEAS1 structure. Supported by FAPESP.

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O-408Structural and biochemical studies show divergentallosteric mechanisms in glycoytic enzymesHugh P. Morgan1, Iain W. McNae1, Matthew W. Nowicki1,Francis O’Reilly1, Paul A. M. Michels2, Linda A. Fothergill-Gilmore1 & Malcolm D. Walkinshaw1

1Centre for Translational and Chemical Biology, Instituteof Structural and Molecular Biology, The Universityof Edinburgh, Michael Swann Building, The King’s Buildings,Mayfield Road, Edinburgh EH9 3JR, UK, 2Welcome trustcentre for cell biology, The University of Edinburgh, MichaelSwann Building, The King’s Buildings, Mayfield Road,Edinburgh EH9 3JR, UK

We have solved a series of eukaryote pyruvate kinase X-raystructures from the parasite Leishmania mexicana (LmPK)and the human M1 (hsM1PK) and M2 (hsM2PK) isoforms.Despite identical active sites and similar overall tetramerarchitecture, the details of allosteric activation are surpris-ingly different.The parasite enzymes use the effector molecule fructose 2,6bisphosphate (F26BP).A series of four LmPYK crystalstructures solved in all possible ligated states (?/- F26BPand substrate ?/- F26BP) shows that enzyme activation isthe result of a conformational change, whereby a concertedrocking motion primes the active sites. This activated state islocked by binding the F26BP effector; this ‘rock-and-lock’mechanisms explains activating role of effector which bindsover 40 A away from the active site.In mammals the M2 isoform is found in rapidly proliferating(e.g. embryonic and cancer) cells and is allosterically regu-lated by F16BP. The M1 isoform is an alternative splicedvariant and is constitutively active and differs in only 22 aminoacids. A direct comparison of the first X-ray crystal structure ofhsM1PK with hsM2PK (crystallised under identical condi-tions) shows how the 22 different amino acids affect thetetramer interface. Together with light scattering and gel fil-tration data we show that allosteric regulation in M2 PK is bestexplained in terms of a monomer-tetramer equilibrium.

P-409DISCO: dichroism, imaging & spectrometryfor chemistry and biologyFrank Wien, Alexandre Giuliani, FredericJamme,ValerieRouam and Matthieu RefregiersSynchrotron SOLEIL, L’Orme des Merisiers, 91192 Gif surYvette Cedex, France

DISCO at SOLEIL Synchrotron (France) is a VUV to visiblebeamline and operational since Sept. 2009. Three endsta-tionsserve the biology and chemistry communities resolvingthe common scientific topic of Bio- macromolecular structureinteractions.

• Synchrotron Radiation Circular Dichroism• Mass spectrometry following VUV photoionisation• Fluorescence imaging with lifetime and spectral

measurements

Here we will present the SRCD experiment. High photon fluxof 1010photons / sec, improved detector performances aswell as user-orientated software developments have proven

to be the garants for successful data collections,which con-siderably increased the information content obtained. Theexploration into the charge transfer region of the peptidebonds is adding specifically new insights. Low sample vol-umes of as little as 2ll per spectra as well as convenientsample chamber handling allow for economic and efficientdata collections. Typical spectra acquisition from 280 to170nm, last for 9 min for three scans with a 1nm stepsize. Prior to high resolution based techniques, SRCDspectrawill answer questions about folding states of macro-molecules including DNA, RNA and sugar macromoleculesas well as their complexes with proteins and speciallymembrane proteins.

O-410Structure of an intercellular channel formedduring sporulation in Bacillus subtilisAmanda McFeat, Elena Blagova,Vladimir Levdikov,Mark Fogg, Richard Bingham, Jen Potts, Keith Wilson,Anthony J. WilkinsonDept. of Chemistry, University of York, York YO10 5YW, UK

Sporulation in Bacillus subtilis begins with an asymmetric celldivision producing a smaller cell called the forespore, whichinitially lies side-by-side with the larger mother cell. In aphagocytosis-like event, the mother cell engulfs the fores-pore so that the latter is internalised as a cell-within-a-cell.Engulfment involves the migration of the mother cell mem-brane around the forespore until the leading edges of thisengulfing membrane meet and fuse. This releases theforespore, now surrounded by a double membrane, into themother cell cytoplasm.Membrane migration during engulfment is facilitated by theinteracting proteins SpoIIQ and SpoIIIAH that are mem-brane-associated and expressed in the forespore and themother cell respectively 1. They interact in the intercellularspace and function initially as a molecular zipper and laterthey participate in a more elaborate complex in whichSpoIIQ and SpoIIIAH are integral components of an inter-cellular channel. This channel is a topic of much currentinterest, having initially been proposed as a conduit for thepassage from the mother cell to the forespore of a specific,but putative, regulator of the RNA polymerase sigma factor,rG 2 and later as a gap junction-like feeding tube 3 throughwhich the mother cell supplies molecules for the biosyn-thetic needs of the forespore. Here we present data on thestructure and interactions of SpoIIQ and SpoIIIAH gleanedfrom biophysical methods and protein crystallography.These data lead to a plausible model for the intercellularchannel.

1. Broder, D. H. & Pogliano, K. (2006). Forespore engulf-ment mediated by a ratchet-like mechanism. Cell 126,917-928.

2. Meisner, J., Wang, X., Serrano, M., Henriques, A. O. &Moran, C. P. J. (2008). A channel connecting the mothercell and forespore during bacterial endospore formation.Proc. Natl. Acad. Sci. 105, 15100-15105.

3. Camp, A. H. & Losick, R. (2009). A feeding tube modelfor activation of a cell-specific transcription factor duringsporulation in Bacillus subtilis. Genes Dev. 23,1014-1024.

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P-411Kinetic studies of vimentin early assembly and filamentelongationStefan Winheim, Norbert Mucke, Harald Herrmann & JorgLangowskiGerman Cancer Research Center, 69120 Heidelberg,Germany

In vitro assembly of intermediate filament (IF) proteins is arapid three-step process. First, tetrameric subunits associatelaterally into unit length filaments (ULFs). In the second stepfilaments elongate by longitudinal annealing via end-to-endfusion of their ends. Third, these filaments compact radiallyinto smooth IFs. Here, we analyzed the first two steps. Therapid lateral assembly kinetics of human recombinantvimentin were studied in a stopped-flow device with lightscattering detection. By comparing wild type vimentin and amutant that remains in ULF state, the contributions of thelateral and longitudinal assembly to the light scattering signalcould be separated. Longitudinal assembly of vimentin IFswas examined by analyzing the time-dependent length dis-tribution by total internal reflection fluorescence microscopy(TIRFM). These profiles are used to develop robust modelsthat describe the time-dependent filament length distributionof IFs. In order to visualize the mechanisms of late elongationdirectly, we mixed short filaments containing either Alexa488- or Alexa 647-labeled vimentin for further assembly. Thestriped structure of the filaments thus obtained unambigu-ously showed that IFs elongate through direct end-to-endannealing of shorter filaments.

Live cell imaging

P-414Dynamics and organization of alpha3-containing glycinereceptors using single molecule detectionKristof Notelaers1,2, Susana Rocha2, Nick Smisdom1, DanielJanssen1, Jochen C. Meier3, Jean-Michel Rigo1, JohanHofkens2 and Marcel Ameloot11Biomedical Research Institute, Hasselt Universityand School of Life Sciences, Transnational University,Limburg, Diepenbeek, Belgium, 2Department of Chemistry,Katholieke Universiteit Leuven, Celestijnenlaan, Heverlee,Belgium, 3RNA Editing and Hyperexcitability DisordersHelmholtz Group, Max Delbruck Center for Molecular,Medicine, Berlin, Germany

The glycine receptor (GlyR) is a chloride permeable ligandgated ion channel and that can mediate synaptic inhibition.Due to a possible involvement in the pathophysiology of tem-poral lobe epilepsy, the different properties of GlyRs containingalpha3L and K subunit isoforms are currently investigated.Previous characterizations of homomeric receptors consistingof these isoforms have shown a difference in their electro-physiological properties and their membrane distribution asobserved by diffraction-limited fluorescence microscopy. Westudied these isoforms, when separately expressed in trans-fected HEK 293T cells, by using single molecule tracking(SMT) in living cells and direct stochastic optical reconstructionmicroscopy (dSTORM) on fixated cells. For both techniques

the GlyRs are stained using a primary antibody directly labeledwith Alexa Fluor dyes. The dSTORM experiments support theobservation that alpha3L GlyR are clustered, while thealpha3K GlyRs are more uniformly spread. The analysis of theshort range diffusion coefficients obtained by SMT reveals thepresence of heterogeneous motion for both isoforms. TheK-isoform has a higher fraction of fast diffusion. In contrast, theL-isoform is more associated with slow diffusion and appearsto undergo hindered diffusion.

P-415Use of fluorescence microspectroscopy to studynanoparticles delivery into living cancer cellsZ. Arsov1,2, I. Urbancic1, M. Garvas1, D. Biglino1,2,A. Ljubetic1, T. Koklic1,2, J. Strancar1,21Jozef Stefan Institute, Ljubljana, Slovenia, 2Centerof Excellence NAMASTE, Ljubljana, Slovenia

Since nanoparticles are suitable for tumor therapy due totheir passive targeting to cancer cells by enhanced perme-ability and retention effect [1], it is important to understandmechanisms of their delivery into the living cancer cells. Inthis respect we have developed a modular spectral imagingsystem based on a white light spinning disk confocal fluo-rescence microscope and a narrow tunable emission filter.Firstly, interaction of polymer nanoparticles and cells labeledwith spectrally overlapping probes was examined. The use offluorescence microspectroscopy (FMS) allowed co-localiza-tion, which showed that the size of polymer nanoparticlesstrongly influences their transfer across the cell plasmamembrane. Next, the delivery of liposomes (composed ofcancerostatic alkylphospholipid (OPP) and cholesterol)labeled with environment-sensitive fluorescent probe wasmonitored. We were able to detect a very small shift inemission spectra of cholesterol-poor OPP liposomes insideand outside the cells, which would not be possible withoutthe use of FMS. This shift implies that the delivery of theseliposomes into cancer cells is based on fusion with the cellmembrane [2].

[1] V. Torchilin, Adv. Drug Deliv. Rev. 63, 131-135 (2011)[2] Z. Arsov et al., submitted.

O-416Engineering the excited state of fluorophores for highresolution imaging of bio- and soft-matterRanieri Bizzarri,1 Giovanni Signore,2 Paolo Bianchini,3

Gerardo Abbandonato,1,2 Antonella Battisti,2 Andrea Pucci,4

Riccardo Nifosı,1 Alberto Diaspro,3 and Fabio Beltram1,2

1NEST, Scuola Normale Superiore and Istituto Nanoscienze– CNR, Pisa, Italy, 2Center for Nanotechnology Innovation@NEST, Italian Institute of Technology, Pisa, Italy,3Nanophysics group, Italian Institute of Technology, GenovaItaly, 4Dipartimento di Chimica e Chimica Industriale,Universita di Pisa, Italy, r.bizzarri@sns.it

High-resolution optical imaging techniques make nowaccessible the detection of nanofeatures in bio-and soft-

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matter by non-ionizing visible radiation. However, high-res-olution imaging is critically dependent by the fluorescentprobes used for reporting on the nano-environment. Onaccount of our long-standing interest in the development offluorescent probes, we set out to design and engineer newfluorescent systems for nanoscale imaging and sensing ofbiological specimens and soft-matter. These fluorophoresreport on fast subtle changes of their nanoscale environmentat excited state and are meant to fulfill these requirements: a)optical responses (intensity, wavelength-shift, lifetime,anisotropy) predictably related to the environmental polarity,viscosity, macromolecular structure, b) high brightnessallowing for single-molecule detection, c) easily conjugable tobiomolecules or macromolecules of interest. Notably, we aimat conjugating these properties with the capability of nanos-copy imaging based on Stimulated Emission Depletion orStochastic Reconstruction Optical Microscopy. In this lecturethe main features and applications of the engineered probeswill be reviewed and future developments in this exciting fieldwill be discussed.

P-417Novel 3D single particle methods reveal new detailsin the foamy virus entry and fusion processesA. Dupont1, K. Stirnnagel2, D. Schupp1, F. Perrotton1,E. Mullers2, D. Lindemann2 and D.C. Lamb11Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universitat, Munich, Germany, 2Instituteof Virology, Technische Universitat Dresden, MedizinischeFakultat ‘‘Carl Gustav Carus’’, Dresden, Germany

Foamy virus (FV) is an atypical retrovirus which sharessimilarities with HIV and hepatitis B viruses. Despitenumerous biochemical studies, its entry pathway remainsunclear, namely membrane fusion or endocytosis. To tacklethis issue, dual color fluorescent viruses were engineeredwith a GFP labeled capsid and a mCherry labeled enve-lope. Using high resolution 3D imaging and 3D single virustracing, we followed the entry of the fluorescent viruses inliving cells with a precision of 30nm in the plane and 40 nmalong the optical axis. To distinguish between the twopossible pathways, we developed a novel colocalizationanalysis method for determining the moment along everysingle trace where the colors separate, i.e. the fusion event.The combination of this dynamical colocalization informationwith the instantaneous velocity of the particle and its posi-tion within the reconstructed 3D cell shape allows us todetermine whether the separation of capsid and envelopehappens at the cell membrane or from endosomes. Wethen compared two types of FV and demonstrated, con-sistently with previous pH-dependency studies, that theprototype FV can enter the cell by endocytosis and mem-brane fusion, whereas the simian FV was only observed tofuse after endocytosis.

P-418Regulation of PIP2 clustering in neuron-like model cellsby synaptic protein palmitoylationMaria J. Sarmento, Sandra N. Pinto, Fabio Fernandes,Manuel PrietoCentro de Quımica-Fısica Molecular and Instituteof Nanoscience and Nanotechnology, Instituto SuperiorTecnico, Complexo I, Avenida Rovisco Pais, 1049-001Lisbon, Portugal

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minorcomponent of the plasma membrane known to a criticalagent in the regulation of synaptic transmission. Clustering ofPI(4,5)P2 in synaptic active zones is important for synaptictransmission. However, PI(4,5)P2 does not spontaneouslysegregate in fluid lipid membranes and another mechanismmust be responsible for the lateral segregation of this lipid inactive zones. Clustering of PI(4,5)P2 is expected to beassociated with lipid-protein interactions and possibly parti-tion towards lipid rafts in the plasma membrane. Here weanalyze the influence of protein palmitoylation on the for-mation of PI(4,5)P2 clusters and on synaptic protein-PI(4,5)P2 interaction by means of Forster resonance energytransfer measurements by fluorescence lifetime imaging(FRET-FLIM) and FRET confocal microscopy.

P-419Imaging SpoIIIE proteins during Bacillus subtilissporulation by in-vivo PALM microscopyJB. Fiche, N. Dieckmann, E. Margeat, P. Dosset,M. Nollmann, D. CattoniCentre de Biochimie Structurale, CNRS UMR 5048 - UM 1 -INSERM UMR 1054, Montpellier, France

Under adverse conditions, B. Subtilis produces stable, dor-mant endospores to enhance its survival (Kay, 1968; Errington,2003). During sporulation an entire chromosome is transferredinto the forespore. This process starts by the formation of anasymmetrically located division septum that leads to the for-mation of two unequally sized compartments: a large mother-cell and a smaller forespore. The septum traps about 30% ofthe chromosome to be transferred into the forespore. Theremaining (*3Mbp) are then translocated from the mother cellinto the forespore by an active mechanism involving the Spo-IIIE DNA translocase. The mechanisms of translocation,particularly the control of the directionality, still remainsunknown and various models have been proposed so far.Since each model predicts very different distribution ofSpoIIIE proteins at the sporulation septum, we used PALMmicroscopy (photoactivated localization microscopy) toinvestigate proteins localization in live-sporulating bacteria.Using this technique, we showed that SpoIIIE proteins areforming a single tight focus at the septum with a character-istic size of around 30nm. More surprising, the focus isusually localized in the mother cell compartment and the

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mean distance between the SpoIIIE focus and the septum is35nm. Our data suggest that during the translocation pro-cess, SpoIIIE proteins are only forming stable complexes onthe mother cell side, allowing then for a control of the chro-mosome translocation from the mother cell to the forespore.

P-420Fast mobility of Bicoid captured by fluorescencecorrelation spectroscopy in live Drosophila embryosAsmahan Abu-Arish1,2, Aude Porcher3, Chris Cabral1,Nathalie Dostatni3, Cecile Fradin11McMaster University, Hamilton, Canada, 2Present address:McGill University, Montreal, Canada, 3Institut Curie, Paris,France

Morphogenetic gradients determine cell identity in a con-centration-dependent manner and do so in a way that is bothincredibly precise and remarkably robust. In order to under-stand how they achieve this feat, one needs to establish thesequence of molecular mechanisms starting with morphogengradient formation and leading to the expression of down-stream target genes. In fruit flies, the transcription factorBicoid (Bcd) is a crucial morphogen that forms an exponentialconcentration gradient along the embryo AP axis and turns oncascades of target genes in distinct anterior domains. Wemeasured Bcd-EGFP mobility in live D. melanogasterembryos using fluorescence correlation spectroscopy andfluorescence recovery after photobleaching. We found thatBcd-EGFP molecules had a diffusion coefficient on the orderof * 7 lm2/s during nuclear cycles 12-14, both in the cyto-plasm and in nuclei. This value is large enough to explain thestable establishment of the Bcd gradient simply by diffusion.On the other hand, in the context of the extremely preciseorchestration of the transcription of the hunchback Bcd targetgene, it is too slow to explain how a precise reading of Bicoidconcentration could be achieved at each interphase withoutthe existence of a memorization process.

P-421Single molecule studies of key processes duringthe initiation of innate and adaptive immune responseKristina Ganzinger1, James McColl1, Ricardo Fernandez2,Kate Irvine3, Clare E. Bryant3, Nicholas J. Gay4,Simon Davis2, David Klenerman11Departement of Chemistry, University of Cambridge,Cambridge, UK, 2Nuffield Department of Clinical Medicineand MRC Human Immunology Unit, University of Oxford,John Radcliffe Hospital, Headington, Oxford, UK,3Department of Veterinary Medicine, Universityof Cambridge, Cambridge, UK, 4Department of Biochemistry,University of Cambridge, Cambridge, UK

The two pillars of the vertebrate immune system are the innateand adaptive immune response, which confer resistance topathogens and play a role in numerous diseases. Here weexploit single molecule fluorescence imaging on live cells tostudy the key molecular processes that underpin theseresponses. The first project looks at the changes in the organi-sation of Toll-like receptor 4 (TLR4) on the cell surface ofmacrophages upon activation via Lipopolysaccharide (LPS), asit is currently not known whether a higher level of TLR4

organisation is required for thesignallingprocess.Macrophagesnatively express TLR4 at a low level which allows for oligo-merisation to be analysed in live cells by dynamic singlemolecule colocalisation (DySCo) using data obtained by total-internal reflection fluorescence (TIRF) microscopy. The experi-ments of the second project aim at determining the critical initialevents in T-cell triggering by labelling key proteins like the TCRreceptor and CD45 on the surface of live T-cells and followinghow their spatial distributionchanges following thebindingof theT-cell to a surface. This enables us to distinguish between thedifferent models of T-cell triggering which are based on aggre-gation, segregation or a conformational change of the TCR.

P-423The study of cells using scanning force microscopyAmy R. Hall, Ana L. Morales Garcıa, Adrian R. Eley, MatthewE. Rogers, Stephen A. Rolfe, Mark GeogheganThe University of Sheffield, Sheffield, UK, London Schoolof Hygiene and Tropical Medicine, London, UK

The poster is focused on an interdisciplinary area of researchconcerning the surface properties of different cells, includingpathogens. The use of Scanning Force Microscopy (SFM) toimage and determine surface characteristics of Chlamydiatrachomatis bacteria and two species each of Rhodococcusbacteria andLeishmania parasiteswill be discussed. Further tothis, research into the adhesive properties of Leishmaniapromastigotes is proposed. This requires fabrication of differentglycopolymer modified surfaces/tips to replicate some of theconditions experienced by the parasite whilst in the insect host.The prokaryotic and eukaryotic organisms considered havedifferent impacts on the environment; C. trachomatis andLeishmania undergo complex multi-stage lifecycles andcause widespread human disease. This gives the researchimportance in a medical context, providing information thatcould be useful for future drug or vaccine research. Thediverse species of genus Rhodococcus have potential foruse as biofilms for environmental bioremediation.The research should result in a combination of adhesion dataand topographic images of the organisms, allowing accurateinvestigation of surface properties both in ambient conditionsand when imaged in aqueous conditions such as under asaline buffer. Functionalization of SFM tips with hydrophilic orhydrophobic groups allows the study of interactions betweenthe functional molecules and the surface of the organism,thus mapping the hydrophobicity of the organism’s surface.

P-424Tracing the microscopic motility of unicellular parasitesAxel Hochstetter1, Eric Stellamanns2, Sravanti Uppaluri2,Niko Heddergott3, Markus Engstler3, Thomas Pfohl1,21Departement Chemie, Universitat Basel, Basel,Switzerland, 2Max-Planck-Institut fur Dynamik undSelbstorganisation, Gottingen, Germany, 3Biozentrum,Universitat Wurzburg, Wurzburg, Germany

The motility of unicellular parasites in mammalians seemsvery interesting, yet very complex. In a world, were inertiacannot be used for propulsion, in a world at low Reynolds

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numbers, most of our everyday strategies of self-propulsiondo not work.One class of parasites that know their way around, the fla-gellate Trypansome, manage not only to survive in the bloodstream, which is a lot faster than its own propulsion velocityand where the Trypanosome is constantly attacked by itshost’s immune response, but also to penetrate the blood-brain-barrier, which actually should be to tight to enter. Eventhough Trypanosomes are known for more than 100 years,their motility behaviour is not completely elucidated yet.Now, using high-speed darkfield-microscopy in combinationwith optical tweezers in microfluidic devices and analyzingthe recorded data, new light has been shed on the motility ofthese parasites. Astonishing results show that Trypano-somes are very well adapted to their hosts environment, theyeven can abuse red blood cells for their self-propulsion anduse the bloodstream itself to drag antibodies bound to theirsurface to their cell mouth, where the antibodies are endo-cytosed and digested.

O-425FRET approaches to signal transduction: from QDbiosensors to live-cell EGFR signalingThomas M. Jovin1, M. Julia Roberti2, SebastianA. Dıaz2, Guillermo O. Menendez2, ElizabethA. Jares-Erijman2, Iwona Ziomkiewicz1,and Donna J. Arndt-Jovin11Laboratory of Cellular Dynamics, Max Planck Institutefor Biophysical Chemistry (MPIbpc), 37077 Gottingen,Germany, 2Departamento de Quımica Organica, Facultad deCiencias Exactas y Naturales, Universidad de Buenos Aires,Ciudad Universitaria, 1428 Buenos Aires, Argentina; PartnerGroup of the MPIbpc (EAJ-E)

The first part of the presentation will discuss nanoparticle(quantum dot, QD) biosensors and nanoactuators that exploitnovel and unusual FRET phenomena in the induction/detec-tion of protein aggregation [1], reversible on-off QDphotoswitching [2], and pH sensing [3]. The second part of thepresentation will feature the application of an integratedchemical biological FRETapproach for the in situ (in/on livingcells) detection of conformational changes in the ectodomainof a receptor tyrosine kinase (the receptor for the growth factorEGF) induced by ligand binding [4]. The measurements wereconducted with a two-photon scanning microscope equippedwith TCSPC detection; novel methods for lifetime analysis adinterpretation were employed to confirm the concerted domainrearrangements predicted from X-ray crystallography.

[1] Roberti M.J., Giordano L., Jovin T.M., and Jares-ErijmanE.A.’’ FRET Imaging by kt/kf’’. ChemPhysChem 12:563-566 (2011).

[2] Dıaz S.A., Menendez G.O., Etchehon M.H., GiordanoL., Jovin T.M., and Jares-Erijman E.A. ‘‘PhotoswitchableWater-Soluble Quantum Dots: pcFRET Based onAmphiphilic Photochromic Polymer Coating’’. ACSNano5:2795-2805 (2011).

[3] Menendez G.O. et al., manuscript in preparation (2011).[4] Ziomkiewicz I. et al., manuscript in preparation (2011).

P-426Detecting protein conformational states in C. elegansin vivo by confocal fluorescence anisotropyVolker Henschel1, Alessandro Esposito2, EugeniaButkevich1, Christoph F. Schmidt 1, Fred S. Wouters3,Dieter R. Klopfenstein1

Drittes Physikalisches Institut, Georg August UniversitatGottingen, Germany, 2Hutchison/MRC Research Centre,MRC Cancer Cell Unit, Cambridge, UK, 3Abteilung Neuro-und Sinnesphysiologie, Zentrum Physiologie undPathophysiologie, Universitat Gottingen, Germany

The study of protein-protein interactions in vivo is oftenhindered by the limited acquisition speed of typical instru-mentation used, for instance, for lifetime imaging microscopy.Anisotropy polarization is alteredby theoccurrenceof FoersterResonance Energy Transfer (FRET) and anisotropy imagingwas shown to be comparatively fast and simple to implement.Here, we present the adaptation of a spinning disc confocalmicroscope for fluorescence anisotropy imaging that allowedto achieve in vivo imaging at high spatial and temporal reso-lution. We demonstrate the capabilities of this system andin-house developed analysis software by imaging livingCaenorhabditis elegans expressing constitutive dimeric andmonomeric proteins that are tagged with GFP.

O-427Measuring intracellular viscosity: from molecular rotorsto Photodynamic Therapy of cancerMarina K. KuimovaDepartment of Chemistry, Imperial College London,Exhibition Road, SW7 2AZ, UK

Viscosity is one of the main factors which influence diffusionin condensed media and is crucial for cell function. However,mapping viscosity on a single-cell scale is a challenge. Wehave imaged viscosity inside live cells using fluorescentprobes, called molecular rotors, in which the speed of rota-tion about a sterically hindered bond is viscosity-dependent[1-3]. This approach enabled us to demonstrate that viscositydistribution in a cell is highly heterogeneous and that thelocal microviscosity in hydrophobic cell domains can be up to1009 higher than that of water. We demonstrated that theintracellular viscosity increases dramatically during lightactivated cancer treatment, called Photodynamic therapy(PDT) [2]. We also demonstrated that the ability of a fluoro-phore to induce apoptosis in cells during PDT [4], or to act asa benign molecular rotor, measuring viscosity, can be con-trolled by carefully selecting the excitation wavelength inviscous medium [5].

[1] M. K. Kuimova, et al J. Amer. Chem. Soc., 2008, 130,6672

[2] M. K. Kuimova, et al, Nature Chem., 2009, 1, 69[3] J. A. Levitt, et al J. Phys. Chem. C, 2009, 113, 11634[4] H. A. Collins, et al Nature Photonics, 2008, 2, 420-425[5] M. K. Kuimova, et al J. Amer. Chem. Soc., 2009, 131,

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P-428Design of structured microenvironments for single celltransfection studiesC. Leonhardt, G. Schwake and J. O. RadlerLudwig-Maximilians-University Munich,Department of Physics and Center for Nanoscience(CeNS), Geschwister Scholl Platz 1, Muenchen,D-80539, Germany

In the field of biophysics and nanomedicine, the cellularreaction and the kinetics of gene expression after transfec-tion of live cells with plasmid DNA or gene-silencing siRNA isof great interest. In a previous study on the transfectionkinetics of non-viral gene-transfer [1] we realised that thedevelopment of single-cell arrays would be a great steptowards easy-to-analyse, high-throughput transfection stud-ies. The regular arrangement of single cells would overcomethe limitations in image-analysis that arise from whole pop-ulations of cells. In addition to that, the analysis of expressionkinetics at the single-cell can help to identify the cell-to-cellvariability within a cell population. In order to develop suitablesingle-cell arrays, we are currently adjusting the differentparameters of such a microenvironment (e.g. size, shape,surface-functionalisation) in order to end up with a definedsurrounding for single-cell transfection studies. In addition tothat, we try to find the optimal uptake pathway for each of thedifferent applications.

[1] G. Schwake et al., Biotechnol. and Bioeng. 105(4) (2010)805.

P-429Single molecule imaging of amyloid-peptideand the prion protein receptor on neuronsJames McColl1, Kristina Ganzinger1, Priyanka Narayan1,Seema Qamar2, Peter St George-Hyslop2,David Klenerman11University of Cambridge, Chemistry Department,Cambridge, United Kingdom, 2Cambridge Institutefor Medical Research, Cambridge, United Kingdom

The neurodegenerative disorder Alzheimer’s disease (AD)causes cognitive impairment such as loss of episodic mem-ory with ultimately fatal consequences. Accumulation andaggregation of two proteins in the brain - amyloid beta andtau - is a characteristic feature. These soluble proteinsaggregate during the course of the disease and assembleinto amyloid-like filaments. Recently it was found that thetoxicity of soluble amyloid beta oligomers must also be takeninto account for the pathogenesis of cognitive failure in AD. Ifoligomers are the predominant toxic species it would bepertinent to determine how they disrupt and impair neuronalfunction. The prion protein (PrP) receptor has been proposedto mediate amyloid beta binding to neuronal cells. We havecharacterised the interaction of the amyloid beta and the PrPreceptor expressed on hippocampal and neuroblastoma cellsat the single-molecule level. We do not detect any colocal-isation of either the 40 or 42 amino acids variants with thePrP receptor.

O-430SFM study of bacterial cell wallsAna L. Morales-Garcıa1, Amy R. Hall1, Talal Althagafi1,Mark Geoghegan1, Stephen A. Rolfe21Physics and Astronomy Department, The Universityof Sheffield, Sheffield, United Kingdom, 2Animal and PlantScience Department, The University of Sheffield, Sheffield,United Kingdom

Bacterial biofilms are of the utmost importance in the studyof environmental bioremediation and the design of materialsfor medical applications. The understanding of the mecha-nisms that govern cell adhesion must be analysed from thephysics point of view in order to obtain quantitativedescriptors.The genus Rhodococcus is widely spread in natural envi-ronments. The species are metabolically diverse and thusthey can degrade a wide range of pollutants. Due to theirhigh hydrophobicity, these cells are very resistant to harshconditions, are able to degrade hydrophobic substances (e.g.oil) and attach to high-contact angle surfaces. The hydro-phobicity of several strains of Rhodococcus is measured andmapped using Chemical Force Microscopy (CFM) in thepresent study.CFM relies on the functionalisation of Scanning ForceMicroscopy (SFM) tips using hydrophobic or hydrophilicgroups. In CFM, the microscope is operated in the force-volume mode, which combines adhesion data with topo-graphic images. The careful control of the tip chemistrypermits the study of interactions between the functionalgroups on the tip and the bacterial surface, thus allowing theassessment of hydrophobicity.In order to perform a CFM study, the cells need to be firmlyanchored to a substrate under physiological conditions (i.e.under a nutrient media or a saline buffer). To this end, severaladhesive surfaces have been tested in order to find the onethat gives the best results.

P-431Super-resolution microscopy in live-cell imagingPer Niklas Hedde1,3, Susan Gayda1, Karin Nienhaus1

and G. Ulrich Nienhaus1,21Institute of Applied Physics & Center for FunctionalNanostructures, Karlsruhe Institute of Technology,D-76131 Karlsruhe, Germany, 2Department of Physics,University of Illinois at Urbana-Champaign, Urbana, IL61801, USA,3Correspondence should be addressed to uli@uiuc.edu

Optical microscopy is arguably the most important techniquefor the study of living systems because it allows 3D imagingof cells and tissues under physiological conditions underminimally invasive conditions. Conventional far-field micros-copy is diffraction-limited; only structures larger than *200nm can be resolved, which is insufficient for many applica-tions. Recently, techniques featuring image resolutions downto *20 nm have been introduced such as localization

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microscopy (PALM, STORM) and reversible saturable opticalfluorescent transition microscopy (RESOLFT, STED). Thesemethods are well suited for live-cell imaging and narrow theresolution gap between light and electron microscopysignificantly.We have used PALM imaging to study the formation anddisassembly of focal adhesions of live HeLa cells in a highresolution pulse-chase experiment using monomeric IrisFP[1]. mIrisFP is a photoactivatable fluorescent protein thatcombines irreversible photoconversion from a green- to ared-emitting form with reversible photoswitching between afluorescent and a nonfluorescent state in both forms. In ourexperiments a subpopulation of mIrisFP molecules isphotoconverted to the red form by irradiating a specifiedregion of the cell with a pulse of violet light. Migration oftagged proteins out of the conversion region can be studiedby subsequently localizing the proteins in other regions of thecell by PALM imaging, now using the photoswitching capa-bility of the red species. Real-time image reconstructiondeveloped in our lab [2] allowed instant control imagingparameters.

[1] J. Fuchs, S. Bohme, S., F. Oswald, P. N. Hedde, M.Krause, J. Wiedenmann, and G. U. Nienhaus, A Photoacti-vatable Marker Protein for Pulse-chase Imaging withSuperresolution, Nature Methods 7, 627-630 (2010).[2] P. N. Hedde, J. Fuchs, F. Oswald, J. Wiedenmann, andG. U. Nienhaus, Online Image Analysis for PhotoactivationLocalization Microscopy, Nature Methods 6, 689-690(2009).

P-432Fluorescence live cell imaging of A549 cells in presenceof PVP-Hypericin: Modifying excitation light dose of blueLEDRozhin Penjweini1, Hans G. Loew1, 2, Maria Eisenbauer3,Paul Breit3 and Karl W. Kratky11University of Vienna, Faculty of Physics, Physicsof Physiological Processes, 2Medical University of Vienna,Department for Therapeutic Radiology, 3Medical Universityof Vienna, Cancer Research Institute, Vienna, Austria

Live cell imaging of cancer cells is often used for in-vitro studiesin connection with photodynamic diagnostic and therapy (PDDand PDT). Especially in presence of a photosensitizer, this livecell imaging canonly beperformedover relatively short duration(at most 1 hour). This restriction comes from the light-inducedcell damages (photodamages) that result from rapid fluores-cence photobleaching of photosensitizer. While these studiesreveal exciting results, it takes several hours to discover thedetailed effects of the photosensitizer on cell damage. Up to ourknowledge, however, there is no general guideline for modifi-cation of excitation light dose to achieve that.In this paper, the relation between excitation light doses,photobleaching of photosensitizer (PVP-Hyperycin) and cellvitality are investigated using human lung epithelial carcinomacells (A549). The strategy of this paper is to reduce the exci-tation light dose by using a low-power pulsed blue LED suchthat the structures are visible in time-lapse images. Fluores-cence signals and image quality are improved by labelling thecells with an additional non-toxic marker called carboxy-fluorescein-diacetate-succinimidyl-ester (CFSE). In total wecollected 2700 time-lapse images (time intervals 2 min) ofdual-marked A549 cells under three different light intensities(1.59, 6.34 and 14.27 mW/cm2) and a variety of pulse lengths(0.127, 1.29, 13, 54.5 and 131 ms) over five hours. We havefound that there is a nonlinear relationship between the amountof excitation light dose and cell vitality. Cells are healthy, i.e.they commence and complete mitosis, when exposed to lowlight intensities and brief pulses of light. Light intensities higherthan 6.34 mW/cm2 together with pulse durations longer than13 ms often cause cell vesiculation, blebbing and apoptosis. Inall other cases, however, we found no cell death. In the future,this striking nonlinearity will be studied in more detail.

P-433Intranuclear visualization and distribution analysisof Pre Integration Complexes in MLV and HIV-1 infectedcellsV. Quercioli1, C. Di Primio1, L. Mulder2 and A. Cereseto31Laboratory of Molecular Biology, Scuola Normale Superioreof Pisa, Piazza dei Cavalieri 7, Pisa, Italy, 2Laboratoryof Microbiology, Mount Sinai School of Medicine, New York,NY, 3Laboratory of Molecular Virology, Centre for IntegrativeBiology (CIBIO), University of Trento, Trento, Italy

Here we report for the first time the visualization of MLV viralparticles in the nuclear compartment of infected cells. We

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developed a fluorescent MLV virus by fusing GFP to the in-tegrase (IN) of MLV. This system provides the possibility toefficiently detect MLV Pre Integration Complexes (PIC) insidethe nuclei of infected cells. A detailed analysis of the spatialdistribution of these fluorescently labelled PICs inside thenucleus has been performed: infected cells have beentransfected with a fluorescent H2B histone or stained withepigenetic markers of heterochromatin (H3K9me3) oreuchromatin (H4K16ac). Moreover, a comparative analysiswas performed relatively to fluorescently labelled HIV-1 PICs(visualized by the trans-incorporation of IN-EGFP into theviral particle). The proposed system allows a detailed char-acterization of the intranuclear distribution of MLV fluorescentviral particles in infected cells and represents a powerful toolto investigate potential interactions between MLV PICs andcellular factors.

O-434Multifunctional ion conductance microscopyfor biophysical studies at nanoscaleAndrew I. ShevchukExperimental Physiology, Division of Experimental Medicine,Department of Medicine, Imperial College London, London,UK (a.shevchuk@ic.ac.uk)

Progressive advances in scanning ion conductance micros-copy (SICM)[1] enabled us to convert ordinary scanningprobe microscope (SPM) in to versatile multifunctional tech-nique. As an imaging tool, ion conductance microscopy iscapable to deliver highest possible topographical resolutionon living cell membranes among any other microscopytechniques[2]. Also, it can visualize surfaces complexity ofthose makes them impossible to image by other SPMs[3]. Ionconductance microscopy combined with a battery of powerfulmethods such as fluorescence resonance energy transfer(FRET) [4], patch-clamp, force mapping, localized drugdelivery, nano-deposition and nano-sensing is unique amongcurrent imaging techniques. The rich combination of ionconductance imaging with other imaging techniques such aslaser confocal and electrochemical [5] will facilitate the studyof living cells and tissues at nanoscale.

References

[1] Hansma PK, Drake B, Marti O, Gould SA, Prater CB. Thescanning ion-conductance microscope. Science 1989; 243:641-643.[2] Shevchuk AI, Frolenkov GI, Sanchez D, James PS,Freedman N, Lab MJ, Jones R, Klenerman D, Korchev YE.Imaging proteins in membranes of living cells by high-reso-lution scanning ion conductance microscopy. Angew. Chem.Int. Ed Engl. 2006; 45: 2212-2216.[3] Novak P, Li C, Shevchuk AI, Stepanyan R, Caldwell M,Hughes S, Smart TG, Gorelik J, Ostanin VP, Lab MJ, MossGW, Frolenkov GI, Klenerman D, Korchev YE. Nanoscalelive-cell imaging using hopping probe ion conductancemicroscopy. Nat. Methods 2009; 6: 279-281.[4] Nikolaev VO, Moshkov A, Lyon AR, Miragoli M, Novak P,Paur H, Lohse MJ, Korchev YE, Harding SE, Gorelik J. Beta2-adrenergic receptor redistribution in heart failure changescAMP compartmentation. Science 2010; 327: 1653-1657.

[5] Takahashi Y, Shevchuk AI, Novak P, Murakami Y, Shiku H,Korchev YE, Matsue T. Simultaneous noncontact topographyand electrochemical imaging by SECM/SICM featuring ioncurrent feedback regulation. J. Am. Chem. Soc. 2010; 132:10118-10126.

P-435Fluorescence of living cells during senescenceand stressNatalja Shkute, Juliana Orlova, Alexandr PetjukevichInstitute of Ecology, Daugavpils University, Daugavpils,Latvia

Fluorescence of cells is an effective method which allows fordefining changes in a set of cell metabolites initially, directlyin living cells even at insignificant damage. Green plants emita red fluorescence (RF, 630-700 nm), a far-red fluorescence(FRF, 700-800 nm), both from chlorophyll, and a blue-greenfluorescence (BGF, 400-630 nm) vacuoles of epidermis thatare related to secondary plant metabolism and UV- protec-tion and piridine [NAD(P)H] and flavine nucleotides, thecofactors of metabolism. The emission spectrum depends onplant species, stage of development, anatomy and environ-mental factors experienced by the plant.First leaves of wheat (Triticum aestivum L.), march penny-wort (Hydrocotyle vulgaris L.) and coleoptiles of wheat(Triticum aestivum L.) seedlings, which were growing in lightand dark conditions, were used to determine fluorescenceof whole cells. Fluorescence emission spectrum was mon-itored by fluorescent microscopy using the spectrometerUSB 4000. Fluorescence intensity F490, F680, F710 andF740 was determined and data was statistically analyzed inANNOVA. We observed that BGF, RF and FRF intensityincreased in the first leaves with the age of the seedlings. Inthe coleoptiles was observed great BGF intensity increasewith the age of the seedlings. In the coleoptiles decreasedRF intensity of the 144 and 196 hours old seedlings, andBGF intensity decreased of 196 hours old seedlings. It wasfound that emission spectrum and fluorescence intensitychanges are induced by the lack of light and salt (NaCl)stress. Analysis of fluorescence spectrum can quickly andaccurately indicate the outset of light and salt stress inplants.There are analogical changes in fluorescence emissionspectrum of plant cells in senescence and stress conditions.It was assumed that environmental stress and senescencehave common mechanisms in plants. This changes can bemonitored by fluorescent microscopy.

O-437Triple-colour super-resolution imaging in living cellsMarkus Staufenbiel, Stephan Wilmes, Domenik Lisse,Friedrich Roder, Oliver Beutel, Christian Richter and JacobPiehlerUniversitat Osnabruck, Fachbereich Biologie, Barbarastraße11, 49076, Germany, markus.staufenbiel@biologie.uni-osnabruck.de

Super-resolution fluorescence imaging techniques basedon single molecule localisation has opened tremendous

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insight into the sub-micrometre organisation of the cell.Live cell imaging techniques such as fluorescence photo-activation localization microscopy (FPALM) are currentlylimited to dual-colour detection due to the restricted avail-ability of red-fluorescent photoswitchable proteins. Weemployed photoswitching of the oxazine dye ATTO655under reducing conditions for super-resolution imaging inthe cytoplasm of living cells. For efficient and specificcovalent labelling of target proteins, we have made use ofthe HaloTag system. ATTO655 was coupled to the HaloTagLigand (HTL) and fast reaction of HTL-ATTO655 with theHaloTag enyzme was confirmed in vitro by solid phasebinding assays. Efficient labelling of the membrane cyto-skeleton using Lifeact fused to the HaloTag was observedand super-resolution imaging was readily achieved. Basedon this approach, we managed to follow the nanoscaledynamics of the actin cytoskeleton as well as clathrin-coated pits using clathrin light chain fused to the HaloTag.We combined this technique with FPALM for triple-colour super-resolution imaging of the spatial distributionof membrane receptors in context of the membraneskeleton.

O-438Simultaneous, multicolour single molecule imagingof the entire ErbB receptor family in live cellsStephen Webb, Christopher Tynan, Laura ZanettiDomingues, Benjamin Coles, Selene Roberts, Daniel Rolfe,Sarah Needham, Michael Hirsch, David Clarke and MarisaMartin-FernandezResearch Complex at Harwell, STFC Rutherford AppletonLaboratory, Didcot, United Kingdom

The ErbB family of receptor tyrosine kinases consists offour transmembrane proteins that transduce signals acrossthe membrane to control cell fate. Growth factor bindingresults in homo- and hetero-interactions between thesereceptors at the membrane. ErbB receptors are implicatedin many cancers, making them a target for therapeuticdrugs. To date, studies of ErbB interactions have beenlimited to individual family members or specific pairs, givingan incomplete picture of the highly complex behaviourcontrolling positive and negative feedback loops and sig-nalling outcomes.To investigate ErbB receptor interactions, we have devel-oped TIRF-based single molecule fluorescencemicroscopes capable of simultaneously imaging three, andsoon five, fluorescence probes in live cells. We have alsodeveloped a catalogue of extrinsic fluorescent probes for1:1 labelling of both endogenous and transfected ErbBfamily members in mammalian cells, plus a Bayesianapproach to the analysis of single molecule data. Thisallows us to track active and inactive ErbB family membersat the basal surface of a model breast cancer cell line thatexpresses physiological levels of all four receptors. Wepresent here initial characterisation of the entire ErbB familytogether in the cell membrane.

P-439Revealing the stoichiometry of endogenous G protein-coupled receptors (GPCRs) at the surface of live cellsusing single molecule imagingLaura Weimann1, James McColl1, James Felce2, SimonDavis2, David Klenerman11Departement of Chemistry, University of Cambridge,Cambridge, UK, 2Nuffield Department of Clinical Medicineand MRC Human Immunology Unit, University of Oxford,John Radcliffe Hospital, Headington, Oxford, UK

The human genome contains more than 800 G protein-coupled receptors (GPCRs); overall, 3-4% of the mamma-lian genome encodes these molecules. Processescontrolled by GPCRs include neurotransmission, cellularmetabolism, secretion, and immune responses. However itis the stoichiometry of these receptors that is the mostcontroversial. The starting point for understanding GPCRfunction was the idea that these receptors are monomeric.On the other hand a lot of recent studies favour the conceptthat GPCR form dimers and are not capable of signalling asindependent monomers. Recent single molecule studies tryto solve this dilemma by suggesting that GPCRs formtransient dimers with a lifetime of *100 ms. Howeverquestions remain about the physiological relevance of thepreparations necessary for these studies, since they havenot been performed on endogenous receptors. Here, wedirectly image individual endogenous receptors using anequimolar mixture of two colour fluorescent Fab fragments.We can then determine the receptors stoichiometry byquantifying its dynamic single molecule colocalisation (Dy-SCo) recorded by total-internal reflection fluorescence(TIRF) microscopy.

Trends in neutron scattering for biology

O-440Function of phosphoglycerate kinase enabled by largedomain movementsR. Biehl1,2, R. Inoue1, T. Rosenkrantz2, J. Fitter2

and D. Richter1,21Julich Centre for Neutron Science, ForschungszentrumJulich, Germany, 2Institute for Complex Systems,Forschungszentrum Julich, Germany, 3Institute for ChemicalResearch, Kyoto University, Kyoto, Japan

The biological function of enzymes is often related to large-scale domain movements. Configurational changes areobserved by methods like x-ray crystallography, which give astatic image of the protein. Are these changes due to thesubstrate binding or are they also related to the crystalconfinement, which favors specific configurations? In solu-tion the protein can have a different configurations andundergo large scale fluctuations.Phosphoglycerate kinase (PGK) has a widely open domainstructure with a hinge near to the active center between the

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two domains. The hypothesis of a substrate-induced con-figuration change, was first proposed by Banks et al. basedon the comparison of crystal structures.We have recently investigated the domain dynamics of PGK(1). Structural analysis by small angle neutron scatteringrevealed that the structure of the holoprotein in solution ismore compact as compared to the crystal structure, butwould not allow the functionally important phosphoryl transferbetween the substrates, if the protein would be static.Brownian large scale domain fluctuations on a timescale of50 ns was revealed by neutron spin echo spectroscopy. Theobserved dynamics shows that the protein has the flexibilityto allow fluctuations and displacements that seem to enablefunction.

[1] Inoue et al., Large Domain Fluctuations on 50-nsTimescale Enable Catalytic Activity in PhosphoglycerateKinase, Biophysical Journal (2010), 99, 2309-2317

O-441Deciphering membrane insertion of the diphtheria toxintranslocation domainAlexandre Chenal1,2,3, Lydia Prongidi-Fix3, Aurelie Perier2,Christopher Aisenbrey3, Gregory Vernier2, StephanLambotte3, Giovanna Fragneto4, Burkhard Bechinger3,Daniel Gillet2, Vincent Forge2, and Michel Ferrand21Institut Pasteur, Paris, France, 2CEA, Grenoble and Gif surYvette, France, 3Institut de Chimie, Strasbourg, France,4Institut Laue Langevin, Grenoble, France,E-mail : alexandre.chenal@pasteur.fr

Many physiological and pathological processes involveinsertion and translocation of soluble proteins into and acrossbiological membranes. However, the molecular mechanismsof protein membrane insertion and translocation remainpoorly understood. Here, we describe the pH-dependentmembrane insertion of the diphtheria toxin T domain in lipidbilayers by specular neutron reflectometry and solid-stateNMR spectroscopy. We gained unprecedented structuralresolution using contrast-variation techniques that allow us topropose a sequential model of the membrane-insertion pro-cess at angstrom resolution along the perpendicular axis ofthe membrane. At pH 6, the native tertiary structure of the Tdomain unfolds, allowing its binding to the membrane. Themembrane-bound state is characterized by a localization ofthe C-terminal hydrophobic helices within the outer third ofthe cis fatty acyl-chain region, and these helices are orientedpredominantly parallel to the plane of the membrane. Incontrast, the amphiphilic N-terminal helices remain in thebuffer, above the polar headgroups due to repulsive elec-trostatic interactions. At pH 4, repulsive interactions vanish;the N-terminal helices penetrate the headgroup region andare oriented parallel to the plane of the membrane. TheC-terminal helices penetrate deeper into the bilayer andoccupy about two thirds of the acyl-chain region. Thesehelices do not adopt a transmembrane orientation. Interest-ingly, the T domain induces disorder in the surroundingphospholipids and creates a continuum of water moleculesspanning the membrane. We propose that this local desta-bilization permeabilizes the lipid bilayer and facilitates thetranslocation of the catalytic domain across the membrane.

O-442Fluorinated surfactants (FSs) for studying membraneproteins (MPs)C. Ebel1, C. Breyton1,2, F. Gabel1, M. Abla3, Y. Pierre2,F. Lebaupain2, G. Durand3, J.-L. Popot2, B. Pucci3

IBS, CEA-CNRS-Univ. Grenoble, France., 2IBPC, CNRS-Univ. Paris-7, Paris, France, 3LCBSM, Univ. Avignon et Paysde Vaucluse, France

The limited stability in vitro of MPs motivates the search ofnew surfactants (1-4). FSs with a polymeric hydrophilichead proved to be mild towards MPs (1).New FSs weredesigned with chemically defined polar heads for structuralapplications. Lac- derivative was efficient in keeping severalMPs water soluble and active but formed elongated rods(2). The Glu- family was synthesized, characterized in bySANS and AUC and for its biochemical interest. The for-mation of rods is related to the low volumetric ratio betweenthe polar head and hydrophobic tail. The surfactant bearingtwo Glucose moieties is the most promising one, leading toboth homogeneous and stable complexes for both BR andthe b6f. It was also shown be of particular interest for thestructural investigation of membrane proteins using SANS(3).

(1) Breyton et al 2004 FEBS Lett 564, 312.(2) Lebaupain et al 2006 Langmuir 22, 8881.(3) Breyton et al 2009 Biophys J 97, 1077.(4) Gohon et al 2008 Biophys J 94, 3523.

O-444Protein dynamics explored by Mossbauer effectand neutron scatteringHans FrauenfelderLos Alamos National Laboratory, Los Alamos, NM, USA

Protein functions are determined by structure and dynam-ics. The naked protein provides the structures that permitthe motions necessary for the functions. The bulk solventand the hydration shell are crucial for the control of themotions through producing two types of fluctuations, calleda and bh. The a fluctuations originate in the bulk solvent,affect the shape of the protein, and open channels. The bhfluctuations originate in the protein’s hydration shell, dependcritically on hydration, but are independent of the solventviscosity. They control internal motions1. To prove theconnection between external fluctuations and proteinmotions tools are needed that measure fluctuations andmotions externally and internally over broad ranges of timeand temperature. The Mossbauer effect and quasielasticneutron scattering are such tools. In proteins both tech-niques show a rapid increase of the mean-square-displacement (msd) above about 180 K. The increase isaccompanied by the appearance of a broad band. Thestandard explanation of the increase, dubbed ‘‘dynamicaltransition’’, is controversial and no satisfactory explanationfor the broad band has been put forward. A new interpre-tation of the Mossbauer effect in proteins proves that thereis no dynamical transition and that the separation into asharp line and a broad band is misleading. The broad bandis inhomogeneous and is composed of sharp lines thathave been shifted by bh fluctuations in the protein’s

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hydration shell and a fluctuations in the bulk solvent. Incrystals the a fluctuations are absent. Using the dielectricspectrum of the bh fluctuations, we predict the shape of theMossbauer spectrum from 80 K to 295 K with one dimen-sionless coefficient. The explanation is important for thedynamics of proteins and for the interpretation of quasi-elastic neutron scattering in complex systems.

1. H. Frauenfelder et al. Proc. Natl. Acad. Sci. USA 106,5129 (2009).

P-445The dynamic landscape in the multi-subunit protein,apoferritin, as probed by high energy resolution neutronspectroscopyMark. T. F. Telling1, 2, Cameron Neylon2, Luke Clifton2,Spencer Howells2, Lambert van Eijck3, and Victoria GarcıaSakai21Department of Materials, University of Oxford, Oxford, UK,2ISIS Facility, STFC, Rutherford Appleton Laboratory,Chilton, UK., 3TU Delft, Delft, The Netherlands

By combining elastic and quasi-elastic neutron scatteringdata, and by applying theory originally developed to investi-gate dynamics in glassy polymers, we have shown that inlyophilised apoferritin above T * 100 K the dynamicresponse observed in the pico- to nano-second time regimeis driven by CH3 dynamics alone, where the methyl speciesexhibit a distribution of activation energies. Our results sug-gest that over the temporal and spatial range studied themain apoferritin peptide chain remains rigid. Interestingly,similar results are reported for other smaller, more flexiblelyophilised bio-materials. We believe this work elucidatesfundamental aspects of the dynamic landscape in apoferritinwhich will aid development of complex molecular dynamicmodel simulations of super-molecules. A detailed apprecia-tion of the relationships between dynamics and biologicalfunction will require analysis based on such models thatrealize the full complexity of macromolecular material.

P-446Dynamics of DPPC vesicles in the presenceof bioprotectantsVictoria Garcıa Sakai1, Hirsh Nanda2,Antonio Faraone2,3, Michihiro Nagao2,41ISIS Facility, STFC, Rutherford Appleton Laboratory,Chilton, UK, 2NIST Center for Neutron Research, NIST,Gaithersburg, MD,USA, 3Dept. of Materials Science &Engineering, University of Maryland, College Park, MD, USA,4Indiana University, Bloomington, IN, USA

Biological systems must often be stored for extended periodsof time. This is done by lyophilisation in the presence oflyoprotectants, such as sugars, which results in stableproducts at ambient conditions.[1] In an effort to understandthe mechanism of preservation and stabilization, the inter-actions between sugars and liposome vesicles, which serve

as a simple membrane model, have been studied exten-sively. Amongst the common sugars, trehalose has superiorpreservative effects [1] and accumulates to high concentra-tions in many anhydrobiotic organisms.Despite many experimental and numerical studies threemechanisms are proposed: vitrification [2], preferentialexclusion [3] and water replacement [4]. To gain more insightinto the stabilization mechanism we have recently investi-gated the effect of trehalose on the bending elasticity of fullyhydrated unilamellar vesicles of 1,2-dipalmitoyl-phosphati-dylcholine (DPPC) in D2O at temperatures below and abovethe lipid melting transition (Tm) using neutron spin-echo. Thedata was analyzed using the Zilman-Granek theory. At alltemperatures measured, trehalose stiffens the bilayer sug-gesting strong interactions between trehalose and the lipid.Trehalose appears to broaden the melting transition but doesnot change the Tm. This agrees with observations usingdifferential scanning calorimetry.

[1] L. Crowe et al, Arch. Biochem. Biophys 242 (1985)240-247.

[2] J. H. Crowe et al, Annu. Rev. Physiol. 6 (1998) 73-103.[3] S. N. Timasheff in Protein-Solvent Interactions (Dekker,

NY, 1995) 445-482.[4] J. H. Crowe et al, in The Roles of Water in Foods

(Chapman & Hall, NY, 1998) 440-455

O-447Influence of macromolecular crowding on proteinstabilityStephane Longeville, Clemence Le CoeurLaboratoire Leon Brillouin, Gif-sur-Yvette, France

Cell interior is a complex environment filled with a variety ofdifferent objects with respect to shape and size. Macromol-ecules are present at a total concentration up to severalhundred grams per litre and the overall occupied volumefraction can reach o & 0.3-0.4. Under crowding environmentprotein- protein interaction play a fundamental role. Thecrowding environment can affect some physical, chemical,and biological properties of biological macromolecules [1,2].Traditionally, protein folding is studied in vitro at very lowconcentration of proteins. Under such conditions, smallglobular single chain proteins can unfold and refold quiterapidly depending mainly to the nature of the solvent. Suchprocesses have been very intensively studied, since foldingof proteins into their native structure is the mechanism, whichtransforms polypeptide into its biologically active structure.Protein misfolding is involved in a very high number of dis-eases [4] (e.g. Alzheimer, Parkinson, and Kreuzfeld-Jacobdiseases, type II diabetes, …).Theoretically, the problem was studied by the introduction ofthe concept of excluded volume [5]. In recent papers [6,7],Minton uses statistical thermodynamic models to address thequestion. He predicted that inert cosolutes stabilize thenative state of proteins against unfolding state mainly bydestabilizing the unfolded state and that the dimension of theunfolded state decreases with increasing the concentrationof cosolute in a measurable way.

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S156 Trends in neutron scattering for biology

Small Angle Neutron Scattering (SANS) is a technique ofchoice for such study because, by using appropriate mix-tures of light and heavy water, it is possible to match thescattering length density of the solvent to the one of thecosolute and thus to measure the conformation of a moleculeat low concentration in a presence of a high concentration ofanother one.We will present a complete experimental study of themechanism that leads to protein stabilization by macromo-lecular crowding [7,8].

[1] R. J. Ellis, Trends in Biochem Sciences 26 (2001)597-604.

[2] A.P. Minton, J Biol Chem 276 (2001) 597-604.[3] R. J. Ellis, Nature 416 (2002). 483-484.[4] C. M. Dobson, Nature 426 (2003) 894-890.[5] Y. Zhou and C.K. Hall, Biopolymers 38 (1996) 273-284.[6] A.P. Minton, Biophysical J 78 (2000) 101-109.[7] A.P. Minton, Biophysical J 88 (2005) 971-985.[8] C. Le Coeur, B. Deme, and S. Longeville, Phys Rev E,

79 (2009) 031910.[9] C. Le Coeur, J. Teixeira, P. Busch, and S. Longeville,

Phys Rev E, 81 (2010) 061914.

P-448Coupled dynamics of protein and hydration waterstudied by inelastic neutron scattering and moleculardynamics simulationHiroshi Nakagawa1,2, Mikio Kataoka1,31Japan Atomic Energy Agency, Tokai, Japan, 2Juelich Centrefor Neutron Science, Forschungszentrum Juelich GmbH,3Nara Institute of Science and Technology, Ikoma, Japan

Proteins work in an aqueous environment at ambient tem-perature. It is widely accepted that the proteins are flexibleand mobile. The flexibility and mobility, that is, proteindynamics are essential for protein functions. Neutron inco-herent scattering is one of the most powerful techniques toobserve protein dynamics quantitatively.Here I will talk about dynamics of protein and its hydrationwater. The structure of a soluble protein thermally fluctuatesin the solvated environment of a living cell. Understandingthe effects of hydration water on protein dynamics isessential to determine the molecular basis of life. However,the precise relationship between hydration water and proteindynamics is still unknown because hydration water is ubiq-uitously configured on the protein surface. We found thathydration level dependence of the onset of the proteindynamical transition is correlated with the hydration waternetwork. Hydration water dynamics change above thethreshold hydration level, and water dynamics control proteindynamics. These findings lead to the conclusion that thehydration water network formation is an essential property

that activates the anharmonic motions of a protein, which areresponsible for protein function.

O-449Nanobiology: membranes and proteins in motionMaikel C. RheinstadterMcMaster University, Hamilton, Ontario, and CanadianNeutron Beam Centre, Chalk River Laboratories, Ontario,Canada, rheinstadter@mcmaster.ca

One of the major challenges of neutron scattering is tocontribute to biology and life-sciences. Modern neutronfacilities and in particular the upcoming European SpallationSource are premier research tools to tackle this importantchallenge. Molecular dynamics and interactions in mem-branes can be studied under physiological conditions usingneutron beams. I will present exciting recent results andpotential applications to demonstrate capabilities of presentand future neutron instrumentation.

O-451Thermal fluctuations of hemoglobin from differentspeciesAndreas M. Stadler,*1 Christopher J. Garvey2, FrancescaNatali3, Ilya Digel4, Gerhard Artmann4, and GiuseppeZaccai31Research Center Julich, Julich Center for Neutron ScienceJCNS-1 and Institute for Complex Systems ICS-1, 52425Julich, Germany, 2Australian Nuclear Scienceand Technology Organisation, Locked Bag 2001, KirraweeDC, NSW 2232, Australia, 3CNR-IOM, c/o Institut Laue-Langevin, 6 rue Jules Horowitz, 38042 Grenoble, France,4Aachen University of Applied Science, IfB, 52428 Julich,Germany

Thermal motions and stability of hemoglobin of three endo-therms (platypus - Ornithorhynchus anatinus, domesticchicken - Gallus gallus domesticus and human - Homo sapi-ens) and an ectotherm (salt water crocodile - Crocodylusporosus) were investigated using neutron scattering and cir-cular dichroism. The results revealed a direct correlationbetween the dynamic parameters, melting -, and body tem-peratures. On one hand, a certain flexibility of the protein ismandatory for biological function and activity. On the otherhand, intramolecular forces must be strong enough to stabilizethe structure of the protein and to prevent unfolding. Our studypresents experimental evidence which support the hypothesisthat the specific amino acid composition of Hb has a significantinfluence on thermal fluctuations of the protein. The amino acidsequence of Hb seems to have evolved to permit an optimalflexibility of the protein at body temperature. Macromolecularresilience was found to increase with body and melting tem-peratures, thus regulating Hb dynamics.

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Trends in neutron scattering for biology S157

Cytoskeleton and cell migration

P-452Optical trapping microrheology in cultured human cellsE.Bertseva1, D.Grebenkov2, S.Jeney1, L.Forro11Laboratory of Physics of Complex Matter, Ecolepolytechnique federale de Lausanne (EPFL), Station 3,CH–1015 Lausanne VD, Switzerland., 2Laboratoire dePhysique de la Matiere Condensee, CNRS – EcolePolytechnique, F-91128 Palaiseau, France

Introduction: We present the microrheological study of thetwo close human epithelial cell lines: non-malignant HCV29and cancerous T24. The optical tweezers tracking wasapplied to extract the trajectories of endogenous lipid gran-ules which were analyzed using a recently proposedequation for mean square displacement (MSD)1.Methods: The set-up is based on a Nd:YAG laser, with thewavelength of 1064 nm. The endogenous granules ofdiameter about 0.4 lm have been chosen as the microrhe-ological tracers, and the tracer trajectory is recorded throughthe back focal plane interferometric position detection.Results: Our 1MHz detection scheme has allowed toobserve the subdiffusion inside the living cells for the lagtimes region 10-5-10-3 s. At long time the MSD becomesinfluenced by the optical potential and tends to a constant.Recently, Desposito and Vinales have derived the analyticalformula for the MSD in the case of subdiffusion influenced byan optical trap1:

Dr2ðtÞ� �

¼ 2kBT

kn 1� Ea �

k

cata

� �� �; ð1Þ

Where k is the trap spring constant, a is the subdiffusionexponent and Ea is the Mittag-Leffler function. Theparameters obtained by fitting this equation to theexperimental MSDs are summarized in Table 1.Discussion and Conclusions:At short lag times we have not found any difference betweenthe two cell types, contrarily to the previous results obtainedby AFM2. For both cell lines the subdiffusion exponent, a wasfound close to �, the value predicted by the theory ofsemiflexible polymers. But the crossover frequency X3, wasfound smaller for the cancerous cells for all datasets. It cor-responds to passage to the confined regime at longer times.

We attribute it to the bigger impact of molecular motors. Thecancerous cells show the larger MSDs at long lag time, whatconfirms the literature results for other cell lines3 and pro-vides the explanation for the AFM results which have beenobtained at low frequency.

References:1M. A. Desposito and A. D. Vinales; 2009; Physical ReviewE; 80: 021111-021117.2S. E. Cross and et al; 2008; Nanotechnology; 19: 384003.3Y. Li, J. Schnekenburger and M. H. G. Duits, Journal ofBiomedical Optics, 2009, 14, 064005

AcknowledgementsThe authors thank Dr. M. Lekka for providing the cell lines,and Dr. J.Lekki for the technical support in the instrumentalpart. We also thank Dr. A.Kulik for useful discussions. Ele-na Bertseva thanks for support the National Swiss FondMHV subsidy PMPDP2_134178/1.

O-453Dynamics of confined actin networks in microchambersS. Deshpande1, D. Steinhauser2, T. Pfohl 1,21Chemistry Department, University of Basel, Switzerland,2Max Plank Institute for Dynamics and Self-Organization,Gottingen, Germany

We study the spatiotemporal evolution of fibrous proteinnetworks present in the intracellular and extracellular matri-ces. Here, we focus on the in vitro actin network dynamicsand evolution. In order to study the hierarchical self-assem-bly of the network formation in a confined environment andprovide external stimuli affecting the system minimally, weintroduce a new microfluidic design.The microfluidic setup consists of a controlling channel towhich microchambers of different shapes and sizes areconnected through narrow channels. This design results inhaving mainly convective transport in the controlling channeland diffusive transport into the microchamber. Rhodaminelabeled actin monomers diffuse into the chamber. Afterpolymerization is induced, they form a confined entanglednetwork. Cross-linking proteins can then be added toincrease the network complexity. Moreover, we can generategradients of reactants across the microchambers and

Table 1 Parameters obtained from fitting Eq. (1) to the MSD of the granules inside cells. The uncertainty is the standarddeviation of parameters between different granules

Trap stiffness (N/m) Cell line 2kBT/k(nm2) a X3 ¼ k

ca

� �1a(Hz)

4�10-6 HCV29 1277±721 0.70±0.07 11±9

T24 1413±669 0.69±0.07 6±3

10-5 HCV29 679±258 0.73±0.02 36±18

T24 487±198 0.67±0.05 10±7

3�10-5 HCV29 186±58 0.73±0.04 172±88

T24 218±52 0.75±0.03 166±103

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S158 Cytoskeleton and cell migration

observe the response of the actin networks. These networksare analyzed for various network properties such as lengthdistribution of links, connectivity distribution of nodes as wellas fluctuations of links and nodes.

P-454Carbon nanotubes as mechanical probes of equilibriumand non-equilibrium cytoskeletal networksNikta Fakhri1, Jorg Enderlein1, Matteo Pasquali2, FrederickC. MacKintosh3, Christoph F. Schmidt11Third Institute of Physics - Biophysics, Georg-August-Universitat, Gottingen, Germany, 2Department of Chemicaland Biomolecular Engineering, Smalley Institutefor Nanoscale Science and Technology, Rice University,Houston, Texas, USA, 3Department of Physicsand Astronomy, Vrije Universiteit, Amsterdam,The Netherlands

Mechanics of cells are determined by the filamentous proteinnetworks of the cytoskeleton. The activity of motor proteinstypically creates strong fluctuations that drive the system outof equilibrium. In order to characterize the networks oneneeds probes that ideally span the characteristic lengthscales between nanometers and micrometers. Single-walledcarbon nanotubes (SWNTs) are nanometer-diameter fila-ments with tunable bending stiffness. On a Brownian energyscale they have persistence lengths of about 20 – 100micrometers and show significant thermal fluctuations on thecellular scale of a few microns. Diffusive motion and localbending dynamics of SWNTs embedded in a polymer net-work reflect forces and fluctuations of the embeddingmedium. We study the motion of individual SWNTs in equi-librium and non-equilibrium networks by near infraredfluorescence microscopy. We show that SWNTs reptate inthe network. We will discuss the possibility of relating thelocal dynamic behavior of SWNTs as multi-scale probes tothe viscoelastic properties of the surrounding network.

P-456Structural and mechanical properties of VASP-mediatedf-actin bundle networksB. Gentry1, S. van der Meulen1, P. Noguera2,J. Plastino2, G. H. Koenderink11Biological Soft Matter Group, FOM Institute AMOLF,Amsterdam, The Netherlands, 2Institut Curie UMR 168 Paris,France

Vasodilator-stimulated phosphoprotein (VASP) is a crucialregulator of actin dynamics. It is important in cellular processessuch as axon guidance and migration, promoting assembly offilopodia and lamellipodia. VASP’s multiple domain structureincreases the range of interactions it has with actin monomers,filaments, and other proteins and it displays multiple bindingmodes both in vitro and in vivo, including barbed end elonga-tion and filament bundling. However, it is not fully understoodhow VASP affects the structural and mechanical properties ofactin networks. We characterize VASP-mediated bundling of

actin networks in a simplified in vitro system using confocalmicroscopy and quantify mechanical properties with rheologymeasurements. We show that the network properties differfrom other actin bundling proteins and reflect VASP’s multipledomain structure, displaying a complex bundling phase spacethat depends upon solution conditions. We observe the for-mation of large bundle aggregates accompanied by areduction in network elasticity at high protein ratios. In addition,we change VASP’s actin binding mode and eliminate bundlingby introducing free actin monomers. Finally, we show pre-liminary results from a biomimetic system that extends therange of actin-VASP interaction.

P-458Cell migration or proliferation? The go or growhypothesis in cancer cell culturesTamas Garay, Eva Juhasz, Jozsef Tımar, Balazs Heged}us2nd Department of Pathology, Semmelweis University,Budapest, Hungary

Background: Cancer related death is constantly growing inthe past decades. The mortality of solid tumors is mostly dueto the metastatic potential of tumor cells which requires a fineadjustment between cell migration and cell proliferation. Asthe metabolic processes in the cell provide a limited amountof available energy (i.e. ATP) the various biological processeslike cell motility or DNA synthesis compete for the ATPavailable. The go or grow hypothesis postulates that tumorcells show either high migration or proliferation potential. Inour study we investigate on a large series of tumor cell lineswhether this assumption stands for malignant cells.Materials and methods: Twenty tumor cell lines derivedfrom malignant mesothelioma (mesodermal origin) andmalignant melanoma (neuroectodermal origin) were sub-jected to three-days-long time-lapse videomicroscopicrecordings. Cell motility and proliferation were characterizedby the probability of cell division within 24 hours and the24-hour migration distance of the cells.Results: We found a wide range in both the cell migratoryactivity and the proliferation capacity in our series. The 24-hourmigration distance ranged from 40 to 300 micron and from 10to 130 micron in mesothelioma and melanoma cells, respec-tively. The lowest 24-hour cell division probability was found tobe 0.21 in both the melanoma and mesothelioma series whilethe highest proliferation activity reached 1.1 and 1.4 in mela-noma and mesothelioma, respectively. Interestingly, in themelanoma cell lines we found a significant positive correlation(r=0,6909; p=0,0186) between cell proliferation and cellmigration. In contrast our mesothelioma cell lines displayed nocorrelation between these two cellular processes.Conclusions: In summary our findings demonstrate that theinvestigated tumor cells do not defer cell proliferation for cellmigration. Important to note the tumor cells derived fromvarious organ systems may differ in terms of regulation of cellmigration and cell proliferation. Furthermore our observationis in line with the general observation of pathologists that thehighly proliferative tumors often display significant invasion ofthe surrounding normal tissue.

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O-459Driving cytoskeletal remodeling by extracellular matrixmechanicsFitzroy J. Byfield, Qi Wen, Shang-You Tee, Paul A. JanmeyUniversity of Pennsylvania, Philadelphia, PA 19104, USA

Many cell types are sensitive to mechanical signals. Onestriking example is the modulation of cell proliferation, mor-phology, motility, and protein expression in response tosubstrate stiffness. Changing the elastic moduli of substratesalters the formation of focal adhesions, the formation of actinfilament bundles, and the stability of intermediate filaments.The range of stiffness over which different primary cell typesrespond can vary over a wide range and generally reflects theelastic modulus of the tissue from which these cells wereisolated. Mechanosensing also depends on the type ofadhesion receptor by which the cell binds, and therefore onthe molecular composition of the specific extracellular matrix.The viscoelastic properties of different extracellular matricesand cytoskeletal elements also influence the response of cellsto mechanical signals, and the unusual non-linear elasticity ofmany biopolymer gels, characterized by strain-stiffeningleads to novel mechanisms by which cells alter their stiffnessby engagement of molecular motors that produce internalstresses. The molecular mechanisms by which cells detectsubstrate stiffness are largely uncharacterized, but simulta-neous control of substrate stiffness and adhesive patternssuggests that stiffness sensing occurs on a length scale muchlarger than single molecular linkages and that the time nee-ded for mechanosensing is on the order of a few seconds.

Janmey, P. A. and R. T. Miller. 2011. Mechanisms ofmechanical signaling in development and disease. J Cell Sci124:9-18.Tee, S. Y., J. Fu, C. S. Chen, and P. A. Janmey. 2011. Cellshape and substrate rigidity both regulate cell stiffness.Biophys J 100:L25-27

P-460Against the wall: microtubule dynamics is regulatedby force and end-binding proteinsSvenja-Marei Kalisch1, Olga Sytina1, Liedewij Laan1,2,Marileen Dogterom1

1FOM-Institute for Atomic and Molecular Physics (AMOLF),Amsterdam, The Netherlands, 2Department of Molecularand Cellular Biology, Harvard University, Cambridge, USA

Microtubule (MT) plus-end dynamics is essential for cell mor-phogenesis. It is regulated by specific end-binding proteins, aswell as by forces generated when MTs grow against obstaclessuch as the cell cortex. However, it is largely unknown whetherend-binding proteins qualitatively or quantitatively change theway MTs respond to force. We therefore investigate dynamicMTs in the presence of the fission yeast proteins Mal3, Tea2,and Tip1 in vitro. Head-on forces are created by growing theMTs against micro-fabricated rigid barriers. Using TIRFmicroscopy we show that the protein complex enhances MTgrowth velocity and induces catastrophes. Moreover, in pres-ence of the proteins and a force catastrophes become even

more frequent. The catastrophe time then shortens by nearlyand order of magnitude. To improve our insight into theunderlying mechanism we need to elucidate the growthvelocities at the barrier with nanometer resolution. This weachieve with force-clamped optical tweezers.

P-461Expression of cytoskeletal protein titin during adaptationto extreme conditionsElena V. KaradulevaInstitute of Theoretical and Experimental Biophysicsof Russian Academy of Sciences, Pushchino, Russia

To explore potential role of cytoskeletal component in car-diomyocyte for adaptation to extreme conditions was carriedout the comparative study of expression of cytoskeletal sar-comeric protein titin in myocardium of ground squirrels duringhibernation and gerbils after spaceflight. We have revealed atwo-fold increase in content of long N2BA titin isoform ascompared to short N2B titin isoform in different heart cham-bers of hibernating ground squirrels. The prevalence of thelong titin isoform is known to determine the larger extensi-bility of heart muscle that promotes, according to Frank-Starling law, the increase in force of heart contractility forpumping higher viscous blood during torpor and adapting themyocardium to greater mechanical loads during awakening.Moreover, titin mRNA level showed seasonal downregulationin which all hibernating stages differed significantly fromsummer active level. It is possible that the decline of mRNAand protein synthesis during hibernation may be regarded asthe accommodation for minimization of energetic expendi-tures. We have not revealed differences in titin mRNA levelsbetween control gerbils and gerbils after spaceflight. But wehave also observed the two-fold growth in the amount ofN2BA titin isoform in left ventricle of gerbils after spaceflightthat is likely to be directed to the restoration of the reducedheart contractility at zero-gravity. These results suggest thatthe increase of the content of the long N2BA titin isoform mayserve as universal adaptive mechanism for regulating ofheart function in response to the extreme conditions.

P-462Interaction of WH2 domain of ABBA with monomericactinRoland Kardos1, Pekka Lappalainen2, Gabor Hild11University of Pecs, Faculty of Medicine, Departmentof Biophysics, 7624 Pecs, Szigeti street 12, Hungary,2University of Helsinki, Institute of Biotechnology, Programin Cell and Molecular Biology, Helsinki, Finnland

ABBA is a novel member of the I-BAR protein family. I-BARproteins play an important role in the formation of membraneprotrusions by deforming the plasmamembrane and recruitingthe components of the polymerising actin filament machinery.They share a similar domain organisation which constitutesthe membrane and F-actin binding IMD domain and theG-actin interacting WH2 domain. The role of the WH2 motif ofthe I-BAR protein family is well characterized, however, thefunction of theWH2motif of ABBAhas remained elusive. In the

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light of this consideration our research focused on the inter-action of WH2 domain of ABBA with G-actin. The affinity ofWH2 motif of ABBA to ADP- and ATP-actin monomer wasinvestigated by applying fluorescence anisotropy measure-ments. It turned out that the WH2 domain preferentially boundto the polymerisation competent ATP-actin monomer. Inkinetic fluorescence assays theWH2domain slowed down therate of nucleotide exchange on actin. This result was sup-ported by fluorescencequenchingexperiments that revealedaclosed conformation for the nucleotide binding cleft of actin inthe presence of ABBA. Our findings suggest that the WH2domain of ABBA behaves similarly to the typical WH2domains, which means that it has a G-actin sequesteringfunction.

O-463Are biomechanical changes necessary for tumorprogression?Josef A. Kas, Anatol Fritsch, Tobias Kiessling, David Nnetu,Franziska Wetzel, Mareike ZinkDivision for Soft Matter Physics, University of Leipzig,Linnestr. 5, D-04103 Leipzig, Germany

With an increasing knowledge in tumor biology an over-whelming complexity becomes obvious which roots in thediversity of tumors and their heterogeneous molecular com-position. Nevertheless in all solid tumors malignantneoplasia, i.e. uncontrolled growth, invasion of adjacent tis-sues, and metastasis, occurs. Physics sheds some new lighton cancer by approaching this problem from a functional,materials perspective. Recent results indicate that all threepathomechanisms require changes in the active and passivecellular biomechanics. Malignant transformation causes cellsoftening for small deformations which correlates with anincreased rate of proliferation and faster cell migration. Thetumor cell’s ability to strain harden permits tumor growthagainst a rigid tissue environment. A highly mechanosensi-tive, enhanced cell contractility is a prerequisite that tumorcells can cross its tumor boundaries and that this cells canmigrate through the extracellular matrix. Insights into thebiomechanical changes during tumor progression may leadto selective treatments by altering cell mechanics. Suchdrugs would not cure by killing cancer cells, but slow downtumor progression with only mild side effects and thus maybe an option for older and frail patients.

P-464Investigating nuclear migration with the help of micro-patterned surfacesAlexa Kiss, Peter Horvath, Ulrike Kutay, Gabor CsucsETH Institute of Biochemistry, Zurich, Switzerland

Nuclear migration is a general term for a non-randommovement of the nucleus toward specific sites in the cell.This phenomenon has been described throughout theeukaryotes from yeast to mammals. The process is howeverstill poorly understood in mammalian cells. By using micro-contact printing we are able to regulate the geometry andspreading of cultured cells. Adhesive micropatterns of

fibronectin provide an attachment surface for the cellswhereas the passivation of the surface by PLL-PEG preventsprotein, thus cell adhesion. Live cell imaging by time-lapsemicroscopy has shown that under these conditions cells gaina bipolar shape, and more interestingly, the nuclei of the cellsshowed auto-reversed motion. Our research tries to under-stand the molecular cues and mechanisms behind theobserved cellular and nuclear movement. We have alreadyshown that the cytoskeleton plays an important role in thisphenomena but the exact players and the detailed mecha-nism remain to be clarified. In order to identify the mostimportant components and their relationship have drugtreatments and siRNA experiments have been applied.Although our research focuses mainly on the motility of thenucleus, it may also help to get a better understanding of thegeneral theme of cell migration.

P-466Modulation of cell internal tension in responseto external geometrical adhesive cuesKalpana Mandal*,Irene Wang, Martial BallandLaboratoire interdiciplinaire de Physique, Universityof Joseph Fourier, Grenoble, France

Mechanotransduction is one of the recent area that bringsattention of both physicists, chemists and biologists. Recentprogress at the interface between those fields have shownthat external physical cues can strongly affect cell behavior.Thus, it has been demonstrated that external physical signalssuch as substrate rigidity can have dramatic impacts oncellular functions such as migration, adhesion, division andalso cell differentiation (A. Engler et all Cell 126: 677-689(2006).).Cells have the ability, via their cytoskeleton (cell internalstructure), to exert traction forces on their micro-environment.These forces are transmitted to cell environment (substrate orneighboring cells) through adhesive contacts. Reversely cellsare also submitted to forces from their environment (breath-ing, muscle contraction etc etc…) Many tools have beendeveloped in order to elucidate this bidirectional biomechan-ical interactions that has been shown to be fundamental fortissue homeostasis. However current techniques have manylimitations related to their difficulties to obtain statisticalquantification regarding cell biomechanical activity.Contrary to classical biological approaches, here I present anovel technique that enable us to quantify cell forces that cellexert on standardized micro-environments. Briefly, by using acombination of Fast Fourier Traction force cytometry (Butleret al. Am. J. Physiol. Cell Physiol. 2002) and MicropatterningTechnique (ChristopherChen et al.,Biotechnol. Prog. 1998,)on soft hydrogel we are able to create regular array of cellsfor which architecture is controlled at the single cell level.Finally, as our micro-chips are designed on soft substrateswe are able to correlate these architecture to the forcesdeveloped by the cells.A/green:actin,blue:nucleus, B/ Paxillin averaged over #19 C/TFM of single cellred:patternsingle cell on Tripod shape;To conclude, my goal is to correlate absolute quantified forcewith cytoskeletal organization.

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P-467Simultaneous quantification of cellular motility, redoxstatus and membrane fluidityG. Maulucci1, M. Papi1, G. Arcovito1, T. Galeotti2, G. Pani2,M. De Spirito11Istituto di Fisica, Universita Cattolica del Sacro Cuore,Roma, 2Istituto di Patologia Generale, Universita Cattolicadel Sacro Cuore, Roma

Cell motility involves a number of strategies that cells use tomove in their environments in order to seek nutrients, escapedanger and fulfil morphogenetic roles. When these pro-cesses become uncontrolled, pathological behaviours, likecancer or metastasis of cancerous cells, can occur. Here wepresent a new method for the contextual quantification ofcellular motility, membrane fluidity and intracellular redoxstate, by using the ratiometric, redox-sensitive protein rxYFPand the ratiometric fluidity-sensitive probe laurdan. We pro-vide evidence that dynamic redox and fluidity changes arecorrelated with signaling processes involved in cellularmotility. These findings may pave the way to novel approa-ches for the pharmacological control of cell invasiveness andmetastasis.

P-468Manipulation of cellular mechanicsAnna Pietuch, Andreas JanshoffGeorg-August University, Tammanstraße 6, 37077Gottingen, Germany, E-mail: anna.pietuch@chemie.uni-goettingen.de

Rheological properties of cells determined by the underly-ing cytoskeleton (cortex) are key features in cellularprocesses like cell migration, cell division, and cell mor-phology. Today it is possible to investigate local cellularelastic properties under almost physiological conditionsusing the AFM. By performing force indentation curves onlocal areas on a cell surface the use of contact mechanicmodels provides the Young’s modulus, comprising infor-mation about the elastic properties of cells. Theadministration of cytoskeleton modifying substances intothe cell is achieved by microinjection.

We are also investigating morphological changes andrearrangements of the cytoskeleton in time resolvedimpedance measurements. Electric cell-substrate imped-ance sensing is a label-free and minimal invasivetechnique which allows monitoring morphological changesof cells in real time. Readout of the impedance is sensitiveto changes in cell-substrate contacts as well as density ofcell-cell contacts yielding important information about theintegrity of the cell layer and changes in the properties ofthe cell membrane. We are studying the cellular responseto modification of the cytoskeleton e.g. by introducingproteins which affect directly the organization of the actinstructure like ezrin.

P-469Mechanical characterization of actin gels by a magneticcolloids techniqueThomas Pujol, Olivia du Roure, Julien HeuvinghPMMH, ESPCI-CNRS-UMPC-P7. Paris, France

The actin polymer is central in cell biology: it is a majorcomponent of cytoskeleton and it plays a fundamental role inmotility, division, mechanotransduction…. Its polymerizationjust beneath the cell membrane generates forces responsiblefor cell movement. Actin filaments form a network whosearchitecture is defined by the nature of the binding proteinsand depends on the location in the cell. For example, in thestructure which leads cell migration, the lamellipodium, thegel is branched due to its interaction with Arp2/3 proteincomplex. Determining the mechanical properties of suchactin network is a crucial interest to understand how forcesare generated and transmitted in living cells. We grow abranched actin network from the surface of colloids using theArp2/3 machinery. The particles are super paramagnetic andthey attract each other via dipole-dipole interaction to formchain. By increasing the magnetic field we apply anincreasing force to the gel and we optically measure theresulting deformation. From those measurements we deducea Young modulus for a large amount of data. We are char-acterizing different networks by varying the concentration ofthe capping and branching proteins and we show howmechanics can be regulated by the different proteins.

O-470Automated image analysis of microtubule dynamicsand maintenance of fission yeast cells morphologyPierre Recouvreux1, Christian Tischer2, Marileen Dogterom1

1FOM Institute for Atomic and Molecular Physics (AMOLF),Amsterdam, The Netherlands, 2European Molecular BiologyLaboratory (EMBL), Heidelberg, Germany

Microtubules (MTs) are central to the organization of theeukaryotic intracellular space and are involved in the control ofcell morphology. In fission yeasts cells MTs transport polarityfactors to poles where growth is located, thus ensuring theestablishment and maintenance of the characteristic sphero-cylindrical shape. For this purpose, MT polymerizationdynamics is tightly regulated. Using automated image

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analysis software, we investigated the spatial dependence ofMT dynamics in interphase fission yeast cells. We evidencedthat compressive forces generated by MTs growing againstthe cell pole locally reduce MT growth velocities and enhancecatastrophe frequencies. In addition, our systematic andquantitative analysis (in combination with genetic modifica-tions) provides a tool to study the role of ?TIPs (plus-endtracking proteins) such as Mal3 and Tip1 in the spatial regu-lation of MT dynamics. We further use this system to decipherhow the linear transport by MT interferes with the feedbackcircuitry that assures the correct spatial distribution of Tea1,the main polarity factor in fission yeast cells.

P-471Modeling of actin cytoskeleton inside giant unilamellarvesiclesEdith Schafer, Andreas JanshoffInstitute of Physical Chemistry, University of Gottingen,Tammannstr. 6, 37077 Gottingen, Germany, E-mail:eschaef@gwdg.de

Actin is one of the most abundant proteins which forms thehighly dynamic cytoskeleton in all eukaryotic cells. It isessential for cell movement processes and also provides thecell membrane with high mechanical strength. For a betterunderstanding of the actin cytoskeleton function as a stabi-lizing but also flexible 3D-network in living cells it is adjuvantto have a suitable model system.For modeling the complex cytoskeleton in a bottom upapproach, we use giant unilamellar vesicles created of arti-ficial lipid bilayers to simulate the cell membrane andencapsulate monomer actin inside the vesicle aqueous core.Afterwards by ion controlled actin polymerization which islocated at the inner leaflet of the vesicles, we induce achange of shape and rigidity of the liposomes.

O-472Control of the fast, slow and reverse gear of the yeastmitotic kinesin-5 Cin8Adina Gerson-Gurwitz1, Christina Thiede2, NataliaMovshovich1, Vladimir Fridman3, Maria Podolskaya3, TsafiDanieli4, Stefan Lakamper2, Dieter R. Klopfenstein2,Christoph F. Schmidt2 and Larisa Gheber1,31Department of Chemistry, Ben-Gurion Universityof the Negev, Beer-Sheva, Israel, 2Drittes PhysikalischesInstitut, Georg-August-Universitat Gottingen, Gottingen,Germany, 3Department of Clinical Biochemistry, Ben-GurionUniversity of the Negev, Beer-Sheva, Israel, 4ProteinExpression Facility, Wolfson Centre for Applied StructuralBiology, Hebrew University of Jerusalem, Jerusalem, Israel

The dynamics of the cytoskeleton are largely driven bycytoskeletal motor proteins. Complex cellular functions, suchas mitotsis, need a high degree of control of these motors.The versatility and sophistication of biological nanomachinesstill challenges our understanding. Kinesin-5 motors fulfillessential roles in mitotic spindle morphogenesis anddynamics and were thought to be slow, processive microtu-bule (MT)-plus-end directed motors. Here we have examinedin vitro and in vivo functions of the Saccharomyces

cerevisiae kinesin-5 Cin8 using single-molecule motilityassays and single-molecule fluorescence microscopy. Invivo, the majority of Cin8 motors moved slowly towards MTplus-ends, but we also observed occasional minus-enddirected motility episodes. In vitro, individual Cin8 motorscould be switched by ionic conditions from rapid (up to 50lm/min) and processive minus-end, to bidirectional, to slowplus-end motion. Deletion of the uniquely large insert in loop8 of Cin8 induced bias towards minus-end motility andstrongly affected the directional switching of Cin8 both in vivoand in vitro. The entirely unexpected in vivo and in vitroswitching of Cin8 directionality and speed demonstrate thatkinesins are much more complex than thought. These resultswill force us to rethink molecular models of motor functionand will move the regulation of motors into the limelight aspivotal for understanding cytoskeleton-based machineries.

P-473Morphological and dynamical changes during TGF-binduced epithelial-to-mesenchymal transitionDavid Schneider1, Marco Tarantola2, and Andreas Janshoff11Institute of Physical Chemistry, Georg-August-University,Gottingen, Germany, 2Max Planck Institute for Dynamicsand Self-Organization, Laboratory for Fluid Dynamics,Pattern Formation and Nanobiocomplexity (LFPN),Goettingen, Germany

The epithelial-to-mesenchymal transition (EMT) is a programof cellular development associated with loss of cell-cellcontacts, a decreased cell adhesion and substantial mor-phological changes. Besides its importance for numerousdevelopmental processes like embryogenesis, EMT has alsobeen held responsible for the development and progressionof tumors and formation of metastases. The influence of thecytokine transforming growth factor 1 (TGF-b1) induced EMTon structure, migration, cytoskeletal dynamics and long-termcorrelations of the mammalian epithelial cell lines NMuMG,A549 and MDA-MB231 was investigated by time-resolvedimpedance analysis and atomic force microscopy (AFM)performing force-indentation measurements. The three celllines display important differences in cellular morphologymirrored in changes of their elastic response (Young modu-lus), as well as their dynamics upon TGF-b1 treatment.Impedance based measurements of micromotility reveal acomplex dynamic response to TGF-b1 exposure which leadsto a transient increase in fluctuation amplitude and long-termcorrelation. Additionally, the investigation of cellular elasticityvia AFM depicts the different cytoskeletal alterationsdepending on the metastatic potential of the used cell type.

P-474Physics of cellular mechanosensitivity studiedwith biomimetic actin-filled liposomesBjorn Stuhrmann, Feng-Ching Tsai, Guido Mul, GijsjeKoenderinkFOM Institute AMOLF, Amsterdam, The Netherlands

Biological cells actively probe the mechanical properties oftheir tissue environment by exerting contractile forces on it,

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and use this information to decide whether to grow, migrate,or proliferate. The physical basis for cell contractility is theactin cytoskeleton, which transmits motor generated stressesto mechanosensitive adhesions sites that anchor the cellto the tissue. The origins of mechanosensing are far fromunderstood due to the complex interplay of mechanicaleffects and biochemical signaling that occurs far from equi-librium. We use a quantitative biophysical approach basedon biomimetic constructs to elucidate physical principles thatunderlie active mechanosensing in biological cells. We havebuilt realistic in vitro models of contractile cells by encapsu-lating cross-linked actin networks together with myosinmotors in cell-sized membranous containers (liposomes).Our method has several advantages over prior methods,including high liposome yield, compatibility with physiologicalbuffers, and chemical control over protein/lipid coupling. I willshow contour fluctuation spectra of constructs and first dataon mechanical response obtained by laser tweezers micro-rheology. Our work will yield novel insights into stressgeneration and stiffness sensing of cells.

P-475Setting up a system to reconstitute cytoskeleton-basedprotein delivery and patterning in vitroNuria Taberner, Liedewij Laan, Marileen DogteromFOM institute AMOLF, Amsterdam, The Netherlands

Keywords: Microtubules, fission yeast, cell polarity, proteinpatterning, plus end binding proteins.Many different cell types, from mobile fibroblasts [1] to fissionyeast cells [2], display non-homogenous protein patterns ontheir cell cortex. In fission yeast the cell-end marker proteinTea1 that among others is responsible for recruiting the actindependent cell-growth machinery, is specifically located atthe growing cell ends. Tea1 travels at the tips of growingmicrotubules and is delivered to the cell ends [2]. We aim toin vitro reconstitute a minimal microtubule plus-end trackingsystem that leads to cortical protein patterning in functional-ized microfabricated chambers. Our model will allow us toperturb microtubule-based transport and diffusion indepen-dently and evaluate the resulting protein patterns.

[1] Heasman, S.J. and A.J. Ridley, Mammalian Rho GTP-ases: new insights into their functions from in vivostudies. Nature Reviews Molecular Cell Biology, 2008.9(9): p. 690-701.

[2] Hayles, J. and P. Nurse, A journey into space. NatureReviews Molecular Cell Biology, 2001. 2(9): p. 647-656.

P-476Effects of the neuronal Br-3 tropomyosin isoformon the actin dynamicsGabor Csaba Talian, Agnes Kokas, Monika Agnes Toth,Katalin Turmer, Miklos NyitraiUniversity of Pecs, Faculty of Medicine, Instituteof Biophysics, Pecs, Hungary

The tropomyosins (TM) are dimeric actin-binding proteinsthat form longitudinal polymers along the actin filament

groove. There is a great variety of isoforms, but the divisionof labour between the individual TMs and their significance ispoorly understood. As in most cell types, also in the neuronsseveral isoforms are present, whose spatio-temporal locali-sation is differentially regulated. The neuron-specific brain 3isoform (TMBr-3) can be found in the axon of the maturecells. We aimed to clone, express and charaterise this pro-tein in terms of its effects on the kinetic parameters of theactin filament. Using a pET28a construct we purified native,tag-free protein, and examined if it influences the rate of actinpolymerisation or the stability of the filaments in the presenceof either gelsolin or latrunculin-A, two depolymerising agents.In cosedimentation experiments the affinity of TMBr-3 toactin was* 3lM, about six times that of skeletal muscletropomyosin. The net rate of actin polymerisation wasreduced by 17% in the presence of TMBr-3. The depoly-merisation induced by gelsolin or latrunculin-A was inhibitedin a concentration-dependent manner. TMBr-3 seems tostabilise actin filaments against disassembly without signifi-cant effect on the net polymerisation.

P-477Cell mobility and metastatic spreading: a studyon human neoplastic cells using optical tweezersF. Tavano1,2, S. Bonin1 E. D’Este2, G. Pinato2,G. Stanta 1, D. Cojoc21ACADEM Department University of Trieste /CattinaraHospital, 2CNR-IOM, National Laboratory TASC, AreaScience Park – Basovizza, Trieste, Italy

The primary causes of death in cancer patients are localinvasion and metastasis but their mechanisms are not yetcompletely understood. Metastatization is accompanied byalterations of the cytoskeleton and membrane structureleading to changes in their biomechanical properties[1]. Inthis study we analyzed by means of Optical Tweezers themechanical properties of two different breast adenocarci-noma cell lines corresponding to different metastaticpotential. OT were used to grab the plasma membrane by a1,5 um silica bead and form a plasma membrane tether. Wemeasured the force exerted by the cell membrane on thebead and drew the force-elongation curves. Fitting data in theKelvin Body model [2]we found out the values for the vis-coelastic parameters influencing the pulling of the membranetethers.The first cell line analyzed, MCF-7, associated to a lowmetastatic potential showed tether stiffness of 153 pN/um inaverage.The second cell line, MDA-MB 231, poorly differentiated witha high metastatic potential had a tether stiffness of 36pN/umin average, that is a four times lower value. These resultsseems to confirm the hypotesis that metastasis prone cellsare softer than less aggressive cancer cells, and support theuse of OT for these measurements for its sub-pN force res-olution and because cells are manipulated without damage.

[1] Suresh S. Biomechanics and biophysics of cancer cells.Acta biomaterialia. 2007 Jul;3(4):413-38.

[2] Scmitz J. The viscoelasticity of membrane tethers andits importance for cell adhesion’’ Biophysics Journal2008 August;95(3): 1448-59

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P-478Moonlighting functions of TPPP/p25N. T}okesi, A. Lehotzky, J. Olah, A. Zotter and J. OvadiInstitute of Enzymology, Hungarian Academy of Sciences,Budapest, Hungary

Tubulin Polymerization Promoting Protein (TPPP/p25) is abrain-specific protein that primary targets the microtubulenetwork modulating its dynamics and stability. TPPP/p25 is adisordered protein with extended unstructured segmentslocalized at the N- and C-terminals straddling a flexibleregion. TPPP/p25 is primarily expressed in oligodendrocyteswhere its multifunctional features such as tubulin polymeri-zation promoting and microtubule bundling activities arecrucial for the development of the projections in the course ofoligodendrocyte differentiation enabling the ensheathment ofaxons with a myelin sheath that is indispensable for thenormal function of the central nervous system.Microtubule network, a major constituent of the cytoskeleton,displays multiple physiological and pathological functions ineukaryotic cells. The distinct functions of the microtubularstructures are attained by static and dynamic associations ofmacromolecules and ligands as well as by post-translationalmodifications. TPPP/p25 is actively involved in the regulationof microtubule dynamics not exclusively by its bundlingactivity, but also by its tubulin acetylation-promoting activity.Atypical histone deacetylases, such as NAD-dependentSIRT2 and histone deacetylase-6, function outside of thenucleus and control the acetylation level of cytosolic proteins,such as tubulin. Acetylation-driven regulation of the micro-tubule network during cellular differentiation is an ambiguousissue. TPPP/p25 has been recently identified as an inter-acting partner and inhibitor of these deacetylases and theirinteraction decreased the growth velocity of the microtubuleplus ends and the motility of the cells. We have establishedcell models for the quantification of the acetylation degree ofmicrotubule network in correlation with its dynamics andstability as well as in relation to aggresome formation, thatmimics the pathological inclusion formation.The intracellular level of TPPP/p25 is controlled at post-transcription level by microRNA and at protein level by theproteosome machinery. Under pathological circumstancesthis disordered protein displays additional moonlightingfunction that is independent of its association with microtu-bule system or deacetylases; it enters aberrant protein-protein interaction with a-synuclein forming toxic aggregateswithin the neuronal and glial cells leading to the formation ofinclusions characteristic for Parkinson’s disease and multiplesystem atrophy, respectively.

P-479Encapsulating active actomyosin networks in giantliposomesFeng-Ching Tsai, Bjorn Stuhrmann, Guido Mul and Gijsje H.KoenderinkFOM Institute AMOLF, Amsterdam, The Netherlands

Keywords: Biomimetic systems, actin cytoskeleton, confine-ment, giant unilamellar liposomes, actomyosin network,membrane

The cell membrane separates the intracellular from theextracellular environment while intimately interacting with thecytoskeleton in numerous cellular functions, including celldivision and motility. Cell shape changes are for a large partmediated by the contractile actomyosin network forming thecortex underneath the cell membrane. To uncover molecularmechanisms of cell shape control based on actin-membraneinteractions, we built a novel biomimetic model system: acell-sized liposome encapsulating an actively contractingactin-myosin network. Our fabrication method is inspiredby a recent report of liposome preparation by swelling oflipid layers in agarose hydrogel films.1 We extensivelycharacterize important liposomal properties, finding diame-ters between 7 and 20 lm, unilamellarity, and excellent anduniform encapsulation efficiency. We further demonstratechemical control of actin network anchoring to the mem-brane. The resulting liposomes allow quantitative tests ofphysical models of cell shape generation and mechanics.

1. Horger, K. S.; Estes, D. J.; Capone, R.; Mayer, M., Filmsof agarose enable rapid formation of giant liposomes insolutions of physiologic ionic strength. J Am Chem Soc2009, 131 (5), 1810-9.

P-480The effect of mDia1-FH2 on the ATPase activity of actinZoltan Ujfalusi and Miklos NyitraiUniversity of Pecs, Department of Biophysics, Pecs, Szigetistr. 12, H-7624, Hungary

Formins are conservative proteins with important roles inthe regulation of the actin based microfilamental system ineukaryotic cells. They have several domains including FH1,FH2, GPB and DAD. In the interaction between actin andformin the FH2 domain plays a key role. The ’mammalianDiaphanous-related 1’ constitutes one of the subfamilies ofthe formins. These mDia1 formin fragments affect the con-formation of the actin filaments in a concentrationdependent manner. In the current work we have investi-gated whether the mDia1-FH2 affects the nucleotideexchange on the actin filaments. Steady-state fluorescenceanisotropy and photometric coupled assay measurementsshowed that the ATP-ADP conversion was accelerated inthe presence of formins, and the effect was stronger atgreater formin concentrations. These observations indicatethat there must be a tight coupling between the rate ofnucleotide exchange on actin protomers and the confor-mational properties of the filaments.

O-481Tropomyosin isoform specific regulation of nucleationfactorsAndrea Vig, Tamas Huber and Beata BugyiUniversity of Pecs, Medical School, Departmentof Biophysics, Pecs, Hungary

In the cohesive structure of the cytoskeleton functionallydistinct actin arrays orchestrate fundamental cell functions ina spatiotemporally controlled manner. Emerging evidences

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emphasize that protein isoforms are essential for the func-tional polymorphism of the actin cytoskeleton. Thegeneration of diverse actin networks is catalyzed by differentnucleation factors, like formins and Arp2/3 complex. Theseactin arrays also exhibit qualitative and quantitative differ-ences in the associated tropomyosin (Tm) isoforms. How themolecular composition and the function of actin networks arecoupled is not completely understood.We investigated the effects of different Tm isoforms (skeletalmuscle, cytoskeletal 5NM1 and Br3) on the activity of mDia1formin and Arp2/3 complex using fluorescence spectroscopicapproaches. The results show that the studied Tm isoformshave different effects on the mDia1-, and Arp2/3 complex-mediated actin assembly. The activity of the Arp2/3 complex isinhibited by skTm and Tm5NM1, while TmBr3 does not haveany effect. All three Tm isoforms inhibited the activity ofmDia1.These results contribute to the understanding of the mech-anisms by which tropomyosin isoforms regulate thefunctional diversity of the actin cytoskeleton.

P-482Effect of collagen type I on human pulmonary arteryendothelial cellsDiana, Zabini1*, Zoltan, Balint2, Horst, Olschewski3, Andrea,Olschewski1,21Department of Anaesthesiology and Intensive CareMedicine, 2Ludwig Boltzmann Institute for Lung VascularResearch, 3Department of Internal Medicine, Divisionof Pulmonology, Medical University of Graz, Graz, Austria.,*Correspondence to: diana.zabini@medunigraz.at;Stiftingtalstrasse 24, 8036 Graz, Austria

Chronic thromboembolic pulmonary hypertension (CTEPH)is a dual pulmonary vascular disorder, which combines majorvascular remodelling with small-vessel arteriopathy. Thepresence of fibroblasts in the clot, occluding the pulmonaryarteries, and its composition create a microenvironment withincreased collagen level, which might affect the local endo-thelial function. In this study, human pulmonary arteryendothelial cells (hPAEC) were exposed to collagen type I toaddress the effect of the thrombotic microenvironment on thevessel wall forming cells.The hPAECs, cultured under standard conditions were trea-ted with 1, 10 and 100lg/ml of collagen type I for 6h and 24h.The changes in the endothelial cell barrier function wereinvestigated by performing permeability and migration test aswell as VE-cadherin staining.Collagen type I treatment led to a decrease in VE-cadherinsignal in hPAEC. The loosening of cell-cell contacts could beproven with a significant increase in permeability after 6h ofcollagen treatment with different concentrations. Besides theloosening of the cell-cell contacts, the hPAEC migration wasalso dose dependently retarded by collagen application overtime.Our data show that collagen-rich microenvironment leads toa disruption of the junctional proteins in hPAECs, indicatingan environmental induced possible alteration in the functionof endothelial cells in the clots of CTEPH patients.

Ion channels: structure and function

O-483Targeting the promiscuous voltage sensor of Kv7.2and TRPV1 channels by non-toxin gating-modifiersPolina Kornilov1, Asher Peretz1, Yana Gofman2,Nir Ben-Tal2 and Bernard Attali11Department of Physiology & Pharmacology, The Sacklerfaculty of medicine, 2Department of Biochemistry, TheGeorge Wise Faculty of Life Sciences, Tel Aviv University,Israel

The pore and gate regions of voltage-gated cation channelshave been often targeted with drugs acting as channelmodulators. In contrast, the voltage sensing domain (VSD)was practically not exploited for therapeutic purposes,though it is the target of gating-modifier toxins. We recentlydesigned novel diphenylamine carboxylates that are pow-erful Kv7.2 channel openers or blockers. Here we show thatNH17 and NH29, two new Kv7.2 channel blocker andopener, respectively, act as gating modifiers. Mutagenesisand modeling data suggest that in Kv7.2, NH29 docks tothe external groove formed by the interface of helices S1,S2 and S4 in a way, which stabilizes the interactionbetween two conserved charged residues in S2, and S4,known to interact electrostatically, in the open state of Kvchannels. Interestingly, NH29 is also a potent blocker ofTRPV1 channels, while the Kv7.2 channel blocker, NH17,activates TRPV1 and sensitizes the TRPV1 current acti-vated by capsaicin. Our data suggest that the VSD ofTRPV1 is also important for the effects of NH29 and NH17.Thus, subtle modifications in the VSD or in the chemicalstructure of the molecule drastically change the attributes ofthe gating-modifier, thereby stabilizing the channel in eitherthe closed or the open state.

P-484Investigation of Channelrhodopsin-2 mutantsusing resonance Raman spectroscopySara Bruun1,2, Hendrik Naumann1, Uwe Kuhlmann1,Claudia Schulz1, Peter Hegemann2 and Peter Hildebrandt11Technische Universitat Berlin, Institut fur Chemie,Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin,Germany, 2Humboldt Universitat zu Berlin, Institut furBiologie, Experimentelle Biophysik, Invalidenstr. 42,D-10115 Berlin, Germany

Channelrhodopsin-2 (ChR2), a membrane-bound cationchannel, is one of the proteins involved in the phototacticresponse of the alga Chlamydomonas reinhardii. Using res-onance Raman spectroscopy, we have investigated theretinal chromophore that is covalently linked via a Schiff baseto the ChR2 protein. The chromophore of the ChR2 H134Rdark state was studied using low laser power and rotating

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cuvette under mixing. With increasing laser power, accu-mulation of the deprotonated state P390 (M-state) in thelaser spot was noted but without any significant increase ofother intermediates. The resonance Raman spectra of thedark state displays symmetric band shapes in the C=C andC=N stretching region indicating the occurrence of only oneisomeric form. The channel activity of ChR2 is most likelyinduced by the all-trans to 13-cis isomerisation of the retinalchromophore, as judged from the similar spectral changesthat occur during transition from dark state to P390 in ChR2and from dark state to M412 in Bacteriorhodopsin. Addi-tionally, we have found that the inactivation of the slowmutant ChR2 C128T is probably caused by hydrolysis of theretinal Schiff base creating a free retinal, due to the almostidentical spectra of the side-chain intermediate and all-transretinal in CCl4.

P-485Architecture of human perforin pore in pure lipid bilayersM. Dalla Serra1, G. Anderluh2, T. Praper2, A. Kladnik2,G. Viero1, A.F. Sonnen3, J.C. Froelich4, R.J. Gilbert31CNR - IBF & FBK, via alla Cascata 56/C, 38123 Trento, Italy,2Dept. Biology, Biotech. Faculty, Uni. Ljubljana, Ljubljana,Slovenia, 3Struct. Biol. Div., Henry Wellcome Bldg Genom.Med., Oxford, UK, 4Dept. Medicine, NorthShore Uni.,Evanston, IL, 60201, USA

Perforin (PFN) is a component of the human innate immunesystem, is released by lymphocytes and its key function is toengineer the delivery of pro-apoptotic granzymes to the targetcell cytoplasm, leading to a controlled suicidal cell death. Todate, it has been unclear whether granzyme delivery is allowedthrough the aperture of pores in the cell membrane, or by someother means. Nevertheless, the ability of PFN to perforate thetarget membranes by stable pores is central to each proposedmodel. Here we present the characterization of functional PFNpores using single-channel conductance and fluorescencemicroscopy.We show that it forms pores heterogeneous in size(up to 20 nm) in planar lipid membranes (PLM) and unilamellarvesicles. FromPLMexperiments we observed that the openingstep and the pore size are dependent on membrane lipidcomposition: disordered membranes favored gradual open-ings of the pore, while in ordered membranes pores opened ina discrete manner.PFN seems capable of triggering an endocytosis-like eventin addition to pore formation, suggesting a new paradigm forits role in delivering granzymes into target cells where theyinduce apoptosis.

O-486Over-expression of human ion channelsand transporters for structural studiesDeclan A. DoyleTrinity College Dublin, School of Biochemistryand Immunology, College Green, Dublin 2, Ireland

The implementation of miniaturisation and high throughputscreening has quickened the pace of protein structure

determination. However, for most proteins the process stillrequires milligram quantities of protein with purity [ 95 %.These amounts are required as a result of unavoidable los-ses during purification and for the extensive screening ofcrystallisation space. For integral membrane proteins (IMPs),one of the initial steps in the structure determination proce-dure is still a major bottleneck - the over-expression of thetarget protein in the milligram quantity range. With a view ofdeveloping guidelines for over-expression of human IMPs, asystematic approach using the three most common labora-tory expression systems (E. coli, S. cerevisiae, Sf9 insectcells) was implemented. Initial expression levels weredetermined by either partial purification using Ni2?-NTA (E.coli), green fluorescence protein (GFP) fluorescence using aC-terminal GFP tagged protein (S. cerevisiae) or FLAG tag-ged partial purification (Sf9 cells). The results show that E.coli is suitable for the over-expression of human IMPs in therequired quantity range however protein size and complexityis an important factor. The yeast system is fast and affordablebut, for the group of human IMPs tested, the expressionlevels were borderline. Finally for the insect cell system, thetimelines are slower and it is in comparison costly to run,however, it can produce relatively large quantities of humanIMPs.

P-487Dipole modifiers affect properties of syringomycinchannels in the presence of large organic anionsSvetlana S. Efimova1, Olga S. Ostroumova1, Valery V.Malev1,2, Ludmila V. Schagina11Institute of Cytology RAS, St. Petersburg, Russia,2St.Petersburg State University, Petergof, Russia

An antifungal cyclic lipopeptide syringomycin E (SRE) fromPseudomonas syringae forms predominantly anion selectivevoltage-gated channels in membranes bathed in solutions ofalkaline metal chlorides [Malev at al., Advances in planar lipidbilayers and liposomes, V. 8, 2008]. Effects of the membranedipole potential (Vd) on the single channel conductance(G) and the steady-state number of open SRE-channels(N) in phosphocholine bilayers bathed in 0.4 M NaAsp andNaGlc (pH 6) were studied. Values of Vd were varied withintroduction into membrane bathing solutions of eitherphloretin reducing Vd, or RH 421 increasing Vd. We foundthat G did not depend on Vd. This fact resulted from a loss ofSRE pore anionic selectivity. A decrease of Vd caused anincrease of N. At the same time, the observed changes in Nwere one order smaller than those of systems containingNaCl [Ostroumova et al., Langmuir, 2008]. Such difference inN values was assigned to specific adsorption of large anionson the bilayer/solution interface. The work was supported inpart by RFBR (# 09-04-00883), SS-3796.2010.4, the Pro-gram of the RAS «MCB» and RSC # P1372 (MES,FTP\\SSEPIR[[).

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P-488Spectroscopic investigation of the structureand function of the copper ATPase CopBof Enterococcus hiraeMagdalena Groß1, Marc Solioz2, Karim Fahmy11Division of Biophysics, Institute of Radiochemistry,Helmholtz-Zentrum Dresden-Rossendorf, PF 510119, 01314Dresden, Germany, 2Department of ClinicalPharamacology,University of Berne, Murtenstrasse 35, 3010Berne, Switzerland, E-mail: m.gross@hzdr.de

The Cu?-ATPase CopB of Enterococcus hirae is a bacterialP-type ATPase involved in resistance to high levels of envi-ronmental copper by expelling excess copper. Themembrane protein CopB was purified from an over-express-ing strain and solubilized in dodecyl-maltoside. By UV circulardichroism the secondary structure is predicted to contain40-50 % a-helices and 10-15% b-sheets in agreement withestimates based on homology with the Ca ATPase SERCA1.We present CD-spectroscopic data on thermal unfolding ofthe protein to address the influence of the binding of the ATPanalogs ATPcS and the fluorescent analog mant-ATP on theprotein stability. Such analogs are used to mimic functionalstates of the ATPase but undergo different interactions withthe binding site that are not well characterized. We propose acompetition-based assay for nucleotide binding using CD-spectroscopy to deduce the occupancy of the nucleotide-binding site by non-fluorescent nucleotides. Alternatively, thechange of intrinsic fluorescence of mant-ATP upon binding tothe ATPase is exploited in these assays. Finally, we showhow the simultaneous measurement of protein CD andnucleotide fluorescence in thermal denaturation experimentsmay help to determine the stability of several functional con-formational states of CopB.

P-489TRPM8 mRNA and protein expression in rat vas deferensK.L. Gulak1, A.P. Kondratskyi1 & Y.M. Shuba1,21International Center for Mol. Physiol., Kyiv, Ukraine,2Bogomoletz Institute of Physiology, Kyiv, Ukraine

The transient receptor potential (TRP) channel TRPM8 is aCa2?-permeable nonselective cation channel activated bycold and the cooling compounds menthol and icilin. TRPM8is expressed in sensory neurons, where its functions lie intemperature detection. TRPM8 is also expressed in variousinternal tissues, e.g. vas deferens, where temperature isstable in physiological conditions, suggesting that TRPM8 isinvolved in other, temperature unrelated processes. RT-PCRanalysis showed the presence of TRPM8 RNA in isolatedtotal RNA from tissue of rat vas deferens. By samplingexclusively smooth muscle cells of the isolated rat vas def-erence PCR analysis showed no amplification of the aimedcDNA. However we found the shortened amplicon, whichcould correspond to TRPM8 splice variant. We performedWestern blot analysis that showed the expression of TRPM8protein in rat vas deference tissues. Our results could sug-gest the probable exon deletion in transmembrane part of thereceptor, resulting in possible expression of electrophysio-logically unfunctional TRPM8 receptor in rat vas deferens.

P-490Computation of Poisson-Nernst-Planck equationsdescribing ion channel on cell membraneHorng, Tzyy-LengDepartment of Applied Mathematics, Feng Chia University,Taichung, Taiwan 40724, Email: tlhorng@math.fcu.edu.tw

Keywords: Ion channel, Poisson-Nernst-Planck equations,Chebyshev pseudospectral method, finite size effect.

Ion channels are proteins with a hole down the middleembedded in cell membranes. Membranes form insulatingstructures and the channels through them allow and controlthe movement of charged particles, spherical ions, mostlyNa?, K?, Ca??, and Cl- Ion channel is extremely importantin physiology. Many diseases are related to the malfunction ofion channels, and the drug market involved with ion channel ishuge. Though important, the mechanism of ion channel stillremains poorly understood nowadays especially about gatingand ion selectivity. One of the reasons is that it is lack of agood mathematical model. The traditional model for ionchannel is Poisson-Nernst-Planck (PNP) equations. ThoughPNP equation can produce some results and agree well withexperiments, this model still can not explain the mechanismsmentioned above. Recently, a modified PNP model consid-ering finite size effect of ions has been derived from energeticvariational analysis. This newmodel may shed some lights onthe explanation of gating and selectivity mechanisms. Thoughprogress is gained in mathematical modeling, few numericalworks were reported. This is because that PNP equations arenot that simple to solve and the complexity of channelgeometry makes the problem even harder. This paper appliesChebyshev pseudospectral method to solve PNP equationunder a 2D axis-symmetric configuration with and withoutconsidering size effect (a Leonard Jones potential nonlocalintegral term). Since we are only interested in steady statemost of time, pseudo-time-dependence is introduced toPoisson equation and turns the original elliptic-parabolicproblem into a pure parabolic one, which saves the cost ofsolving Poisson equation in each time step. The resultsshowing the steady-state distribution of electric potential,ionic concentrations are obtained efficiently. Channel current,a summation of drift and diffusive currents, can be furthercomputed from the flux of ionic concentrations. The influenceof finite size effect will be also addressed.

O-491Effect of cholesterol and cytoskeleton on KV10.1membrane distributionJimenez-Garduno AM1,2, Pardo LA2, Ortega A1, Stuhmer W2

1UNAM, Mexico-City, Mexico, 2MPI-EM, Gottingen,Germany

The potassium channel KV10.1 is expressed nearly exclusivelyin the central nervous system. Besides its function as an ionchannel, KV10.1 has also been associated with non-canonicalsignaling functions. Various membrane proteins associatedwith cholesterol-sphingolipids enriched microdomains areinvolved in signaling pathways. In this work we studied themembrane distribution of KV10.1 in highly purified brain-tissueplasmamembranes as a function of cholesterol content versus

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cytoskeletal proteins. The results show that one fraction ofKV10.1 associates to cholesterol-rich domains or DetergentResistant Membranes (DRM) and another fraction to non-DRMdomains. The KV10.1 fraction inserted in DRM is dependenton cholesterol as well as on cytoskeleton proteins. Depletion ofcholesterol leads to a doubling of KV10.1 current density. Wesuggest that KV10.1 coexists in two different populations: onewhere the transmembrane domain fits cholesterol enrichedmembranes and another able to fit into a less packed lipidbilayer. The importance of this distribution on signaling pro-cesses needs to be further investigated.

P-492Effect of charge fluctuations on the ionic escape ratefrom a single-site ion channelI. Kh. Kaufman1, R. Tindjong1, D. G. Luchinsky1,2,P. V. E. McClintock11Department of Physics, Lancaster University, Lancaster,LA1 4YB, UK, 2NASA Ames Research Center, MS 269-3,Moffett Field, CA, 94035, USA

We analyse the permeation of an open, single-site, ionchannel by use of Brownian Dynamics (BD) simulations atdifferent concentrations. We show for the first time by mod-elling that the ionic escape rate increases significantly withconcentration due to electrostatic amplification of chargefluctuations at the channel mouth.We use the reduced model of an axis-symmetric water-filledchannel whose protein wall has a single charged site. Thechannel length, radius and fixed charge are selected to matchexperimental data forGramicidinA. The ioncurrent, occupancyand escape rate are simulated by the 1D self-consistent BDtechnique with account taken of the electrostatic ion-ion inter-action. The bath with non-zero ion concentration on one side ofthe channel is modelled via the Smoluchowski arrival rate.It is shown that: a) The occupancy saturates with Michaelis-Menten kinetics. b) The escape rate starts from the Kramersvalue at small concentrations and then increases with con-centration due to the electrostatic amplification of chargefluctuations. The resulting dynamics of the current can bedescribed by modified reaction rate theory accounting forionic escape over the fluctuating barrier [1].

[1] D. G. Luchinsky et al, J. Stat. Mech. (2009), 01010

P-493Functional reconstitution of membrane proteinsby isothermal titration calorimetryNadin Jahnke,1 Oxana Krylova,1 Carolyn Vargas,2 SandroKeller,21Biophysics of Membrane Proteins, Leibniz Instituteof Molecular Pharmacology (FMP), Robert-Rossle-Str. 10,13125 Berlin, Germany, 2Molecular Biophysics, Universityof Kaiserslautern, Erwin-Schrodinger-Str. 13, 67663Kaiserslautern, Germany

Many membrane-protein functions are amenable to bio-physical and biochemical investigation only after the proteinof interest has been reconstituted from a detergent-solubi-lised state into artificial lipid bilayers. Unfortunately, functionalreconstitution has remained one of the main bottlenecks in

the handling of numerous membrane proteins. In particular,gauging the success of reconstitution experiments has thusfar been limited to trial-and-error approaches. To address thisproblem, we have established high-sensitivity isothermaltitration calorimetry (ITC) as a powerful method for monitoringthe reconstitution of membrane proteins into liposomes. ITChas previously been employed for characterising liposomesolubilisation and reconstitution in the absence of protein.Here we show that ITC is also excellently suited for trackingthe complex process of membrane-protein reconstitution in anon-invasive and fully automated manner. The approach isexemplified for the prokaryotic potassium channel KcsA,which we first purified in detergent micelles and then recon-stituted into stable proteoliposomes at very high proteindensities. Electrophysiological experiments performed inplanar lipid membranes confirmed that KcsA regained itsfunctional activity upon ITC-guided reconstitution.

O-494Gating currents of low-voltage-activated T-type calciumchannels familyMaria Karmazınova, Lubica LacinovaInstitute of Molecular Physiology and Genetics, SAV,Bratislava, Slovak Republic

T-type calcium channels are distinguished by relatively lowvoltage threshold for an activation and steep voltagedependence of activation and inactivation kinetics just abovethe activation threshold. Kinetics and voltage dependence ofmacroscopic inward calcium current through CaV3 channelswas described in a detail. In contrast, very little information isavailable on gating current of these channels. Therefore wecompared gating currents measured from all three CaV3.1,CaV3.2 and CaV3.3 channels.Voltage dependencies of chargemovement differ dramaticallyfrom those for macroscopic current. First, their slope factorsare several-fold bigger that slope factors of macroscopic cur-rent activation. Second, activation mid-point for CaV3.3channels on-gating is shifted to more positive membranepotentials by about 20 mV compare to CaV3.1 and CaV3.2channels, whose activationmid-points are similar. The same istruth for off-gating voltage dependences. Kinetics of both on-and off-gating is remarkably faster for CaV3.1 and CaV3.2channels compare to CaV3.3 channels. Further, more chargeis moved per unit of macroscopic current amplitude in CaV3.3channels compare to CaV3.1 and CaV3.2 channels.

AcknowledgementSupported by APVV-0212-10 and VEGA 2/0195/10.

O-495Differential modulation of inactivated statesby hydrophilic and hydrophobic sodium channel blockersPeter Lukacs, Rene Cervenka, Xaver Koenig, Agnes Mike,Michael Kovar, Touran Zarrabi, Karlheinz Hilber, Hannes TodtMedical University of Vienna, Center for Physiologyand Pharmacology, Vienna, Austria

The local anaesthetic lidocaine (LID) is generally believed toreach its binding site in the intracellular vestibule of thevoltage-gated sodium channel via the cell membrane. QX222

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(QX) is a permanently charged, quaternary amine analogueof LID, that can access this binding site via a hydrophilicroute across the channel protein. The mutation I1575E of theadult rat muscle-type sodium channel (rNaV1.4) opens sucha hydrophilic pathway.When bound to the internal vestibule, LID stabilizes both fastand slow inactivated states. We wondered whether QX, oncebound to the internal vestibule, exerts a similar modulatoryaction on inactivated states as LID. The construct I1575Ewas expressed in tsA201 cells and studied by means of thepatch-clamp technique.When applied from the extracellular side 500 lM QXstabilized the slow but not the fast inactivated state inI1575E. When applied internally, QX entered the channel,but stabilization of inactivated states could not beobserved.These results suggest that binding site for use-dependentblock is in the inner vestibule of the channel, fast inactivationis modulated only by the hydrophobic form of LID, and thebinding site for modulation of slow inactivation by QX is onlyaccessible from the extracellular side of the channel.

P-496Membrane composition effects on pore-forming abilityof two cholesterol-dependent cytolysinsM. Marchioretto1, V. Antonini1, L. Lunelli1,2, R. Dallapiccola2,T. Praper3, G. Anderluh3, M. Dalla Serra11Istituto di Biofisica, Consiglio Nazionale delle Ricerche &Fondazione Bruno Kessler, via alla Cascata 56/C, Trento,Italy, 2Fondazione Bruno Kessler, Via Sommarive 18, Trento,Italy, 3Dept. Biology, Biotech. Faculty, Uni. Ljubljana,Ljubljana, Slovenia

Perfringolysin O (PFO) and Listeriolysin O (LLO) are twoCholesterol-Dependent Cytolysins (CDCs) produced byGram-positive bacteria. Their main task is to facilitate theinfection disrupting plasma or endosomal membranes.Even if CDCs have been well studied, their pore-formingmechanism is not clear yet. Two models are pro-posed: pre-pore to pore transition and arc-shaped poreformation.Based on this, we investigated the electrophysiologicalproperties and structures of the two toxins pores in differentmembrane compositions (i.e. phospholipid acyl chains andcholesterol concentration) using Planar Lipid Membrane(PLM) technique and Atomic Force Microscopy (AFM). Ouranalysis identified two distinct pore-forming abilities. OnPOPC-CHO 50% membrane, both proteins form well definedpores with conductance values mainly around 10-15 nS,visible by AFM as complete ring inserted into the membraneof about 30 nm diameter. On the other hand on DOPC-CHO20% membrane, PFO and LLO form pores with heteroge-neous conductance (from pS to nS) but in most of the cases,pores are difficult to be sorted out both by PLM and AFManalyses.Collectively, our results support the idea that prevalence ofpre-pore or arc-shaped oligomeric intermediates are pro-moted by different membrane lipid compositions.

P-497Concerted or sequential conformational transitionsin Kv7.1 channel activation gating?Eshcar Meisel and Bernard AttaliDepartment of Physiology & Pharmacology, The SacklerFaculty of Medicine, Tel Aviv University, Israel

The assembly of Kv7.1 with KCNE1 produces the IKS K?

current that is crucial for the repolarization of the cardiacaction potential. Mutations in these subunit genes producethe long QT syndrome, a life-threatening ventricular arrhyth-mia. Despite numerous studies performed on Shaker-like Kvchannels, very little is known about the mechanisms under-lying Kv7.1 channel activation. Do the voltage-inducedconformational changes in the Kv7.1 tetrameric complexgives rise to cooperativity in channel function? If so, do theyinvolve concerted or sequential conformational transitions insubunit interactions along Kv7.1 channel activation? Todetermine the nature of subunit interaction, we introducedone voltage sensor domain (VSD) mutation, R231W, intoone, two, three, or four subunits by using a concatenatedtetrameric channel construct. When present in all 4 VSDs,the S4 R231W mutation produces instantaneous and volt-age-independent K? currents by ‘‘locking’’ the channel in theopen state. When present in only one VSD, the R231Wmutation does not produce an instantaneous current butcauses a left shift in the channel activation curve. The othercombinations of mutated VSDs suggest a sequential con-formational transition in Kv7.1 channel gating.

P-498Different chemical properties predict resting andinactivated affinity of sodium channel inhibitorsArpad Mike, Nora Lenkey, Sylvester Vizy E., Laszlo FodorInstitute of Experimental Medicine, Departmentof Pharmacology, Budapest, Hungary

Affinity of inhibitor compounds to resting and inactivated con-formations of sodium channels (Kr and Ki) were investigated:Their correlations with chemical descriptors of inhibitors werestudied, in order to deduce the nature of chemical interactionsinvolved in binding to both conformations. Two separateapproaches were used: (1) We performed a literature search,calculated Kr and Ki values, and created a database of alto-gether 204 Kr-Ki pairs obtained from 73 publications. (2) Wecarried out a comparative electrophysiological study of 35drugs using rNav1.2 expressing HEK 293 cells and the QPatchautomatic patch-clamp instrument.We observed that lipophilicity (quantified by the logarithm ofthe calculated water-octanol partition coefficient, logP) isimportant in determining both Kr and Ki, but had a greatereffect on Ki. Distribution coefficients (logD) discriminatedbetter between Kr and Ki than partition coefficients (logP).The ratio of positively charged/neutral forms (quantified by theacidic dissociation constant, pKa) was a significant determi-nant of resting affinity: predominantly charged compoundstended to be more potent against resting channels, whileneutral compounds tended to be more state-dependent.Aromaticity was more important for inactivated state affinity.

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S170 Ion channels: structure and function

P-499The Na1/H1 exchanger NHE7, biochemical features,intracellular localization and biological functionNina Milosavljevic, Laurent Counillon and Mallorie PoetTransport Ionique Aspects Normaux et PathologiquesUniversite de Nice – Sophia Antipolis, and CNRS UMR6097,Nice, France

Keywords: Acidification of intracellular compartments, Na?/H? exchanger, somatic cell genetics, kinetic measurements,neurological diseases

The acidification of intracellular compartments is critical for awide range of cellular processes. A recent candidate for pHregulation within early endosomes and TGN is the highlyconserved intracellular Na?/H? exchanger 7 isoform (NHE7),whose mutation leads to neurological syndromes in humanpatients. However, due to its intracellular localization, NHE7biochemical features are still poorly characterized and itsbiological function remains elusive.We have developed somatic cell genetic techniques thatenable the selection of variant cells able to resist H? killingthrough plasma membrane expression of H? extruders.This enabled us to obtain stable cell lines with forcedplasma membrane expression of NHE7. We used them tomeasure its functional and pharmacological parameters withhigh accuracy, using fast transport kinetics. To summarize,this exchanger displays unique features within the NHEfamily, especially with respect to its affinity for its substrates,lithium, sodium and protons and for its guanidine-derivedinhibitors. Taken together with our results on the subcellularlocalization of the native NHE7, these unique biochemicalfeatures provide new insights on the biological function andpathological implications of this intracellular Na?/H?

exchanger.

P-500Analysis of the collective behaviour of ion channelsJ. Miskiewicz, Z. Trela, S. PrzestalskiWrocław University of Environmental and Life Sciences,Physics and Biophysics Department, ul. Norwida 25, 50-375Wrocław, Poland

A novel approach to the analysis of the ion currentrecordings is proposed. The main goal of the standardpatch clamp technique is to measure single channelactivity (however the whole cell configuration is also usedin various researches). In the presented study the ionchannels time series recordings were several (up to four)ion channels were present are analysed and the collectivebehaviour of ion channels is investigated. The time ioncurrent time series are converted into dwelltime series andthe channel activity is analysed. The hypothesis of col-lective ion channels behaviour is verified and the influenceof organolead compounds (Met3PbCl) on collective ionchannel activity is measured. The analysis is performed onthe SV cation channels of vacuolar membrane of Betavulgaris.The research was financed by Polish Ministry of Science andHigher Education by grant No. NN305 336434.

P-501On motor and electrical oscillations in urinary tract:computer evaluationEva Neu1,2, Daniele Martin1,2, Viktor Foltin1,2, Erich Gornik2,Rumen Stainov3,4, Tanya Zlateva4, Walter Seidenbusch1,2,Michael Ch. Michailov1,21Inst. Umweltmedizin c/o Int. Council Sci. Dev./Int. Acad.Sci., Austria, Bulgaria, Germany, Slovakia & Univ. Bratislava,Univ. Innsbruck, Univ. Erl.-Nurnberg. ICSD e.V. Postfach(POB) 340316, D-80100 Munchen, Germany, 2Fac. Physics,Inst. Exp.Physics, TU & Univ. Vienna-Innsbruck, Austria,3Comp. Sci. Dept., Univ. Fulda-Sofia, Germany-Bulgaria,4Comp. Sci. Dept., Boston Univ., USA

Introduction: Motor/electrical activity of urogenital tract isfundamental for patho-physiology (incontinence, reflux, etc.can lead to renal hypertonia).Method: Parameters: Motor patterns (guinea-pig) – fre-quency/F, amplitudes/A (% init. length, isot. & intracell. rec.)of spontaneous phasic/SPC & tonic/STC contractions, alsoelectrical spikes/S, bursts/B, burst plateaus/BP (Neu et al.Biophys.J. 125/6a/Jan2008; Eur.Biophys.J. 36/S1,S150/2007 & 34/6,765/2005; J.Physiol.Sci. 59/S1,249/2009;Faseb J. 19/4,139.4/2005).Results: SPC-pyelon F:5.4±6.1/min, A:4.7±2.7%, SPC-ure-ter F:1.4±0.7, A:5.3±3.2, SPC-detrusor F:3.8±0.9,A:18.7±5.3, STC-trigone F:0.3±0.1, A:74.6±14.8; ureter-S:F:18.0±4.8/min, A:42.8±5.5mV, -BP: F:7.2±4.8, A:47.5±3.0,-BP-duration 1.3±0.9s; detrusor-S: F:15.0±7.8, A:47.6±7.3; -BP: F:1.1±0.9, A:52.3±9.0, -BP-duration 9.9±3.0s (n=327,p\0.01). Stretch (3-80mN), K-/Ca-influence induced specificchanges in motor/electrical parameters. Special computerprogramme reflects exactly biophysical parameters.Conclusion: Acc. to earlier/recent results mechano-sensi-tive Ca??-activated K?-channels participate in electricaloscillations of detrusor/ureteral myocytes. Further experi-ments/evaluations incl. computer analysis are necessary toprove correlation with motor patterns.

O-502Molecular rearrangements during slow inactivationof the Shaker-IR potassium channelT.G. Szanto, F. Papp, F. Zakany, Z. Varga, T. Pian*,C. Gajewski*, C. Deutsch*, and G. PanyiDepartment of Biophysics and Cell Biology, Universityof Debrecen, Debrecen, Hungary and *Departmentof Physiology, University of Pennsylvania, Philadelphia,Pennsylvania 19104, USA

Crosstalk between the activation and slow inactivation gatesin Shaker potassium channels is now well-established. Theactivation gate perceives the conformation of the inactivationgate (Panyi and Deutsch, 2006, 2007). Closure of the inacti-vation gate speeds opening and slows closing of theactivation gate, i.e., stabilizing the gate in the open configu-ration. If this coupling involves movement of the S6transmembrane segment, then we predict state-dependentchanges in accessibility of residues lining the channel cavity.We engineered cysteines, one at a time, at positions 470,

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471, 472, 473, and 474 in a T449A Shaker-IR backgroundand determined modification rates for the cysteine modifyingreagents, MTSET and MTSEA, in the open, closed, andinactivated state of the channel. Neither reagent, applied fromthe intracellular side, modifies cysteines at 470-474 in theclosed state. Both 470C and 474C are rapidly modified in theopen state and at approximately one-tenth this rate in theinactivated state. In contrast, 471C is not modified in the openstate but can be modified by MTSEA but not MTSET in theinactivated state. Residue 472C cannot be modified in any ofthe three states. Mutant 473C did not express current. Ourfindings are consistent with a rotation of S6 in the inactivatedstate, which increases the accessibility of residue 471 whilesimultaneously decreasing accessibility of residues 470 and474. Any model of C-type inactivation in the Shaker Kvchannel must conform to these experimental observations.[Supported by NIH grant GM 069837 (CD) and OTKA K75904 (GP)].

P-503Mapping of an IKS channel opener reveals interactionsbetween KCNE1 and the Kv7.1 voltage sensorYoni Haitin, Nataly Menaker, Asher Peretz and Bernard AttaliDepartment of Physiology & Pharmacology, The SacklerFaculty of Medicine, Tel-Aviv University, Israel

In the heart, assembly of Kv7.1 with KCNE1 generates therepolarizing K? current IKS. We and others recently sug-gested a strategic location of KCNE1 wedged between twoadjacent Kv7.1 voltage sensing domains (VSD) and nearbyhelix S6 of another Kv7.1 subunit. Here we show that the IKSchannel opener, diisothiocyanostilbene-2’,2’-disulfonic acid(DIDS) acts on IKS to convert the time- and voltage-depen-dent channels into nearly voltage- and time-independentcurrents. The two isothiocyanate functionalities are crucial forthe potent activating effect of DIDS on IKS, since SITS thathas only one of these groups and DNDS, which lacks iso-thiocyanate groups do not activate IKS currents. DIDS doesnot affect Kv7.2 and has a weaker effect when KCNE1 is co-expressed with a chimeric Kv7.1 bearing a Kv7.2 VSD pad-dle. Mutagenesis data suggest that DIDS may activate IKS byinteracting to an external pocket, formed at the interface ofthe superficial boundary of the KCNE1 transmembranesegment and the VSD of Kv7.1. DIDS interaction at the Kv7.1VSD-KCNE1 interface reveals the importance of two lysineresidues, K41 in KCNE1 and K218 in Kv7.1 S3-S4 linker andunderscores the proximity of KCNE1 to the VSD of Kv7.1.

P-504An in-silico approach to primaquine bindingto Trp756 in the external vestibule of sodiumchannel Nav 1.4Eduardo M Salinas-Stefanon1, Angel Islas1, Thomas Scior2,Evelin Martinez-Morales1, Lourdes Millan31Instituto de Fisiologıa, 2Departamento de Farmacia, 3Centrode Quımica Instituto de Ciencias, B. Universidad Autonomade Puebla, Puebla, Puebla, Mexico

The aim of our computed study was to examine the pos-sible binding site of primaquine (PQ) using a combined

homology protein modeling, automated docking andexperimental approach. The target models of wild-type andmutant-types of the voltage-dependent sodium channel inrat skeletal muscle (rNav 1.4) were based on previous workby Tikhonov and Zhorov. Docking was carried out on theP-loop into the structure model of rNav 1.4 channel, in theopen state configuration, to identify those amino acidicresidues important for primaquine binding. The three-dimensional models of the P-loop segment of wild typesand mutant types (W402. W756C, W1239C and W1531A atthe outer tryptophan-rich lip, as well as D400C, E755C,K1237C and A1529C of the DEKA motif) helped us toidentify residues playing a key role in aminoquinolinebinding. In good agreement with experimental results, a1000-fold inhibition loss was observed, tryptophan 756 iscrucial for the reversible blocking effects of PQ. As a result,W756C abolished the blocking effect of primaquine in volt-age-clamp assays.

P-505Hydrogen bond formation accelerates channel openingof the bacterial mechanosensitive channel MscLYasuyuki Sawada and Masahiro SokabeDepartment of Physiology, Nagoya University GraduateSchool of Medicine, Nagoya, Japan

The bacterial mechanosensitive channel MscL is constitutedof homopentamer of a subunit with two transmembrane innerand outer (TM1, TM2) a-helices, and its 3D structure of theclosed state has been resolved. The major issue of MscL is tounderstand the gating mechanism driven by tension in themembrane. To address this question, we performed molecu-lar dynamics (MD) simulations for the opening of MscLembedded in the lipid bilayer. In the closed state of MscL,neighboring TM1 inner helices are crossed each other nearthe cytoplasmic side and Leu19 and Val23 in the constrictedpart form a stable hydrophobic environment called gate. Uponmembrane stretch, Phe78 in TM2 outer helices was draggedby lipids, leading to an opening of MscL. Thus Phe78 wasconcluded to be the major tension sensor. During opening,TM1inner helices were also dragged and tilted, accompaniedby the outward sliding of the crossings. This led to a slightexpansion of the gate associated with an exposure of oxygenatoms of the backbone to the inner surface of the gate. Thisallows water penetration in the gate and formation of hydro-gen bonds between water and the exposed oxygen, which inturn weakened the hydrophobic interaction at the crossings,causing a further opening of the gate and water permeation.

O-506Molecular characterization of BKCa channel in cardiacmitochondriaToro Ligia, Singh Harpreet, Lu Rong, GardeazabalRodrıguez Pedro F., Bopassa Jean C., Wu Yong, and StefaniEnricoUniversity of California, Los Angeles, Los Angeles, CA90095-7115, USA

Mitochondrial BKCa, mitoBKCa has been proposed to becardioprotective and formed by proteins of *50 to *125

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S172 Ion channels: structure and function

kDa. Thus, we investigated the molecular characteristics ofthis channel in isolated mitochondria from murine heart.Labeling of adult mouse cardiomyocytes with plasmalemmaBKCa antibodies, mitotracker, and wheat germ agglutininyielded remarkable mitochondrial but not plasma membranelocalization. Nanoscale fluorescence microscopy (StimulatedEmission Depletion) revealed 7 to 15 of *40-50 nm BKCa

clusters per mitochondria. Further, Western blot analysis ofpurified mitochondria showed the presence of a full length*125 kDa protein. Systematic RT-PCR exon scanning ofisolated cardiomyocyte mRNAs were consistent with a fulllength *125 kDa alpha-subunit protein and revealed theexpression of three splice inserts. Insertless-BKCa robustlylocalized to the plasma membrane of CHO cells but when aC-terminal splice insert was present BKCa was readily tar-geted to the mitochondria (protein proximity index was *1.0indicating 100% colocalization). Hence, cardiac mitoBKCa iscomposed by full-length BKCa protein but with splice insertswhich may facilitate its targeting to mitochondria. Supportedby NIH and AHA.

P-507Modulation of slowly activating (SV) channels in red beet(Beta vulgaris L.) taproots by organo-lead and –tin.Z. Trela1, Z. Burdach2, S. Przestalski1 and W. Karcz21Department of Physics and Biophysics, Wrocław Universityof Environmental and Life Sciences, Norwida 25, PL-50-375Wrocław, Poland, 2Department of Plant Physiology, Facultyof Biology and Environmental Protection, Universityof Silesia, Jagiellonska 28, PL-40-032 Katowice, Poland

Patch-clamp technique was used to examine effect of tri-methyl-lead and -tin on the SV channel activity in the redbeet (Beta vulgaris L.) taproot vacuoles. It was found thatthe addition of both investigated compounds to the bathsolution inhibit, in a concentration-dependent manner, SVcurrents. When single channel properties were analyzed,only little channel activity can be recorded in the presence of100 lM of organometal. Compounds investigated decreasedsignificantly (by about one order of magnitude) the openprobability of single channels. The recordings of singlechannel activity obtained in the presence and in the absenceof organometal showed that compounds only slightly (by ca.10%) decreased the unitary conductance of single channels.It was also found that organometal diminished significantlythe number of SV channel openings, whereas it did notchange the opening times of the channels. Taken together,these results suggest that organometal binding site is loca-ted outside the channel’s selectivity filter and that theinhibitory effect of both compounds investigated on SVchannel activity probably results from organometal-induceddisorder in compatibility between membrane lipids andmembrane proteins.The research was financed by Polish Ministry of Science andHigher Education by grant No. NN305 336434.

P-508Electrophysiological investigation of the hVDAC1 ionchannel in pore-spanning membranesConrad Weichbrodt, Claudia SteinemGeorg-August-Universitat Gottingen, IOBC, Tammannstr. 2,D-37077 Gottingen, Germany, E-mail: cweichb@gwdg.de

The human Voltage-Dependent Anion Channel 1 (hVDAC1)plays an important role in cell life and apoptosis since it is themain porin of the outer mitochondrial membrane. AshVDAC1 is believed to play a pivotal role in apoptosis-relateddiseases such as stroke, Alzheimer, Parkinson and cancer,the alterations of its electrophysiological properties underdifferent conditions are of great value.To perform different investigations, refolded hVDAC1 isreconstituted in artificial membranes which typically consistof DPhPC with a cholesterol fraction of 10–30%. They areprepared via the Muller-Rudin-technique on a functionalizedporous alumina substrate containing pores with a diameter of60 nm. The quality of these so-called nano-Black-Lipid-Membranes (nano-BLMs) is verified via electrochemicalimpedance spectroscopy (EIS), hVDAC1 is reconstituted andsingle channel recordings are made. Membranes are alsoprepared by spreading proteoliposomes on hydrophobizedporous silicon nitride with pores of 1 lm diameter.Information about altered gating-characteristics and relatedconductivities is gained by application of holding potentialsup to ±100 mV and evaluation of the resulting currents.

AcknowledgmentThe hVDAC1 was a kind gift of Prof. C. Griesinger, MPIBPC,Gottingen.

P-509MD simulations reveal mutation induced reorientationof binding residues in the hERG K1 channelKirsten Knape1, Tobias Linder2, Anna Stary-Weinzinger21Institute for Theoretical Chemistry, Vienna, Austria,2Department for Pharmacology and Toxicology, Vienna, Austria

Pharmacological inhibition of cardiac hERG K? channels isassociated with increased risk of arrhythmias. Many drugsbind directly to the channel, thereby blocking ion conduction.Ala-scanning mutagenesis identified residues important fordrug block. Two aromatic residues Y652 and F656 werefound to be crucial for block of most compounds. Surpris-ingly, some cavity blocking drugs are only weakly affected bymutation Y652A. In this study we provide a structural inter-pretation for this observation. MD simulations on the Y652Amutant suggest side chain rearrangements of F656 locatedone helical turn below Y652. Loss of p-p stacking inducesreorientation of F656 from a cavity facing to a cavity liningconformation, thereby substantially altering the shape of thebinding site. Docking studies reveal that due to their rigidshape and compactness Y652 insensitive drugs can stillfavorably interact with the reoriented F656 aryl groups, whilemolecules with more extended geometries cannot.

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P-510Clues to understanding the voltage regulation of TRPA1Vlastimil Zıma1, Lucie Sura2, Ivan Barvık1, Viktorie Vlachova21Institute of Physics, Faculty of Mathematics and Physics,Charles University, Prague 2, Czech Republic, 2Instituteof Physiology, Academy of Sciences of the Czech Republic,Prague 4, Czech Republic

The ankyrin transient receptor potential channel TRPA1 is atransmembrane protein that plays a key role in the trans-duction of noxious chemical and thermal stimuli innociceptors. In addition to chemical activation, TRPA1 can beactivated by highly depolarizing voltages but the molecularbasis of this regulation is unclear. The transmembrane part ofthe tetrameric TRPA1 is structurally related to the voltage-gated K? channels in which the conserved charged residueswithin the fourth transmembrane region (S4) constitute partof a voltage sensor. Compared to these channels, the volt-age-dependence of TRPA1 is very weak (apparent numberof gating charges * 0.4 versus 12 in K? channels) and itsputative voltage-sensing domain most likely lies outside theS4 because TRPA1 completely lacks positively chargedresidues in this region. In the present study we usedhomology modelling and molecular dynamics to createmodels of the transmembrane part and the proximal cyto-plasmic C terminus of TRPA1. In combination withelectrophysiological data obtained from whole cell patch-clamp measurements we were able to point out severalpositively charged residues which mutation strongly alter thevoltage sensitivity of TRPA1 channel. These may be candi-dates for as yet unrecognized voltage sensor.

Photosynthesis

P-511Action of double stress on Photosystem 2Aliyeva Samira A., Gasanov Ralphreed A.Institute of Botany, Azerbaijan National Academyof Sciences, Baku, Azerbaijan

The simultaneous effect of photoinhibitory illumination andtoxic action of heavy metals ions (Cd2? and Co2?) on activityof PS2 in vitro measuring by millisecond delayed fluores-cence (ms-DF) of chlorophyll A was studied. During action onchloroplasts only of Cd2? (10-3M) the fast component of ms-DF, which originates via radiative recombination of reactioncenter with the CaMn4-cluster or YZ on donor side of PS2, isinhibited stronger than at action of only Co2?. The steady-state level at Cd2? treatment is remain stable, while at Co2?

action it is increased. Simultaneous action of Cd2? and pho-toinhibitory illumination (4000 lmol photons m-2 s-1) haveshown that fast component of ms-DF was inhibited faster withtime than in case of action of Co2? and excess light. Resultindicates that damage sites of action Cd2? and Co2? aredonor and acceptor side of PS2, accordingly. We assume thatbinding site of Cd2? is YZ or CaMn4-cluster, one of therecombination partners with P680

? on the donor side of PS2.Thereby, action of Cd ions on donor side of PS2 leads mainlyto development of mechanism of donor-side photoinhibition.

P-512Field instrument for determination of the photosyntheticcapacity of intact photosynthetic bacteriaE. Asztalos1, Z. Gingl2 and P. Maroti11Department of Medical Physics and Informatics, Universityof Szeged, Hungary, 2Department of Experimental Physics,University of Szeged, Hungary

A combined pump and probe fluorometer and spectropho-tometer with high power laser diodes has been constructed tomeasure fast induction and relaxation of the bacteriochloro-phyll fluorescence yield and light-induced absorption changesin intact cells of photosynthetic bacteria. The construction isthe upgraded version of our previous set up1 with better timeresolution (5 ls). The compact design of the mechanics,optics, electronics and data processing makes the deviceeasy to use as outdoor instrument or to integrate into largermeasuring systems. The versatility and excellent performanceof the apparatus will be demonstrated on different fields: 1)organization and redox state of the photosynthetic apparatusof the whole cells under different growth conditions deducedfrom fluorescence characteristics including the lag phase, theamplitude and the rise time of the variable fluorescence, 2)electron transfer in the reaction center, cytochrome bc1 com-plex and in between obtained from relaxation of thefluorescence and 3) re-reduction kinetics of the oxidized pri-mary donor of the reaction center and energetization andrelaxation of the intracytoplasmic membrane tracked byabsorption changes at 798 and 525 nm, respectively.

(1) Kocsis et al. Photosynth Res (2010) 105:73–82.

P-514Role of carotenoid and aggregation in energy dissipationof IsiA from cyanobacteriaSandrine D’Haene, Jan P. DekkerDepartment of Biophysics, Faculty of Sciences,VU University, Amsterdam, The Netherlands

Previous work has established that the Iron Stress Inducedprotein A (IsiA) synthesized by cyanobacteria under stressconditions, has at least two functions: light harvesting [1]and photoprotection [2]. Under prolonged iron starvationIsiA becomes the main chlorophyll-binding protein in the celland occurs without a photosystem association. These IsiAaggregates have a strong ability to dissipate light energyand there is evidence of carotenoid participation in thequenching mechanism via downhill energy transfer fromchlorophyll to the S1 state of a carotenoid [3]. In the presentwork we have measured the temperature dependence ofthe fluorescence of carotenoid depleted mutants (echine-none and/or zeaxanthin) and IsiA monomers in order toinvestigate the role of carotenoid and aggregation in thequenching process. Pigment analysis confirms the absenceof the carotenoid mutated in its biosynthesis but shows thatit is mainly replaced. The monomers are lacking twocarotenoids, echinenone and one of the two ß-carotenesfound previously in IsiA aggregates. Temperature depen-dent fluorescence shows that quenching properties areaffected in the monomers and the mutants lackingzeaxanthin.

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References:

[1] Andrizhiyevskaya E.G., Schwabe T.M.E., Germano M.,D’Haene S., Kruip J., van Grondelle R., Dekker J.P. -Biochim. Biophys. Acta, 2002, 1556, 265-272

[2] Ihalainen J.A., D’Haene S., Yeremenko N., van Roon H.,Arteni A.A., Boekema E.J., van Grondelle R., MatthijsH.C.P., Dekker J.P. -Biochemistry, 2005, 44, 10846-10853

[3] Berera R., van Stokkum I.H.M., D’Haene S., KennisJ.T.M., van Grondelle R. and Dekker J.P. - BiophysicalJournal, 2009, vol 96 (6), 2261-2267

O-515Photosynthetic water oxidation: from biophysics to solarfuelsHolger Dau, Ivelina Zaharieva, Michael Haumann, LazloGerencser, Andre Klauss, Bjoern Suess, Marcel RischFree University Berlin, Dept. of Physics, Berlin, Germany

Soon exhausted oil resources and global climate change havestimulated research aiming at production of alternative fuels,ideally driven by solar energy. Production of solar fuels needs toinvolve the splitting of water into protons, energized electronsand dioxygen. In photosynthetic organisms, solar-energy con-version and catalysis of water splitting (or water oxidation)proceed in an impressive cofactor-protein complex denoted asphotosystem II (PSII). The heart of biological water-oxidation isa protein-bound manganese-calcium complex working attechnically unmatched efficiency. In an attempt to learn fromnature, the natural paragon is intensely studied using advancedbiophysical methods. Structural studies by X-ray spectroscopywith synchrotron radiation play a prominent role in this endea-vor. Time-resolved methods provide insights in the formation ofintermediate states of the reaction cycle. An overview is pre-sented focusing on (i) the efficiency of solar energy usage inPSII, (ii) the interrelation between electron transfer and protonrelocations, and (iii) the mechanism of water oxidation. As anoutlook, new results on water oxidation by biomimetic manga-nese and cobalt oxides, which may become a key element infuture solar-fuel systems, are presented.

P-516Energy harvesting in photosynthetic antennas:structural and dynamical characterization of non-equivalent peridinins in different forms of the Peridinin-Chlorophyll-ProteinG. Di Paolo1 and L.Guidoni1,21Department of Physics, La Sapienza–University of Rome,Italy, 2Department of Chemistry, Chemical Engineeringand Materials, University of L’Aquila, Italy

The Peridinin-Chlorophyll-Protein (PCP) is a light-harvestingcomplex (LHC) that works as antenna in the photosyntheticprocess of dinoflagellates. The protein contains both chloro-phylls and carotenoids molecules, the latter being responsibleto extend the spectral range of captured light to regions wherechlorophylls are transparent. PCP crystal structures [1] revealthat each chlorophyll is surrounded by 3 or 4 molecules of thecarotenoid peridinin, located in non-equivalent positions. Thedifferent protein environment of the sites might be responsibleof a spectral shift of the pigments with the functional role toextend the absorption spectra of the complex and enhance its

light harvesting capabilities. High resolution X-ray diffractiondata on a reconstructed PCP, the refolded peridinin-chlorophylla-protein (RFPCP) [2], and on the less common high salt-PCP(HSPCP) opened the way to the mechanistic understanding ofperidinin spectral tuning, peridinin chlorophyll energy transferand photoprotective mechanism [3]. The two PCP forms differin various features: spectral properties,molarmass, aminoacidsequence and, above all, pigment stoichiometry, the peridi-nin:chlorophyll ratio being 4:1 for the RFPCP and 3:1 for theHSPCP [4]. In the present work we perform classical moleculardynamics simulations of the RFPCP and the HSPCP in explicitwater solution. We analyse the structure and dynamics of theproteins and of their pigments to characterize the differentperidinin sites in both PCP forms in terms of quantities that canaffect the chromophore spectra, such as distorsion, fluctua-tions and nature of the protein environment. The comparisonbetween the data suggests correspondences between thepigments of the two forms. Quantum and mixed quantum/classical molecular dynamics simulations are also under pro-gress to investigate the effect of the protein environment on theelectronic and optical properties of the PCP pigments.

[1] F. P. Sharples, P. M. Wrench, K. Ou, R. G. Hiller,Biochimica et Biophysica Acta 1276, 117 (1996)

[2] T. Schulte, D. M. Niedzwiedzki, R. R. Birge, R. G. Hiller,T. Polivka, E. Hofmann, and H. A. Frank, PNAS 106,20764 (2009)

[3] A. Damjanovic, T. Ritz and K. Schulten, BiophysicalJournal, 79, 1695 (2000)

[4] R. P. Ilagan, S. Shima, A. Melkozernov, S. Lin, R.E. Blankenship, F. P. Sharples, R. G. Hiller, R. R. Birgeand H. A. Frank, Biochemistry, 43,1478 (2004)

P-518Porous silicon/photosynthetic reaction center hybridnanostructureK. Hajdu1, Cs. Gergely2, M. Martin2, L. Zimanyi3, V. Agarwal4,G. Palestino4, D.S. Leza4, K. Hernadi1, Z. Nemeth1, L. Nagy11Departments of Medical Physics and Informaticsand Department of Applied and Environmental Chemistry,University of Szeged, Hungary, 2Laboratoire CharlesCoulomb, UMR 5221 CNRS-Universite Montpellier 2,Montpellier, France, 3Institute of Biophysics, BRC, Szeged,Hungary, 4CIICAP- Universidad Autonoma del Estado deMorelos, Col Chamilpa, Cuernavaca and Facultad deCiencias Quımicas, Universidad Autonoma de San LuisPotosı, Mexico

Multifunctional self-assembled nanoscale materials based onbio-matter are of special interests because of promising

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peculiar applications, like in optoelectronics, biosensors,photovoltaics. Among the existing carrier matrices conduc-tive metal oxides (e.g. indium tin oxide, ITO), carbonnanotubes, graphenes, silicon (Si) are the most frequentlyused materials because of their unique characteristics suchas good conductivity, good optical properties and excellentadhesion to substrates. In our work we combined purifiedphotosynthetic reaction center protein (RC) and porous sili-con (PSi) investigating the morphology and optoelectronicproperties of the bio-nanocomposite material. FTIR spec-troscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy indicated the binding of theprotein to the PSi. Specular reflectance spectra showed ared shift in the characteristic interference peak of the PSimicrocavity which was saturated at higher concentration ofthe protein. The binding was more effective if the function-alization was done by the Si-specific oligopeptide comparedto the classical covalent binding via aminopropyl-triethox-ysilane (APTES). Excitation by single saturation flashesindicated that the RC still exhibited photochemical turnoverafter the binding.

P-519Effects of singlet oxygen elicitors on plant leaves – arethese all caused by ROS?Eva Hideg 1, 2, Ferhan Ayaydin 3, Julia Tandori 4, TamasKalai 5, Laszlo Kovacs 1

1Institute of Plant Biology, Biological Research Center,Szeged, Hungary, 2Institute of Biology, University of Pecs,Hungary, 3Cellular Imaging Laboratory, Biological ResearchCenter Szeged, Hungary, 4Department of Medical Physicsand Informatics, University of Szeged, Hungary, 5Instituteof Organic and Medicinal Chemistry, University of Pecs,Hungary

The role of reactive oxygen species (ROS) in plant stress,both as damaging agent and as potential signal molecule isoften assessed in experiments using photosensitized elicitordyes. For these studies, it is essential to know how efficientlythese chemicals generate ROS, whether they are specificROS sources, as well as their cellular localization and addi-tional side effects. The present study addresses these issuesusing a variety of dyes known and traditionally applied assinglet oxygen (1O2) sources. Rose Bengal (RB), MethyleneViolet (MVI), Methylene Blue (MB), Neutral Red (NR) andIndigo Carmine (IC) were studied as putative ROS sources intobacco leaves.ROS products of photosensitized dyes were measured invitro, using spin trapping EPR spectroscopy. Dye concentra-tions and irradiation concentration leading to equal absorbedexcitation quanta were determined spectrophotometrically. Invivo studies were carried out using tobacco leaves infiltratedwith water solutions of the putative 1O2 sources. Cellularlocalizations were identified on the basis of the dyes’ fluo-rescence. RB, NR and MVI reached into mesophyll cells andwere used to study the effects of these dyes on photosyn-thesis. Photochemical yields and quenching processes werecompared before and after photosensitization of the elicitordyes inside the leaf samples.

Supported by the Hungarian Scientific Research Fund(OTKA-NKTH K67597) and by the National DevelopmentAgency (TAMOP-4.2.1./B-09/1/KONV-2010-005).

O-520Chlorophyll-chlorophyll charge transfer quenching isthe main mechanism of non-photochemical quenchingin higher plantsAlfred R. HolzwarthMax-Planck-Institut fur BioanorganischeChemie, Stiftstraße34-36, D-45470 Mulheim a.d.Ruhr, Germany

Non-photochemical quenching (NPQ) in plants protectsagainst photochemical destruction of the photosyntheticapparatus under excess light conditions.While one location ofthe NPQ process has been shown to be centered on themajorlight harvesting complex II (LHCII) (Q1 type or qEquenching),an additional quenching center responsible for qI type (iden-tical to Q2 center) quenchinghas been suggested to belocated on the minor light-harvesting complexes upon accu-mulation ofzeaxanthin (Zx), in particularon CP24 and CP29We have performed femtosecond transient absorption andtime-resolved fluorescence measurements of NPQ quench-ing in intact leaves of higher plants, on isolated lightharvesting complexes in the minor (non-aggregated)lightharvesting complex CP29 reconstituted with violaxanthin(Vx) or Zx, and in the isolated major LHC II complex in theaggregated state. In all of these situations we find the for-mation of Chl-Chl charge transfer (CT) states to be thedominant quenching mechanism. The yield of formation ofcarotenoid cation states and/or carotenoid S1 state is eitherextremely low or absent, thus excluding their involvement inNPQ quenching as a major quenching mechanism.

P-521Fluorescence dynamics of plant light harvestingcomplexes studied by single molecule spectroscopy1Cristian Ilioaia, 1Tjaart Kruger, 2Matthew P. Johnson, 3PeterHorton, 2Alexander V. Ruban and Rienk van Grondelle1Department of Physics and Astronomy, VU University,Amsterdam, The Netherlands, 2School of Biologicaland Chemical Sciences, Queen Mary University of London,London, United Kingdom, 3Department of Molecular Biologyand Biotechnology, University of Sheffield, Sheffield, UnitedKingdom

Single-molecule spectroscopy (SMS) is a powerful techniquethat allows investigation of fluorescence properties fromsingle fluorescing systems. This technique enabled us toinvestigate the dynamics of the fluorescence intensity andspectral profiles of single, isolated light harvesting complex(LHC) on timescales of milliseconds to seconds, duringcontinuous laser illumination. We were able to observe howeach complex can rapidly switch between different emissionstates [1,2] and to characterise the intensity and the spectraldynamics of major and minor antenna complexes fromplants, in two different environments, mimicking the lightharvesting and the light dissipating state, respectively. Theresults will be discussed with respect to the current modelsfor nonphotochemical quenching (NPQ) mechanisms [3,4,5],

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a vital photoprotection mechanism during which the LHCs ofplants switch between a state of very efficient light utilisationand one in which excess absorbed excitation energy isharmlessly dissipated as heat.

[1] T.P.J. Kruger, V.I. Novoderezhkin, C. Ilioaia, R. vanGrondelle (2010) Biophys. J. 98, 3093–3101

[2] T.P.J. Kruger, C. Ilioaia, L. Valkunas, R. van Grondelle(2011) J. Phys. Chem. B, in press

[3] A.V. Ruban et al. (2007) Nature 450, 575–U22[4] A.R. Holtzwarth, Y. Miloslavina, M. Nilkens, P. Jahns

(2009) Chem. Phys. Lett. 483, 262-267[5] T.K. Ahn et al. (2008) Science 320, 794-797

P-522Characterization of the light harvesting antenna systemin the diatom Phaeodactylum tricornutumJidnyasa Joshi, Matthias Schmidt, Claudia BuchelInstitute of Molecular Biosciences, Johann WolfgangGoethe-University, Frankfurt am Main, Germany

Phaeodactylum tricornutum is one of the most utilized modelorganisms in diatom photosynthesis research mainly due toavailability of its genome sequence (1). It‘s photosyntheticantennae are the Fucoxanthin Chlorophyll a/c binding Pro-teins (Fcps) which share a high degree of homology withLhcs of higher plants and green algae (2). For detailedinvestigation of the antenna system of P.tricornutum, atransgenic strain expressing recombinant His-tagged FcpAprotein was created which simplified the purification of aspecific stable trimeric Fcp complex consisting of FcpA andFcpE proteins. Excitation energy coupling between fuco-xanthin and chlorophyll a was intact and the existence of achlorophyll a/fucoxanthin excitonic dimer was demonstrated(3). We investigated in detail the existence of specificantenna system for PSI and PSII in P.tricornutum as in caseof higher plants. Our studies indicated that at least the mainlight harvesting proteins FcpA and FcpE are most probablyshared as a common antenna by both PSI and PSII.

References:

1. Bowler C., et al. 20082. Green BR. 2003.3. Joshi-Deo J., et al. 2010

O-523Effect of hydrophobic mismatch on the light-inducedstructural changes in bacterial reaction centersS. S. Deshmukh, H. Akhavein, and Laszlo KalmanDepartment of Physics, Concordia University, Montreal,Quebec, Canada

Light-induced structural changes are reported near the pri-mary electron donor of bacterial reaction centers (BRC)dispersed in detergent micelles and in liposomes from lipidswith different fatty acid chain lengths. In this study we presentevidence for the correlation between the light-inducedincrease of the local dielectric constant, determined by theanalysis of the electrochromic absorption changes, and the

lifetime of the charge-separated state at physiologically rel-evant temperatures. The increase of the local dielectricconstant induced a significant decrease of the oxidationpotential of the primary electron donor and a slow protonrelease, which appears to be the rate limiting step in theoverall process. Systematic selection of the head groupcharges of detergents and lipids, as well as the thickness ofthe fatty acid chains of the liposome forming lipids canincrease the lifetime of the charge-separated state by up to 5orders of magnitude. Such extensions of the lifetime of thecharge-separated state were reported earlier only at cryo-genic temperatures and can provide new opportunities toutilize the BRC in energy storage.

P-524Ontogenesis of photosynthetic bacteria trackedby absorption and fluorescence kineticsM. Kis, E. Asztalos, P. MarotiDepartment of Medical Physics and Informatics, Universityof Szeged, Hungary

The development of photosynthetic membrane ofRhodobactersphaeroides was studied by absorption spectroscopy and fastinduction of bacteriochlorophyll fluorescence in different pha-ses of the growth, under various growing conditions (oxygencontent, light intensity etc.) and in synchronous cell population.The results are: 1) The newly synthesized components of themembranes were imbedded immediately into the proteinousscaffold independently on the age of the cell (no ,,transient’’membranes were observed). 2) Under aerobic conditions, thepigments were bleached and under anaerobic conditions thepigment systems showed greening. The relative variablefluorescence (Fv/Fmax) had small age-dependent (but not cell-cycle-related) changes. The fluorescence induction kineticswas sensitive marker of the aerobiosis: the Fv/Fmax ratiodropped from 0.7 to 0.4 and the photochemical rate constantfrom 5�104 s-1 to 3�104 s-1 with an apparent halftime of about4-5 hours after change from anaerobic to aerobic atmosphere.3) The electrogenic signal (absorption change at 525 nm)reflected the energetization of the membrane which showedcell-cycle dependent changes. That included periodic pro-duction and arrangement of protein-lipid components of themembrane synchronized to the cell division.

P-525Functional domain size of the plant light-harvestingantenna in native membranes and artificial aggregatesPetar H. Lambrev1,3*, Franz-Josef Schmitt1,2, SabineKussin1, Max Schoengen2, Zsuzsanna Varkonyi3, HansJoachim Eichler2, Gy}oz}o Garab3, Gernot Renger1,*1Max-Volmer-Laboratory for Biophysical Chemistry PC 14,Berlin Institute of Technology, Strasse des 17. Juni 135,10623 Berlin, Germany, 2 Institute of Optics and AtomicPhysics, Berlin Institute of Technology, Strasse des 17. Juni135, 10623 Berlin, Germany, 3Institute of Plant Biology,Biological Research Center, Hungarian Academyof Sciences, Temesvari krt. 62, 6726 Szeged, Hungary

The degree of energetic connectivity, or functional domainsize, was studied in artificial aggregates of the main light-

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harvesting complex II (LHCIIb) from spinach or in nativethylakoid membranes by picosecond time-resolved fluores-cence. The domain size was estimated by monitoring theefficiency of added exogeneous singlet excitation quenchers– phenyl-p-benzoquinone (PPQ) and dinitrobenzene (DNB).The fluorescence decay kinetics of the systems under studywere registered without quenchers and with quenchersadded in a range of concentrations. Stern-Volmer constants,K0SV and KSV - for aggregates (membranes) and detergent-solubilized complexes, respectively, were determined fromthe concentration dependence of the ratio of the mean fluo-rescence lifetimes without/with quencher (s0, s). The ratioK0KSV • s0/s00 was suggested as a measure of the functionaldomain size. Values in the range of 15-30 were found forLHCII macroaggregates and 12-24 for native thylakoidmembranes, corresponding to domain sizes of 500-1000chlorophylls. Although substantial, the determined functionaldomain size is still orders of magnitude smaller than thenumber of physically connected pigment-protein complexes;thus our results imply that the physical size of an antennasystem beyond these numbers has little or no effect onimproving the light-harvesting efficiency.

P-526Photosynthetic reaction center/carbon nanotube hybridnanostructuresM. Magyar1, K. Hajdu1, K. Hernadi2, E. Horvath3, A. Magrez3,K. Nagy4, Gy. Varo4, L. Forro3 and L. Nagy11Departments of Medical Physics and Informatics, Universityof Szeged, Hungary, 2Department of Appliedand Environmental Chemistry, University of Szeged,Hungary, 3Institute of Physics of Complex Matter, EcolePolytechnique Federale de Lausanne, Switzerland, 4Instituteof Biophysics, HAS Biological Research Center, Szeged,Hungary

The interaction between photosynthetic reaction centerproteins (RCs) purified from purple bacterium Rhodobactersphaeroides R-26 and functionalized and non-functionalized(single (SWNT) and multiple (MWNT) walled) carbonnanotubes (CNT-s) has been investigated. Both structural(AFM, TEM and SEM microscopy) and functional (flashphotolysis and conductivity) techniques showed that RCscan be bound effectively to different CNTs. Both physicalsorption and binding through –NH2 or –COOH groups gavesimilar results. However, it appeared that by physicalsorption some sections of the CNTs were covered by mul-tiple layers of RCs. After the binding the RCs kept theirphotochemical activity for a long time (at least for threemonths, even in dried form) and there is a redox interactionbetween the CNT and RCs. The attachment of RC to CNTsresults in an accumulation of positive and negative chargesfollowed by slow reorganization of the protein structure afterexcitation. In the absence of CNT the secondary quinoneactivity decays quickly as a function of time after drying theRC onto a glass surface. The special electronic propertiesof the SWNT/protein complexes open the possibility forseveral applications, e.g. in microelectronics, analytics orenergy conversion and storage.

O-527Relaxation of bacteriochlorophyll fluorescence in intactcells of photosynthetic bacteriaP. Maroti and E. AsztalosDepartment of Medical Physics and Informatics, Universityof Szeged, Hungary

The decay of the high fluorescence state generated byactinic illumination of different durations was measured inwhole cells of various strains and mutants of photosyntheticpurple bacteria. Although similar method is used in higherplants, its application in photosynthetic bacteria is novel andhighly challanging. The available data are restricted and onlythe re-oxidation of the reduced primary quinone (QA

– ? QA) isusually blamed for the decay kinetics. Here, we will analysethe complexity of the kinetics over a very broad time range(from 5 ls to 5 s) and show that the dark relaxation of thebacteriochlorophyll fluorescence reflects the overlap of sev-eral processes attributed to the intra- and interproteinouselectron transfer processes of the reaction center (RC) andcytochrome bc1 complex of the bacterium. In the shorter (\1ms) time scale, the dominating effect is the re-reduction ofthe oxidized primary donor (P? ? P) that is followed by there-oxidation of the acceptor complex of the RC by the cyto-chrome bc1 complex. As the life times and amplitudes of thecomponents depend on the physiological state of the pho-tosynthetic apparatus, the relaxation of the fluorescence canbe used to monitor the photosynthetic capacity of the pho-tosynthetic bacteria in vivo.

P-528Anisotropic circular dichroism signatures of orientedthylakoid membranes and lamellar aggregates of LHCIIYuliya Miloslavina1, Petar H. Lambrev1, Tamas Javorfi2,Zsuzsanna Varkonyi1, Vaclav Karlicky1, Joseph S. Wall3,Geoffrey Hind3 and Gy}oz}o Garab11Institute of Plant Biology, Biological Research Center,Szeged, Hungary, 2Diamond Light Source Ltd., DiamondHouse, Chilton, Didcot, U.K., 3Biology Department,Brookhaven National Laboratory, Upton, NY, USA

Circular dichroism (CD) spectroscopy is an indispensabletool to probe molecular architecture. At the molecular levelchirality results in intrinsic CD, pigment-pigment interactionsin protein complexes give rise to excitonic CD, whereas ‘‘psi-type’’ CD originates from large densely packed chiralaggregates. It has been well established that the anisotropicCD (ACD), measured on samples with defined orientation,carries specific information on the architecture of molecules.However, ACD can easily be distorted by linear dichroism ofthe sample or instrumental imperfections – which might bethe reason why it is rarely studied in photosynthesis research.Here we present ACD spectra of isolated intact and washed,unstacked thylakoid membranes, photosystem II membranes(BBY), and tightly-stacked lamellar macroaggregates of themain light-harvesting complex II (LHCII). We show that theACD spectra of face- and edge-aligned stacked thylakoidmembranes and LHCII lamellae exhibit profound differencesin their psi-type CD bands. Marked differences are also seenin the excitonic CD of BBY and washed thylakoid mem-branes. Thus ACD provides an additional dimension to the

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analysis of complex biological structures as compared toisotropic CD.

P-529Conformation changes after primary charge separationin photosynthetic reaction centersL. Nagy1, A. Borzsonyi2, K. Osvay2, K. Nagy3,Gy. Varo3, P. Maroti1, M. Terazima41Department of Medical Physics and Informatics, Universityof Szeged, Hungary, 2Department of Optics and QuantumElectronics, University of Szeged, Hungary, 3Instituteof Biophysics, BRC, Szeged, Hungary, 4Kyoto University,Graduate School of Science, Department of Chemistry,Kyoto, Japan

Light induced conformation changes of quinone depletedphotosynthetic reaction centers (RCs) purified from thecarotenoid-less Rhodobacter sphaeroides R-26 were inves-tigated by transient absorption (TA) and grating (TG)methods. Surprisingly, the decay of the TA signal measuredat 860 nm was divided into a 15 ns and a 40 ls components.The latter coincides with the life time of the TG signal, whichwas assigned earlier [Nagy et al. (2008) FEBS Lett. 582,3657-3662] to spectrally silent conformational changes. Thenature of the 40 ls phase was investigated further. Although,the probability of chlorophyll triplet formation under ourmeasuring conditions was small, possible contribution of thetriplet states was also studied. The presence of carotenoid inthe wild type RCs eliminated the 40 ls component indicatingthe role of carotenoid in the energy transfer within the RCs.There was no significant effect of the molecular oxygen onthe TA. This fact may be explained if the chlorophyll tripletsinside the protein have reduced accessibility to molecularoxygen. A differential effect of osmotic potential and viscosityon conformation changes accompanying the primary chargeseparation was measured by the effect of ficoll, glucose andglycerol as compared the TA to the TG signals.

O-530Variable chlorophyll fluorescence: in part a yield changedue to light-induced conformational changeGert Schansker1, Szilvia Z. Toth1, Laszlo Kovacs1,Alfred R. Holzwarth2 and Gy}oz}o Garab11Institute of Plant Biology, Biological Research Center,Hungarian Academy of Sciences, Szeged, Hungary, 2Max-Planck-Institut fur Bioanorganische Chemie, Mulheim an derRuhr, Germany

On a dark-to-light transition the chlorophyll fluorescence risesfrom a minimum intensity (F0) to a maximum intensity (FM).Conventionally, this rise is interpreted to arise from thereduction of the primary quinone acceptor, QA, of photosys-tem II - although this cannot explain all presently availableobservations. In untreated leaves, at room temperature, thefluorescence rise follows the reduction of the electron trans-port chain (ETC). Once induced, *30-40 % of the variablefluorescence intensity relaxes within 100 ms in darkness andcan be re-induced within 3 ms as long as the ETC remainsreduced. Analyzing the fluorescence relaxation kinetics, ?/-DCMU, *30% of the amplitude cannot be explained by QA

-

re-oxidation. Special properties of this phase determined onDCMU-inhibited samples: at cryogenic temperatures (below -40�C), where the QA

- /S2 recombination is blocked, it stillrelaxes and it exhibits a strong temperature dependence withan apparent Ea & 12 kJ/mol, whereas the reduction of QA isnearly temperature insensitive. A fluorescence yield change,driven by light-induced conformational change in the reactioncenter complex, can explain all these observations.

P-531Tuning function in bacterial light-harvesting complexesKatia Duquesne1, Edward O’Reilly2, Cecile Blanchard1,Alexandra Olaya-Castro2 and James N. Sturgis11LISM, CNRS and Aix-Marseille University, Marseille,France, 2Department of Physics, University College, London,UK

Purple photosynthetic bacteria are able to synthesize avariety of different light-harvesting complexes, sometimesreferred to as LH2, LH3 and LH4. Here we have investigatedthe structural origins of these different forms and the mannerin which the sequence tunes the absorption spectrum of thelight-harvesting system. We then consider the functionalconsequences of this tuning for the organization of the lightharvesting system and on the ecology of the organisms.Specifically by spectroscopic techniques, in particular circulardichroism and resonance Raman spectroscopy, we have beenable to obtain information on the organization of the bacterio-chlorophyll binding sites in the unusual LH4 of Roseobacterdenitrificans. This provides a picture of how different peripherallight-harvesting complexes are able tomodulate the absorptionspectrum. The structure and organization of this complex is theput in the context of the the recently published variability of thelight-harvesting complexes. In particular the observation of theirability to form mixed complexes containing different polypep-tides. We examine quantitatively the possible reasons formaintaining such variability by considering the transport prop-erties ofmembranes containing either pure ormixedcomplexesand show that mixed complexes can permit light-harvesting tocontinue during adaptation. We then consider the differentconstraints thatmaybebehind this typeofadaptation indifferentbacteria and the conditions under which different types ofantenna system might be optimal Finally we integrate this intothe evolutionary context of adaptation to variable light intensityand the ecological niches where such organisms are found.

P-532Interaction between photosynthetic reaction centersand ITOT. Szabo1, G. Bencsik2, G. Kozak1, Cs. Visy2,Z. Gingl3, K. Hernadi4, K. Nagy5, Gy. Varo5 and L. Nagy11Departments of Medical Physics and Informatics, 2PhysicalChemistry and Materials Science, 3Technical Informaticsand of, 4Applied and Environmental Chemistry, Universityof Szeged, Hungary, 5Institute of Biophysics, HAS BiologicalResearch Center, Szeged, Hungary

Photosynthetic reaction center proteins (RC) purified fromRhodobacter sphaeroides purple bacterium were deposited onthe surface of indium-tin-oxide (ITO), a transparent conductive

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oxide, and the photochemical/-physical properties of thecomposite was investigated. The kinetics of the light inducedabsorption change indicated that the RC was still active in thecomposite and there was an interaction between the proteincofactors and the ITO. The electrochromic response of thebacteriopheophytine absorption at 771 nm showed anincreased electric field perturbation around this chromophoreon the surface of ITO compared to the one measured insolution. This absorption change is associated with the charge-compensating relaxation events inside the protein. Similar lifetime, but smaller magnitude of this absorption change wasmeasured on the surface of borosilicate glass. The lightinduced change in the conductivity of the composite as afunction of the concentration showed the typical sigmoid sat-uration characteristics unlike if the chlorophyll was layered onthe ITO which compound is photochemically inactive. In thislater case the light induced change in the conductivity wasoppositely proportional to the chlorophyll concentration due tothe thermal dissipation of the excitation energy.

P-533The supramolecular organization of photosystem II invivo studied by circular dichroism spectroscopyTunde Toth1,2, Herbert van Amerongen2,3, Gy}oz}o Garab1,Laszlo Kovacs11Institute of Plant Biology, Biological Research Center,Hungarian Academy of Sciences, Hungary, 2WageningenUniversity, Laboratory of Biophysics, Wageningen, TheNetherlands, 3MicroSpectroscopy Centre, WageningenUniversity, Wageningen, The Netherlands

The light reactions of photosynthesis in higher plants takeplace in granal chloroplast thylakoid membranes, which con-tain chirally organized macrodomains composed ofphotosystem II (PSII) supercomplexes associated with lightharvesting antenna complexes (LHCIIs). The physiologicalrelevance of this hierarchic organization, which often manifestitself in semicrystalline assemblies, has not been elucidatedbut the diversity of the supramolecular structures and theirreorganizations under different conditions indicates its regula-tory role. The present work focuses on the structural andfunctional roles of different components of LHCII-PSII super-complexes. We used various growth conditions, influencingthe protein composition, and different Arabidopsis mutants(KoCP24, KoCP26, KoPsbW, KoPsbX, Dgd1), with alteredorganization of the membranes, and measured their circulardichroism (CD) spectra as well as their chlorophyll fluores-cence kinetics to characterize the chiral macro-organization ofthe chromophores and the functional parameters of themembranes, respectively. We show that the formation of chiralmacrodomains require the presence of supercomplexes. Ourdata also reveal specific functions of some of the protein or lipidcompounds in the light adaptation processes of plants.

O-534Excitation energy transfer and non-photochemicalquenching in photosynthesisRienk van GrondelleDepartment of Physics, VU University, De Boelelaan 1081,1081HV, Amsterdam, The Netherlands

The success of photosynthesis relies on two ultrafast pro-cesses: excitation energy transfer in the light-harvestingantenna followed by charge separation in the reactioncenter. LHCII, the peripheral light-harvesting complex ofPhotosystem II, plays a major role. At the same time, thesame light-harvesting system can be ‘switched’ into aquenching state, which effectively protects the reactioncenter of Photosystem II from over-excitation and photo-damage. In this talk I will demonstrate how LHCII collectsand transfers excitation energy. Using single moleculespectroscopy we have discovered how LHCII can switchbetween this light-harvesting state, a quenched state and ared-shifted state. We show that the switching propertiesbetween the light-harvesting state and the quenched statedepend strongly on the environmental conditions, where thequenched state is favoured under ‘NPQ-like’ conditions. It isargued that this is the mechanism of non-photochemicalquenching in plants.

P-535Photobiology in the soil: arrested chlorophyllbiosynthesis in pea epicotyl sectionsBeata Vitanyi, Katalin Solymosi, Annamaria Kosa,Bela BoddiEotvos University, Institute of Biology, Department of PlantAnatomy, Pazmany P. s. 1/C, H-1117 Budapest, Hungary

The key regulatory step of chlorophyll (Chl) biosynthesis isthe NADPH:protochlorophyllide oxidoreductase (POR) cata-lyzed reduction of protochlorophyllide (Pchlide)which is lightactivated in angiosperms. This process is usually describedon artificially dark-grown plants.In this work, we studied epicotyl segments developed underthe soil surface, which were dissected from pea plants grownunder natural light conditions. Using 77 K fluorescencespectroscopy, pigment analyses, electron microscopy andfluorescence microscopy, we found that upper segmentsshowed transitional developmental stages, i.e. Chl appearedbesides Pchl(ide) and etio-chloroplasts were typical. Inregions under 5 cm depth, however, the characteristics of thesegments were similar to those of plants germinated artifi-cially in complete darkness, i.e. only Pchl(ide) and etioplastswere present.The results of this work prove that these latter symptoms mayoccur in shaded tissues of fully developed, photosyntheticallyactive plants grown under natural conditions.

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Electron and proton transfer, bioenergetics

O-537Computational Bioenergetics: From pure electrontransfer in cytochromes to chemistry in catalasesV. Tipmanee1,2, M. Breuer1, P. Zarzycki3, M. Alfonso-Prieto4,C. Rovira4, K. M. Rosso3, J. Blumberger11University College London, Department of Physicsand Astronomy, Gower Street, London WC1E 6BT, UK,2University of Cambridge, Department of Chemistry,Lensfield Road, Cambridge CB2 1EW, UK, 3PacificNorthwest National Laboratory, Richland, Washington, USA,4Computer Simulation & Modeling Laboratory, Parc Cientıficde Barcelona, Baldiri Reixac 4, 08028 Barcelona, Spain

In this overview talk it will be shown how atomistic compu-tations can complement experimental measurements in ourquest to understand biological electron and proton transferreactions. At first the molecular simulation methods for cal-culation of important electron transfer parameter such asreorganization free energy, electronic coupling matrix ele-ments and reduction potentials will be explained. Then threeapplications will be discussed where such computations helpinterpret experimental data on a molecular level. The firstexample concerns electron tunneling between heme a andheme a3 in the proton pump cytochrome c oxidase. Thisreaction is very fast, occurring on the nano-second timescale, and it is unclear if this is due to an unusually lowreorganization free energy or due to high electronic coupling.Carrying out large-scale all-atom molecular dynamics simu-lation of oxidase embedded in a membrane, we do not findevidence for unusually small values of reorganization energyas proposed previously, implying that the nanosecond tun-neling rate between heme a and a3 is supported by veryefficient electronic coupling. The second example is onelectron transport in a deca-heme ‘wire’-like protein, used bycertain anaerobic bacteria to transport electrons from theinside of the cell to extracellular substrates. The crystalstructure of such a protein has been solved recently for thefirst time. However, it is unclear if and in which direction thewire structure supports electron transport. Here we presentresults of heme reduction potential calculations that help usreveal the possible electron flow in this protein. In a thirdexample we explain how quantum mechanical/molecularmechanical methods (QM/MM) recently helped us under-stand why the catalase from H. Pylori is prone to undergo anundesired protein radical migration reaction during catalysis.

P-539Proton pumping activity of purple and brownmembranes regenerated with retinal analoguesK. Bryl1, K.Yoshihara21University of Warmia and Mazury, Department of Physicsand Biophysics, Olsztyn, Poland, 2Suntory Institutefor Bioorganic Research, Wakayamadai, Osaka 618, Japan

The retinal protein bacteriorhodopsin (BR) acts as a light-drivenproton pump in the purple membrane (PM) of Halobacteriumsalinarium (H.s.). Theaimof thesestudieswas toclarifywhetherthe specific crystalline structure of protein and protein-substrate

interactions are significant for H? transfer into the aqueous bulkphase. Two membrane systems were prepared: purple mem-branes (BR arranged in a two-dimensional hexagonal lattice)and brownmembranes (BR not arranged in a crystalline lattice)were regenerated with 14-Fuororetinals. Light-induced protonrelease and reuptake as well as surface potential changesinherent in the regenerated systems reaction cycles weremeasured. Signals of optical pH indicators residing in theaqueousbulkphasewerecomparedwith signalsof pH indicatorcovalently linked to the extracellular surfaceof proteins andwithsurfacepotential changesdetectedby thepotentiometricprobe.The energies of activation of proton transfer have been calcu-lated. Experimental results and thermodynamic parameters(energies of activation) suggest the different mechanism ofproton transfer into the aqueous bulk phase in these two sys-tems. The implications for models of localized- delocalizedenergy coupling by proton gradients will be discussed.

P-540Electrochemical behaviour of Dps—a mini-ferritinCristina M. Cordas, Joana Wilton, Tania Cardoso, FilipeFolgosa, Alice S. Pereira, Pedro TavaresREQUIMTE-Departamento de Quımica, CQFB, Faculdadede Ciencias e Tecnologia, Universidade Nova de Lisboa,Quinta da Torre, 2829-516 Caparica, Portugal,E-mail: cristina.cordas@dq.fct.unl.pt

Iron regulation is a vital process in organisms and in most ofthem it is accomplished through the metal solubilisation andstorage by ferroxidase enzymes of the Ferritin family, whichhave the ability of sequester, oxidize and mineralize ferrousions using oxygen or hydrogen peroxide as substrate. DNA-binding proteins from starved cells (Dps) belong to this Fer-ritin’s family. Dps belongs to the sub-type designated as mini-ferritins and, besides iron storage and release capability, isresponsible for hydrogen peroxide resistance showing theability to form stable complexes with DNA. The preferable co-substrate of this enzyme is H2O2 although the reaction canoccur in the presence of oxygen with lower rate [1, 2]. In thiswork, the electrochemical behaviour of the recombinant Dpsfrom Pseudomonas nautica, was assessed as a functionof metal content in anaerobic environment with H2O2 asco-substrate. The obtained electrochemical results togetherwith spectroscopic studies allowed inferring some newhypothesis on the Dps iron uptake mechanism.

[1] T. Haikarainen, et al, Cell. Mol. Life Sci., 2010, 67, 341.[2] E. Chiancone, et al, Biochim. Biophys. Acta-Gen. Subj.

2010, 1800, 798.

P-541Myoglobin at Au/SAM junctions: temperature, highpressure and solvent kinetic isotope effectsTinatin D. Dolidze, Dimitri E. Khoshtariya and Rudi van EldikLEPL Life Science Research Centre, 0160, Tbilisi, Georgia,Institute for Biophysics and Bionanosciences, Departmentof Physics, I. Javakhisvili Tbilisi State University, 0128,Tbilisi,Georgia, Department of Chemistry and Pharmacy,University of Erlangen-Nurnberg, 91058 Erlangen, Germany

We applied electrochemical methodology to study electronexchange and accompanying medium reorganization effects

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for myoglobin electrostatically immobilized on Au-depositedmixed self-assembled monolayers (SAMs) of the composition:–S–(CH2)11–COOH/–S–(CH2)11–OH. Our approach allows fora soft switching of the haem group charge state and accurateprobing of the accompanying reorganizational dynamics ofconformational (quasi-diffusional) and quantum (e.g. proton-related) modes. The electron transfer rate constants weredetermined with H2O or D2O as solvent, under the variabletemperature (288-328 K) or pressure (5-150 MPa) conditions,revealing the overall reorganization free energy of 0.5±0.1 eV,activation volume of -3.1±0.1 cm3 mol-1 and inverse solventkinetic isotope effect of 0.7±0.1 (25 �C). On the grounds of anextended charge-transfer theory, we propose specific proton-coupled ET mechanism additionally coupled to the slow con-formational dynamics of the protein matrix accompanied bytranslocation(s) of haem-adjacent water molecule(s).

P-542Proton gradients across pore spanning membranes:towards on-chip energy conversionDaniel Frese, Claudia SteinemInstitute for Organic and Biomolecular Chemistry, Georg-August-University Goettingen, Germany

In cell organelles, chemiosmotic potentials resulting from pro-ton gradients across membranes are widely used to fixchemical energy in forms of ATP. The high efficiency of thisprotein-mediated energy conversion raises interest for artificialproton gradient setups. To investigate proton transport acrossartificial membranes, we prepared pore spanning membranes(PSMs) on porous silicon substrates via painting technique.This allowed us to trap aqueous content of well-defined com-position and volume inside the substrates microcavities.Nigericin, a peptide that acts as an H?-K?-antiporter andbacteriorhodopsin, a transmembrane protein which is wellknown to be a light driven proton pump, were reconstituted intothe pre-formed PSMs to achieve proton transport from oneaqueous compartment to the other. Changes of proton con-centrations inside the pores were monitored by means ofconfocal laser scanning microscopy (CLSM). Therefore, poreswere filled with pyranine, a pH-sensitive fluorescence dye andvariations in intensity were measured to analyze protontranslocation. We were able to show that both, nigericin andbacteriorhodopsin are capable of building up a proton gradientacross PSMs and plan to co-reconstitute ATP synthases foron-chip energy conversion by formation of ATP.

P-543Application of the Gibbs free energy profilesto sequential biochemical reactionsPeter Farkas, Tamas Kiss and Eugene HamoriDepartment of Biological Physics, Eotvos Lorand TudomanyEgyetem, Budapest, Hungary, and Departmentof Biochemistry, Tulane University, New Orleans, La., USA

The full understanding of the energetic details of complexmetabolic reaction sequences requires a step-by-step analysisof theGibbs FreeEnergy (G) changes of the ‘‘parasystem’’ (i.e.,a collection of atoms comprising all the molecules participatingin a given reaction) as it gradually changes from its initial-reactants state to its final-products state along the reaction

pathway. Knowing the respective equilibrium constants of eachof the participating reaction steps and also the actual in vivoconcentrationsof themetabolites involved, a free-energyprofilecan be constructed that will reveal important information aboutthe progress of the reaction asdrivenby thermodynamic forces.This approach will be illustrated on some biochemical reactionsincluding the glycolytic/gluconeogenetic pathways. Further-more, the often misleading text-book representation ofenzymatic catalysis will be reexamined and explained in ther-modynamic terms using the free-energy profiles of both thenon-catalyzed and the enzyme-catalyzed reactions.

O-544Mechanistic insights for redox-active proteinsfunctionalized at Au/SAM junctionsDimitri E. Khoshtariya, Tinatin D. Dolidze, David H. Waldeckand Rudi van EldikInstitute for Biophysics and Bionanosciences, Departmentof Physics, I. Javakhisvili Tbilisi State University, 0128,Tbilisi,Georgia, LEPL Life Science Research Centre, 0160,Tbilisi, Georgia, Department of Chemistry, Universityof Pittsburgh, Pittsburgh, PA 15260, USA, Departmentof Chemistry and Pharmacy, University of Erlangen-Nurnberg, 91058 Erlangen, Germany

Redox-active proteins can be diversely functionalized at metal-deposited self-assembled monolayers (SAMs) of a widelyvariable composition and thickness. The voltammetric meth-odology in combination with the advanced data processingprocedures allow for comprehensive kinetic data within thecongruent series of nano-devices and the subsequent calcu-lation of the key physical parameters, such as the mediumreorganization energy of ET, the donor-acceptor electroniccoupling, effective relaxation time (related to the protein’s andenvironment’s fluctuational dynamics), etc. In our studies the‘‘model’’ redox protein, cytochrome c (CytC), was either freelydiffusing to SAM terminal groups (mode 1), or attached toSAMs through the electrostatic interaction (mode 2), or specific‘‘wiring’’ (mode 3). Another redox-active protein, azurin, wasconfined at terminal SAM groups through the hydrophobicinteraction (mode 4). Diverse experimental strategies includingthe variation of SAM thickness, solution viscosity temperatureand hydrostatic pressure, allowed for a severe demonstrationof the full adiabatic and nonadiabatic control (thinner andthicker SAMs, respectively), and the intermediary regime, in anice agreement with the major theoretical predictions.

O-545Proton transfers in a light-driven proton pumpJ.K. LanyiDept. Physiology & Biophysics, University of California,Irvine, USA

Illumination of bacteriorhodopsin causes isomerization of all-trans retinal to 13-cis,15-anti and a cyclic reaction ensues, inwhich the protein and the chromophore undergo conforma-tional changes with an overall ten millisecond turn-over timeand a proton is transported from one membrane side to theother. With crystal structures of six trapped intermediatestates and plausible structural models for the remaining twointermediates, structures are now available for the initial

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bacteriorhodopsin state and all intermediates. They revealthe molecular events that underlie the light-induced transport:protonation of the retinal Schiff base by Asp85, protonrelease to the extracellular membrane surface, a switchevent that allows reprotonation of the Schiff base from thecytoplasmic side, side-chain and main-chain motions initi-ated in the cytoplasmic region, formation of a single-file chainof hydrogen-bonded water molecules that conducts theproton of Asp96 to the Schiff base, and reprotonation ofAsp96 from the cytoplasmic surface. The observed changescan be summarized as a detailed atomic-level movie in whichgradual relaxation of the distorted retinal causes a cascadeof displacements of water and protein atoms results in vec-torial proton transfers to and from the Schiff base.

P-546Studying electron transfer in oligopeptides through lSRL. Nuccio1, V. Fodera2, F.L. Pratt3, A.M. Donald2, A.J. Drew1

1Queen Mary University of London, School of Physics,London, UK, 2University of Cambridge, CavendishLaboratory, Cambridge, UK, 3ISIS Pulsed Neutron and MuonSource, Rutherford Appleton Laboratory, Chilton, Didcot, UK

Electron transfer (ET) processes are fundamental in photo-synthesis, respiration and enzyme catalysis. The relativeimportance of superexchange and sequential mechanisms inbiological ET is still a matter of debate. The identification ofany ‘‘stepping stones’’ necessary for electron hopping is akey point in the understanding of long range ET. Hence, thestudy of a single event in the sequence of reactions occurringin these phenomena is a fundamental but formidable task.Muon spin relaxation (lSR) has been shown to be sensitiveto charge transport on a molecular lengthscale. The muon isa very sensitive probe of electron transport, as any changesto the electronic density sampled by the muon can change itsspin polarization, which can easily be measured. In thiscontext, a very useful tool is the detection of the so-calledAvoided Level Crossing (ALC) resonances [1]. Theenhancement in the loss of polarization of the muon’s spin onthese resonances dramatically increases sensitivity.We show that a laser pump - lSR probe technique canmeasure ET processes at particular, and most importantlyknown, sites within the amino acids chain, and thereforetrack the time evolution of the electron over the molecule.

[1] S. Blundell, Chem. Rev. 104, 5717-5735 (2004)

P-547Involvement of histidine in electron and proton transferin bacterial photosynthetic reaction centerDelphine Onidas1,2, Joanna M. Stachnik3, Sven Brucker3,Steffen Kraetzig3, Sabine Hermes3, Klaus Gerwert31Laboratoire de Chimie-Physique, CNRS UMR 8000,Universite Paris-Sud, F-91405 Orsay, France, 2UFRBiomedicale, Universite Paris Descartes, F-75006 Paris,France, 3Lehrstuhl fur Biophysik, Ruhr-Universitat, D-44780Bochum, Germany

Keywords: Photosynthesis, reaction center, electron transfer,proton transfer, Fourier transform infrared, L210DN, isotopiclabeling, band assignment, histidine, mechanism

In photosynthesis, the central step in transforming lightenergy into chemical energy is the coupling of light-inducedelectron transfer to proton uptake. In the photosyntheticreaction center (RC) of Rhodobacter sphaeroides, fast for-mation of the charge separated state P?QA

- is followed by aslower electron transfer from the primary quinone QA to thesecondary quinone QB and the uptake of a proton from thecytoplasm to QB. Previous Fourier transform infrared (FTIR)measurements on RC suggested an intermediate X in theQA

-QB to QAQB- transition. Mutation of the amino acid

AspL210 to Asn (L210DN mutant) slows down proton uptakeand oxidation of QA

- . Using time-resolved FTIR spectroscopywe characterized this RC mutant and proposed specific IRbands that belong to the intermediate X. To study the role ofthe iron–histidine complex located between QA and QB, weperformed fast-scan FTIR experiments on the L210DNmutant marked with isotopically labelled histidine. Weassigned IR bands of the intermediate X between 1120 cm-1

and 1080 cm-1 to histidine vibrations. These bands show theprotonation of a histidine, most likely HisL190, during theQA

-QB to QAQB- transition. Based on these results we pro-

pose a new mechanism of the coupling of electron andproton transfer in photosynthesis.

O-548Sodium influence on energy transduction by bacterialcomplexes IAna P. Batista, Bruno C. Marreiros, Manuela M. PereiraInstituto de Tecnologia Quımica e Biologica, UniversidadeNova de Lisboa, Av. da Republica (EAN), 2781-901 Oeiras,Portugal

Complex I of respiratory chains is an energy transducingenzyme present in most bacteria and in all mitochondria. It isthe least understood complex of the aerobic respiratorychain, even though the crystallographic a-helical structures ofbacterial and mitochondrial complexes have been recentlydetermined [1-2]. This complex catalyses the oxidation ofNADH and the reduction of quinone, coupled to cationtranslocation across the membrane.Rhodothermus marinus complex I, our main model system, isa NADH:menaquinone oxidoreductase and has beenextensively characterized. We have made an exhaustivestudy in order to identify all the subunits present in thecomplex [3]. The nature of the coupling charge of R. marinuscomplex I was investigated using inside-out membranevesicles, which were active with respect to NADH oxidationand capable of creating and maintain an NADH-drivenmembrane potential (DW) positive inside. It was observedthat this bacterial complex I is able of H? and Na? transport,although to opposite directions. The coupling ion of thesystem was shown to be the H? being transported to theperiplasm, contributing in this way to the establishment of theelectrochemical potential difference, while Na? is translo-cated to the cytoplasm [4]. The sodium ion extrusion from themembrane vesicles was due to the activity of complex I,since it was sensitive to its inhibitor rotenone, and it was stillobserved when the complex I segment of the respiratorychain was isolated by the simultaneous presence of cyanideand external quinones. Additional studies have shown thatalthough neither the catalytic reaction nor the establishment

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of the DpH requires the presence of Na?, the presence of thision increased the proton transport. Combining all theseresults, a model for the coupling mechanism of complex Iwas proposed, suggesting the presence of two differentenergy coupling sites, one that works only as a proton pump(Na? independent), and the other functioning as a Na?/H?

antiporter (Na? dependent) [4]. This model was reinforced byfurther studies performed in the presence of the Na?/H?

antiporter inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA)[5].A deeper inside into the coupling mechanism of this enzymewas provided by studying the influence of sodium ions onenergy transduction by complexes I from Escherichia coliand Paracoccus denitrificans. It was observed that the Na?/H? antiporter activity is not exclusive of R. marinus complexI, since the E. coli enzyme is also capable of such a trans-port, but is not a general property given that the P.denitrificans enzyme does not performed sodium transloca-tion [6]. Due to the fact that R. marinus and E. coli enzymesreduce menaquinone while P. denitrificans complex I reduceubiquinone, it is suggested that the Na?/H? antiporter activitymay be correlated with the type of quinone used assubstrate.

[1] Efremov, R.G., Baradaran, R. and Sazanov, L.A. (2010)Nature 465(7297) 441-5.

[2] Hunte, C., Zickermann, V. and Brandt, U. (2010)Science 329(5990) 448-51.

[3] Batista, A.P., et al., (2010) Anal. Biochem. 407(1)104-10.

[4] Batista, A.P., et al., (2010) Biochim Biophys Acta,1797(4) 509-15.

[5] Batista, A.P., Marreiros, B.C. and Pereira, M.M. (2011)ACS Chem Biol DOI: 10.1021/cb100380y

[6] Batista, A.P. and Pereira, M.M. (2011) Biochim BiophysActa 1807(3) 286-92.

O-549Mechanism of proton transfer in nitric oxide reductase:computational studyAndrei PisliakovRIKENAdvancedScience Institute,Wako-shi, 351-0198JapanE-mail: apisliakov@riken.jp

Under anaerobic conditions some bacteria can use nitrateinstead of oxygen in a process called denitrification. Duringdenitrification, the reduction of NO to N2O is catalyzed by amembrane-bound enzyme nitric oxide reductase (NOR). Thisenzyme is an important step in the evolution of a respiratorysystem: NOR belongs to the superfamily of O2-reducingheme-copper oxidases and is assumed to be the evolutionaryancestor of cytochrome c oxidase. The understanding of NORfunctioning was limited by the lack of structural information,but recently the first structures (cNOR type from Ps. aerug.and qNOR type from G. stearoth.) were solved [1-2].We will present results of the first computational studies ofNOR (both cNOR and qNOR types) [2-3]. The studiesinclude: (i) large-scale all-atom MD simulations of proteins intheir natural environment (i.e. embedded in membrane andsolvent), which were performed to describe water dynamics

inside the protein and to identify potential proton transferpathways, and (ii) free-energy calculations by the empiricalvalence bond (EVB) method [4] for the explicit proton trans-locations along the pathways established by MD. Amongimportant findings are new proton pathways, which were notpredicted from the X-ray structure and could be identifiedonly by means of computer simulations. Simulations alsoreveal that, despite a high structural similarity between cNORand qNOR, these enzymes utilize strikingly different protonuptake mechanisms. Our results provide insights into thefunctional conversion between NO and O2 reductases, andinto evolution proton transfer mechanisms and respiratoryenzymes in general.

[1] T. Hino et al, Science 330 (2010) 1666.[2] Y. Matsumoto et al, submitted.[3] A.V. Pisliakov et al, submitted.[4] A.V. Pisliakov et al, PNAS 105 (2008) 7726.

P-550Functional and structural characterizationof cytochrome c6-like from a nonphotosyntheticorganismMarta S. e Sousa, Marta A. Silva, Leonor Morgado, Carlos A.SalgueiroRequimte-CQFB, Departamento de Quımica, Faculdade deCiencias e Tecnologia, Universidade Nova de Lisboa,2829-516 Caparica, Portugal

The genome of the bacterium Geobacter sulfurreducens (Gs)encodes for 111 c-type cytochromes (1). Genetic studiesusing cytochrome deficient Gs strains and proteomic studiesidentified cytochromes that were produced under specificgrowth conditions (2-4). A putative outer-membrane cyto-chrome, OmcF, is crucial for Fe3? and U6? reduction and alsofor microbial electricity production (4). OmcF is a monohemec-type cytochrome with sequence similarity to soluble cyto-chromes c6 of photosynthetic algae and cyanobacteria (4).The structure of oxidized OmcF was determined (5) being thefirst example of a cytochrome c6-like structure from a non-photosynthetic organism. The structural features of OmcFhinted a different function compared to cytochromes c6 fromphotosynthetic organisms, being an excellent example of howstructurally related proteins are specifically design by Natureto perform different physiological functions. In order to eluci-date OmcF structural-functional mechanism, isotopic labeledprotein (15N and 13C) was produced and its structure in thereduced form determined by NMR. Single point-mutations atkey residues were produced by site-directed mutagenesisand their impact on the structural and functional properties ofOmcF will be presented.

1. Methe, B.A. et al. (2003) Science 302, 1967.2. Shelobolina, E.S. et al. (2007) BMC Microbiol. 7, 16.3. Ding, Y. H., et al. (2008) Biochim Biophys Acta 1784,

1935.4. Kim, B.-C. et al. (2008) Bioelectrochemistry 73, 70.5. Pokkuluri, P.R. et al. (2009) Proteins 74, 266.

AcknowledgmentsThis work was supported by project grant PTDC/QUI/70182/2006 (CAS) and doctoral grants SFRH/BD/61952/2009

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(MAS) and SFRH/BD/37415/2007 (LM) from Fundacao paraa Ciencia e a Tecnologia (Portugal). We thank Dr. M. Schifferfor providing the OmcF coding plasmid.

P-551The no-synthase paradoxJerome Santolini1, Pierre Dorlet1, Albane Brunel1, Jean-LucBoucher3, Cecile Roselli-Sicard2, Sophie Bernad21LSOD, iBiTec-S, C. E. A. Saclay, 91191 Gif-sur-YvetteCedex France, 2LCP, Bat. 350, Universite Paris-Sud, 91405Orsay Cedex France, 3UMR 8601, CNRS, 45 rue des SaintsPeres, 75270 Paris cedex 06 FranceE-mail: jerome.santolini@cea.fr

Keywords: Nitric Oxide, NO-Synthase, Molecular Mecha-nism, Biophysics, Kinetics

In the early 90s, the search for the source of nitrogen mon-oxide (NO) production in mammals led to the discovery ofthree major isoforms of NO-Synthases (NOS): the neuronalNOS (nNOS), the inducible NOS (iNOS), and the endothelialNOS (eNOS) ((1)). 20 years later, based on genomic analy-sis, numerous NOS-like proteins have been identified in thegenome other organism and in particular of several bacteria(Bacillus Anthrax, Staphylococcus aureus… (2)).In spite of superimposable 3D structures and the ability tocatalyse NO production, all these enzymes are carrying dif-ferent (if not opposite) physiological activities includingcGMP signalling, cytotoxic activities, anti-oxidant defence,metabolism… Moreover, NOSs become increasingly asso-ciated to oxidative-stress related pathologies ranging fromneurodegenerative disorders, cardiovascular and inflamma-tory diseases, diabetes, cancers (3)….This apparent paradox seems related to the belief that thestrong similarity of sequence and structure of NO-Synthasesmust lead to a unique and identical functioning (NO pro-duction) for all isoforms. This is blatant for bacterial NOS-likeproteins that are lacking the essential components requiredfor NO biosynthesis but remain considered as genuine NOsynthases. This approach might remain an obstacle to theunderstanding of NOS actual biological role and could pre-vent the design of efficient NOS-targeted therapeuticstrategies.To elucidate this ‘‘NOS paradox’’ our group has initiated amultidisciplinary approach that aims to relate the widediversity of NOS biological activities to variations in the cat-alytic mechanism of NOSs, to modifications of theirregulation patterns and to adaptations to their physiologicalenvironment. In this context we have been investigating themechanism of bacillus subtilis NOS-like proteins with aspecial focus on the features that are specific to NOSmechanism: i) electron and proton transfers and the role ofthe substrate and the pterin cofactor ii) oxygen activation andthe role of the proximal ligand iii) the very molecular mech-anism and the variations in the nature of reactionintermediates.For that matter we have been using a combination of radio-lytical techniques (cryoreduction with 60Co y-irradiation,

pulse-radiolysis with the ELYSE electron accelerator), state-of-the-art spectroscopies (EPR, ATR-FTIR and resonanceRaman, and picoseconds UV-Visible absorption spectroso-copies), organic synthesis (synthesized substrates andcofactors analogues) biochemistry and molecular biology(site-directed mutagenesis).We will present our results on the coupling of electron andproton transfers, on the tuning of the proximal ‘‘push effect’’and we will discuss the conditions that favour for each NOSisoform NO production versus other reactive nitrogen andoxygen species.

1. Alderton, W. K., Cooper, C. E., and Knowles, R. G.(2001) The Biochemical journal 357(Pt 3), 593-615

2. Sudhamsu, J., and Crane, B. R. (2009) Trends inmicrobiology 17(5), 212-218

3. Pacher, P., Beckman, J. S., and Liaudet, L. (2007)Physiological reviews 87(1), 315-424

4. Stuehr, D. J., Santolini, J., Wang, Z. Q., Wei, C. C., andAdak, S. (2004) The Journal of biological chemistry279(35), 36167-36170

O-552Molecular characterization of photosynthetic ironoxidationIvo H. Saraiva1, Lina Bird2, Eduardo Calcada3, Carlos A.Salgueiro3, Dianne K. Newman4, Ricardo O. Louro11Instituto de Tecnologia Quımica e Biologica, UniversidadeNova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras,Portugal, 2Massachusetts Institute of Technology, 02139Cambridge MA, USA, 3Requimte-CQFB, Departamento deQuımica, Faculdade de Ciencias e Tecnologia, UniversidadeNova de Lisboa, Campus Caparica, 2829-516 Caparica,Portugal, 4Division of Biology, California Instituteof Technology and Howard Hughes Medical Institute, 1200East California Boulevard, Pasadena, CA 91125, USA

Photosynthetic Iron Oxidation (PIO) is an ancient form ofphotosynthesis with relevant consequences in the shaping ofthe planet. This form of metabolism may have been involvedin the deposition of geological structures known as BandedIron Formations, which hold key information regarding theco-evolution of photosynthesis and Earth. Rhodopseudo-monas palustris TIE-1 and Rhodobacter ferroxidans SW2both use ferrous iron as an electron donor to support pho-tosynthetic growth (i.e. photoferrotrophy). The SW2 foxEYZoperon can stimulate light-dependent iron oxidation by otherbacteria. It codes a two-heme cytochrome, a pyrroloquinolinequinone protein and an inner membrane transporter,respectively. In TIE-1 the pioABC operon is required forphotoferrotrophy. It codes for a ten-heme cytochrome, anouter membrane beta-barrel and a high potential iron-sulfurprotein (HiPIP) respectively. Here we present functional andstructural characterization of proteins involved in PIO. Thismolecular characterization is essential for understanding thismode of bioenergetic metabolism, and may one day aid thedevelopment of biotechnological applications like microbialfuel cells and bioremediation.

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P-554Anoxia induces increased activity of alternative oxidasein fungus Phycomyces blakesleeanusM. Stanic1, M. Zivic2, M. Hadzibrahimovic3, M. Zizic1

and J. Zakrzewska 4

1Institute for Multidisciplinary Research, Kneza Viseslava 1,11030 Belgrade, Serbia, 2Faculty of Biology, Universityof Belgrade, Takovska 4, 11000 Belgrade, Serbia,3Department of Biology, State University of Novi Pazar, VukaKaradzica bb, 36300 Novi Pazar, Serbia, 4Instituteof General and Physical Chemistry, Studentski trg 12-16,11000 Belgrade, Serbia

Alongside classical, cytochrome respiratory pathway, Phyc-omyces blakesleeanus possess alternative, cyanide-resistant respiration (CRR) facilitated by alternative oxidase(AOX). In order to study role of oxygen in regulation of CRR,the effects of cyanide on respiration of 24h old mycelia inaerated (control), hypoxic and anoxic conditions were mea-sured. Mycelium was incubated in these conditions for 1.5h,3h and 5h. After 1.5h, AOX activity was increased only inspecimens incubated in anoxic conditions (13.6%). After 3h,increase in AOX activity was significant in both hypoxic andanoxic specimens (18.9% and 18.8%, respectively), witheven greater increase after 5h, 20.7% for hypoxic and 23.3%for specimens in anoxic conditions. Mycelia treated for 5hwas then oxygenated for 10 minutes. This induced decreasein AOX activity of 17% in anoxic and even 23.9% in hypoxicmycelia. AOX is recognized as one of the mechanisms formaintaining low levels of reduced ubiquione which canfunction in conditions in which cytochrome chain is disabled,such as anoxia. This is in concordance with results obtainedon P. blakesleeanus, where AOX levels rise in hypoxic andanoxic conditions and decrease close to control level shortlyafter introduction of oxygen into the system.

P-555Influence of Escherichia coli F0F1-ATPaseon hydrogenase activity during glycerol fermentationK. Trchounian1,2, G. Sawers2, A. Trchounian11Department of Biophysics, Yerevan State University, 0025Yerevan, Armenia, 2Institute for Microbiology, Martin-LutherUniversity Halle-Wittenberg, 06120 Haale, Germany

E. coli encodes four hydrogenases (Hyd); only three of these,Hyd-1, Hyd-2 and Hyd-3 have been well characterized. Hyd-2has been shown recently to reversibly evolve hydrogen dur-ing glycerol fermentation at pH 7.5 [1]. Proton reduction wasinhibited by N,N’dicyclohexylcarbodiimide suggesting a linkwith the proton-translocating F0F1-ATPase. Indeed, at pH 7.5in an E. coli mutant (DK8) lacking F0F1 overall Hyd-activitywas reduced to approximately 50% of the wild type activity;Hyd-2, but not Hyd-1, was detected in an in-gel activity assay.F0F1 is therefore suggested to be required for Hyd-1 activity.At pH 6.5 in glycerol medium Hyd-activity in DK8 was *10%of wild type activity and Hyd-1 and Hyd-2 exhibited only weakactivity. This indicated a significant F0F1 contribution towardsHyd-activity as pH decreased. Furthermore, at pH 5.5 Hyd-activity was negligible and only a very weak activity bandcorresponding to Hyd-2 could be observed. These resultssuggest that F0F1-ATPase is essential for hydrogenaseactivity during glycerol fermentation at pH 5.5. Taken together,

the results suggest an interdependence between Hyd-1, Hyd-2 and F0F1-ATPase activity.

[1] Trchounian K, Trchounian A. (2009) Int. J. HydrogenEnergy, 34, 8839-8845.

O-557Characterization of a vacuolar cytochrome b561by redox titration and spectrum analysisLaszlo Zimanyi and Alajos BercziMetalloprotein Biophysics Laboratory, Instituteof Biophysics, Biological Research Centreof the Hungarian Academy of Sciences,Szeged, Hungary

Cytochromes b561 (Cyts-b561) of invertebrates, vertebratesand plants are predicted to consist of six trans-membranehelices, with four conserved histidines involved in the coor-dination of two hemes. They are generally accepted tocatalyze ascorbate-driven trans-membrane electron trans-port. One isoform from the tonoplast of plant cells (TCytb)has been expressed in yeast and the characterization of therecombinant TCytb has just been started1,2.We have titrated the Q-band spectra of the wild type, H83A/H156A, and H83L/H156L TCytb at room temperature withdifferent reducing agents. Earlier results have proven thatthese mutants contained only one heme. The series ofspectra were analized by SVD. Fitting the V vectors by theNernst equation allowed us (1) to determine the differencespectra characterizing each of the two hemes, and (2) toshow that the double mutant TCytb-s have only the lowpotential heme, in good agreement with earlier resultsobtained at low temperature and/or by different spectroscopytechniques1,2.

[1] Berczi, A., Su, D., Asard, H. 2007. FEBS Lett. 581:1505-1508.

[2] Desmet, F., Berczi, A., Zimanyi, L., Asard, H. and VanDoorslaer, S. 2011. FEBS Lett. 585:545–548.

Supported by the Hungary-Romania Cross Border Cooper-ation Program of the EU, grant HURO/0901/219.

Ion channels and disease

O-558hERG1 channels: from antitargets to novel therapeutictargets in oncology.Annarosa ArcangeliDepartment of Experimental Pathology and Oncology,University of Florence and Istituto Toscano Tumori (ITT),Florence, Italy

Ion channels and transporters control many facets of cancercell biology1 and blocking the activity of these impairs tumorcell growth in vitro and in vivo. This new paradigm hasopened new opportunities for pharmaceutical research inoncology1,2. We have contributed to this field showing that

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Kv11.1 (hERG1) channels are aberrantly expressed in sev-eral human cancers where they control different aspects ofthe neoplastic cell biology such as proliferation and apopto-sis, invasiveness and angiogenesis, the latter through theregulation of VEGF secretion (reviewed in1). The hERG1-dependent effects were shown in vitro and, more recently, invivo. In preclinical models of both leukemia1 and colorectalcancer3, hERG1 overexpression confers a higher malig-nancy to neoplastic cells. Moreover, hERG1 blockers havetherapeutic potential, since preclinical tests showed thattreatment with specific hERG1 blockers overcame chemo-resistance in acute leukemias4 as well as reduced GI cancergrowth, angiogenesis and metastatic spread3. The overallmessage emerging from our data is that the hERG1 proteinrepresents a novel biomarker and drug target in oncology. Upto now, hERG1 was considered an ‘‘antitarget’’ due to thecardiac side effects that many (but not all) hERG1 blockersproduce and that result in lengthening the electrocardio-graphic QT interval. We report here recent studies on knownand newly developed hERG1 blockers that exhibit no car-diotoxicity and are more specific for the hERG1 channelsexpressed in cancer cells.

1. Arcangeli et al., Curr Med Chem 16: 66-93 (2009);2. Wulff et al., Nature Rev Drug Discovery 8: 982-1001

(2009);3. Crociani et al., submitted; Arcangeli et al., 30th Anniver-

sary Conference AICR,7-9 April 2010, abstract;4. Pillozzi et al., Blood, 117: 902-914, 2011.

This work was supported by the Italian Association forCancer Research (AIRC), the Istituto Toscano Tumori andthe Associazione Noi per Voi, Firenze.

O-559Analysis of the K1 current in human T cellsin hypercholesterinaemic stateAndras Balajthy, Zoltan Denes Peth}o, Zoltan Krasznai,Sandor Somodi, Peter HajduDepartment of Biophysics and Cell Biology, DebrecenMedical and Health Science Centre, University of Debrecen,98 Nagyerdei krt., Debrecen, Hungary, H-4012

We reported previously that the increase of the cholesterolcontent of the cell membrane (in vitro) modified the bio-physical parameters of the gating of Kvl.3 K? ion channels inhuman T-lymphocytes. In our present study we aimed todetermine the effect of hypercholesterolemia on the bio-physical parameters of Kv1.3gating and the proliferation of Tcells.T-lymphocytes were isolated from the peripheral blood ofpatients with cholesterol level considered normal (\5.2mmol/l,control group) and patients with hypercholesterina-emia (hc). Whole-cell K? currents were measured in patch-clamped T cells and the kinetic (activation and inactivationkinetics) and equilibrium parameters (voltage-dependenceof steady-state activation) of Kv1.3 gating were determined.Lymphocyte proliferation was measured using CFSEstaining with and without anti-CD3 and anti-CD28stimulation.

Our results indicate that the biophysical parameters of Kv1.3gating are similar in the control group and in the ’hc’ samples.The CFSE-based assay showed that hypercholesterolemic Tcells had higher spontenaous activation rate compared tocontrol group. However, T cells from high cholesterol levelpatients challenged by anti-CD3 and anti-CD28 exhibitedlower proliferation rate than control cells.

P-560Endothelial cells involved in chronic thromboembolicpulmonary hypertension have altered calciumhomeostasisZ. Balint1,2,6, D. Zabini1, W. Klepetko3, I. Lang4,H. Olschewski5 and A. Olschewski1,21University Clinic of Anaesthesia and Intensive CareMedicine, Medical University of Graz, Graz, Austria, 2LudwigBoltzmann Institute for Lung vascular Research, Graz,Austria, 3Department of Heart and Thoracic Surgery,University Clinic of Surgery, 4Department of InternalMedicine, Division of Cardiology, Medical Universityof Vienna, Austria, 5Department of Internal Medicine,Division of Pulmonology, Medical University of Graz, Graz,Austria, 6Stiftingtalstrasse 24, 8010 Graz, AustriaE-mail: zoltan.balint@lvr.lbg.ac.at

Chronic thromboembolic pulmonary hypertension (CTEPH)is a rare and late possible consequence of venous throm-boembolism. In this study, cellular properties of endothelialcells isolated from surgical material of CTEPH patients(CTEPH-hECs) were compared with control human pul-monary artery endothelial cells (hPAEC).The purified endothelial cells from CTEPH surgical tissueand the control hPAECs were cultured under standard con-ditions. CTEPH-hECs and hPAECs were stained for vonWillebrand factor and vascular-endothelial cadherin. Toaddress the intracellular calcium changes, Fura-2/am loadedcells were challenged with 10lM histamine, intracellularcalcium stores were depleted with 15lM BHQ and the acti-vation of transient receptor potential canonical channels(TRPCs) were followed.CTEPH-hECs and hPAECs both express similar pattern ofvon Willebrand factor and vascular-endothelial cadherin.CTEPH-hEC show no significant differences in basal calciumlevel (140,89±51,40 mM Ca2? for CTEPH-hEC, n=221;128,61±49,16 for hPAEC, n=245) as well as in the hista-mine-induced calcium peak (278,78±21,97 for CTEPH-hEC,n=110; 242,40±7,65 for hPAEC, n=133) in comparison tocontrol hPAECs. Interestingly, the challenge with BHQ leadsto a significant increase in calcium store depletion signal(505,27±37,51 for CTEPH-hEC, n=111; 200,67±25,77 forhPAEC, n=112). By adding back the calcium to the cells asignificant difference in the calcium influx occurs(652,39±46,88 for CTEPH-hEC, n=111; 322,84±19,33 forhPAEC, n=112).The isolated and purified endothelial cells from surgicalmaterial of CTEPH patients present the characteristic endo-thelial phenotype, but they show altered calciumhomeostasis at the stored calcium and store operated cal-cium influx level.

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P-561Identification of an intramolecular disulphide bondin the Na-channel b1-subunitR. Barbieri, D. Baroni, O. MoranCNR, Institute of Biophysics, Via De Marini 6, 16149 Genoa,Italy

Generalized epilepsy with febrile seizures plus (GEFS?,OMIM 604233) is a childhood genetic epilepsy syndromecorrelated to mutations in the ancillary b-subunit of neuro-nal voltage-gated sodium channels (NaChs). b1-subunit isnon-covalently associated with NaCh a- subunits, servingas modulator of channel activity, regulator of channel cellsurface expression and as cell adhesion molecule. The firstand best characterized GEFS? mutation is the C121W.This mutation changed a conserved cysteine residue into atryptophan, disrupting a putative disulphide bridge thatshould normally maintain an extracellular immunoglobulin-like fold.In this study, we investigated the presence of this putativedisulphide bond using 2-D-diagonal-SDS-PAGE, where theproteins were separated in the first dimension in absence ofa reduction agent and in presence of it in the seconddimension. This method allows to visualize the protein abovethe diagonal experimentally confirming that the disulphidebond is intramolecular. The protein spot was in-gel digestedand identified by MS/MS. This data support the hypothesis ofa loss of the S-S bond causing a molecular defect on GEFS?b1-mutation.Supported by Fondazione Carige, Italy.

O-562Calcium channels of vascular remodellingDavid J BeechInstitute of Membrane & Systems Biology, Facultyof Biological Sciences, University of Leeds, Leeds, LS2 9JT,UK

Vascular remodelling in physiology and disease is substan-tially due to vascular smooth muscle cells and endothelialcells that switch to phenotypes characterised by proliferation,migration, secretion, invasion etc. We have sought tounderstand ion channels in this context, particularly in humandiseased tissue. Data are consistent with roles of thepotassium channels KCa3.1 and Kv1.3. Most of our recentwork has, however, focused on calcium-permeable channelsthat are chemically activated. The data suggest that theTRPC1 channel subunit has an important role and is up-regulated in remodelling. We showed, for example, thatTRPC1, operating with TRPC5, is stimulated by oxidizedphospholipids (AL-Shawaf et al 2010, ATVB 30, 1453-).Intriguingly TRPC1 inhibits the function of other TRPCchannels and so it is unclear how it acts to drive calciumentry. Other types of calcium channel also contribute: Datasuggest roles for Orai1 and STIM1 as subunits/regulators ofCRAC channels. Growth factor (PDGF and VEGF) signallingshows striking dependence on such channels, conferringfunctional significance for cell migration and angiogenesis (Liet al 2011, Circ Res published on-line). These and otherrecent findings will be discussed. Financial support is pro-vided by Wellcome Trust, BHF, MRC.

P-564LQTS acquired by pentavalent antimony treatmentin guinea pigsA.C.A. Carvalho, L.F. Rodrigues-Junior, P.C. Arantes,M. Marocolo, C.C. Lores, J.H.M. Nascimento,Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

Pentavalent antimony (PA) is the mainstream agent of choicefor Leishmaniasis treatment. In therapeutic doses the PAtreatment has cardiac side effects that include QT interval pro-longation, torsade de pointes arrhythmias and sudden death.The objective of this study was to investigate arrythmogenicproperties of PA in treated guinea-pig, analyzing ECG param-eters, ventricular action potential and IKr, Iks ionic currents. Theguinea-pigs received daily 20mg/kg PA or not for 15 days. PAgroup had an increased QRS complex (control=10 ± 3 andPA=20±6, p\0,01), QTc interval duration (control=270±5 andPA=305±5, p\0,01) and QT dispersion (control=29±6 andPA=54±5, p\0,05) on ECG. The AP analysis demonstratedtriangulation (control=41±9 and PA=83±15, P\0,01) and pro-longation at 30% (control=55±1 and PA=85±3, p\0,05) and90% (control=100±3 and PA=168±5) AP with PA treatment.Also, the AP treatment reduced markedly IKr density (con-trol=2,1±0,7 and PA=0,6±0,2, p\0,01) and do not alter IKsdensity (control=5,5±1,5 and PA=5,9±1,4, p\0,01). The PAtreatment was lethal in 30% of the animals. Our results sug-gested that PA is a proarrhythmic drug that upon chronic usemay improve the mortality and cause repolarization distur-bances that characterized acquired long QTsyndrome.

O-565Voltage-gated ion channel dysfunction precedescardiomyopathy development ion the dystrophic heartX. Koenig1, S. Dysek2, S. Kimbacher1,2, M. Kovar1,A.K. Mike1, R. Cervenka1, P. Lukacs1, B.D. Xuan1, K. Nagl1,H. Todt1, R.E. Bittner2 and K. Hilber11Center for Physiology and Pharmacology, MedicalUniversity of Vienna, Vienna, Austria, 2Center for Anatomyand Cell Biology, Medical University of Vienna, Vienna, Austria

Duchenne muscular dystrophy (DMD) is associated with severecardiac complications. Recent research suggests that impairedvoltage-gated ion channels in dystrophic cardiomyocytesaccompany cardiac pathology. It is, however, unknown if ionchannel defects are primary effects of dystrophic genemutations,or secondary effects of the developing cardiomyopathy. Here, westudied Na and Ca channel impairments in dystrophic neonatalcardiomyocytes, derived from DMD mouse models, prior to car-diomyopathy development. Dystrophin-deficiency reduced Nacurrent density. In addition, extra utrophin-deficiency altered Nachannel gating. Moreover, also Ca channel inactivation wasreduced, suggesting that ion channel abnormalities are universalprimary effects of dystrophic gene mutations. To assess devel-opmental changes, we also studied Na channel impairments indystrophic adult cardiomyocytes, and found a stronger Nacurrent reduction than in neonatal ones. The described Nachannel impairments slowed the action potential upstroke inadult cardiomyocytes, and only in dystrophic adult mice, theQRS interval of the ECG was prolonged. Ion channel impair-ments precede pathology development in the dystrophic heart,andmay be considered cardiomyopathy triggers. Supported byAustrian FWF (P19352).

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P-566Exercise does not prevent cardiac autonomicdysfunction in rats treated with anabolic steroidsMoacir Marocolo1,2, Ana Carolina Carvalho1, Paulo CesarArantes1, Cassia Loares1, Luiz Fernando Rodrigues1, SilvioMarques Neto1, Jose Hamilton Matheus Nascimento11Federal University of Rio de Janeiro/UFRJ - Carlos ChagasFilho Biophysics Institute/IBCCF, Rio de Janeiro, Brazil,2Federal University of Triangulo Mineiro/UFTM – SportSciences Department, Uberaba, Brazil

Keywords: Anabolic androgenic steroids, regular exercise,heart rate variability, cardiac autonomic dysfunctionThe aim of this study was to evaluate the changes in HRVinduced by administration of nandrolone decanoate (ND),with or without exercise during 8 weeks(W). 40 male ratswere divided into four groups: sedentary (DS) and trained(DT) treated with ND; sedentary (CS) and trained (DT)treated with vehicle. Exercised were carried 5 days/W. ECGwas recorded at 0, 4th and 8thW. Time- and frequency-domain analysis of heart rate variability was performed.SDNN and RMSSD were reduced in DT (7.81±2.85ms) andDS (6.21±1.13ms) compared to CS (9.6±2.6ms) after 4th

and 8thW (DT:7.02±3.17 ms, DS:4.6±1.12 ms vsCS:8.4±1.1 ms); pNN5 was reduced only after 8thW(DT:23.3±5.7ms, DS:14.3±6.3ms vs CS:33.2±8.5ms). CTshowed increase in RMSSD and pNN5% after 8W, com-pared to CS (RMSSD=10.47±2.36ms vs 8.38±1.1ms;pNN5%=43.2±9 vs 33.2±8.5). High frequency(HF) of DSand DT was reduced compared to CS after 4th (6.65±6ms2,4.9±1.7ms2 vs 12.8±2.63ms2) and 8th (5.34±3.39ms2,4.16±0.93ms2 vs 11.54 ± 1.6ms2) while LF/HF ratio increasedafter 4th (2.1±1.3, 1.95±0.83 vs 0.9±0.2) and 8th (2.08±1.0,2.5±0.44, 0.85±0.14). CT had a rise in HF compared to CS on8thW (14.75±2.44ms2 vs 11.5±1.6ms2). A Supraphysiologicaldose of ND induces cardiac autonomic dysfunction and regularexercise does not prevent this effects.

P-567Mg21-inhibitable TRPM7-like cation current in humanatrial myocytesI. Martisiene, V. Gendviliene, D. Zablockaite,R. MacianskieneLithuanian University of Health Sciences,Kaunas, Lithuania

The aim of our work was to identify the presence of TRPM7-like current in human atrial myocytes, and to investigate thepotential role of these channels in the electrical activity ofatrial cells during atrial fibrillation (AF). Myocytes were iso-lated from right atria biopsies from 72 adult patients in sinusrhythm (SR) and 49 patients in AF. We demonstrate that inboth patient groups under voltage clamp, with voltage-dependent and other ion channels inhibited, cells dialyzedwith low intracellular free [Mg2?]i produced outward-rectifyingTRPM7 currents that could be suppressed by raising extra-cellular [Mg2?]o or by application of Gd3? and 2-APB (at 100lM), known to block TRP channels. The current was absentin cells dialyzed with physiological free [Mg2?]i or MgATP, butthat did not protected from the spontaneous run-up in diva-lent free external solutions. In addition, we established that

the density of activated TRPM7-like current in cells frompatients with AF was higher as compare with those with SR(5.30±0.39 and 3.62±0.23 pA/pF, respectively, at ?80mV,p\0.001). In conclusion: this is the first study, whichdescribes TRPM7-like current in native human atrial cardio-myocytes that operates under pathophysiological conditions.AcknowledgementsSupported by RCL Nr.MIP-78/2010.

O-568Clustering of voltage-gated sodium channel NaV1.8in lipid rafts is essential for action potential propagationin nociceptive unmyelinated axonsKenji Okuse, Amber Finn, Alessandro PristeraDivision of Cell & Molecular Biology, Faculty of NaturalSciences, Imperial College London, London, UK

It has been over 15 years since sensory neuron-specificsodium channel NaV1.8 was identified. Since then NaV1.8 hasbeen shown to play a crucial role in pain pathways, and itbecame a prominent drug target for novel pain killers. In con-trast to myelinated neurons, mechanisms that target voltage-gated sodium channels to the unmyelinated C-fibre axons arelargely unknown.We investigated the localisation of NaV1.8 inunmyelinated primary sensory neurons. NaV1.8 was found tobe clustered in lipid rafts on unmyelinated axons. When thelipid rafts are disrupted, remarkable reduction in both theconduction velocity and the number of cells responsive tomechanical stimuli to the unmyelinated axons were seen.Using a compartment culture system, we also found that dis-ruption of rafts in the middle region of the sensory axonscaused a significant reduction in the responsiveness of theneurons to chemical stimuli to nerve endings. This is due to thefailure of action potential propagation through the axons.These data suggest that clustering of NaV1.8 in lipid rafts ofunmyelinated fibres is a key factor for the functional propertiesof the channel, which may due to a change in the voltagethreshold. Disruption of the NaV1.8 cluster and modifying lipidrafts in primary sensory neurons may be a useful newapproach to control the excitability of nociceptive neurons.

P-569Effect of ion channel blockers on T-lymphocytesstimulated with anti-CD3 and anti-CD28Zoltan Denes Peth}o, Andras Balajthy, Zoltan Krasznai,Zoltan Varga, Gyorgy PanyiDepartment of Biophysics and Cell Biology, Medicaland Health Science Center, University of Debrecen, Hungary

Ion currents in are crucially important for activation ofT-lymphocytes. Our aim was to investigate how the blockageof various ion channels in isolation or in combination affectsmitogen-dependent activation and proliferation of T-cells.We activated human peripheral blood lymphocytes usingmonoclonal antibodies against the TCR-CD3 complex andCD28. We applied specific channel blockers inhibiting themajor ion channels of the T-cell: either the Kv1.3 (TEA orAnuroctoxin), IKCa1 (TRAM-34), or the CRAC channel (2-APB) alone or in combination. Five days after the stimulus wemeasured the change in cell size and cellular granulation with

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flow cytometry alongwith the proportion of dividing cells, usingCFSE (carboxyfluorescein succinimidyl ester) dilution assay.Our measurements indicated that the ion channel blockerssuppressed the proportion of dividing cells in a dose-dependent manner. Increasing the strength of the stimulationreduced the potency of the blockers to inhibit cell proliferationand eventually the blockers were ineffective in decreasinglymphocyte proliferation. We experienced the greatestamount of inhibition using the combination of the blockers,which indicates synergy in the regulation pathway of thevarious ion channels.

P-571Quantitative NaPi2b transporter expression in differenthistomorphological types of ovarian cancerM.A. Shyian1, 2, V.S. Gryshkova1, O.I. Kostianets1, 2,V.V. Filonenko1, R.G. Kiyamova11Institute of Molecular Biology and Genetics, NAS of Ukraine,Kyiv, 03680, Ukraine, 2Taras Shevchenko National Universityof Kyiv, Kyiv, 01601, UkraineE-mail: m.a.shyyan@gmail.com

Recently, sodium dependent phosphate transporter NaPi2bwas revealed as potential marker for breast, thyroid andovarian cancer. In vivo, NaPi2b is involved in maintenance ofphosphate homeostasis and mutations or aberrant expres-sions of its gene (SLC34A2) are associated with severaldiseases including cancer. However, data about NaPi2bmRNA expression in different types of cancer and corre-spondent normal tissues are controversial and restricted.We investigated SLC34A2 gene expression level in normalovarian tissues and different histomorphological types ofovarian tumors using real-time PCR analysis. It was foundthat SLC34A2 gene was highly expressed in well-differenti-ated endometrioid and papillary serous tumors, but was notexpressed in low-differentiated tumors, benign tumors and inmost normal tissues. mRNA expression of SLC34A2 in se-rouse and endometrioid ovarian tumors accurately correlatedwith protein expression that was detected in these tumorsamples by Western blot analysis and immunohistochemistryin our previous investigation.Upregulation of SLC34A2 gene expression in well-differen-tiated tumors may reflect cell differentiation processes duringovarian cancerogenesis and could serve as potential markerfor ovarian cancer diagnosis and prognosis.

O-572Membrane-permeant inhibitors of the potassium channelKv1.3 induce apoptosis in cancer cell linesLuigi Leanza1, Brian Henry3, Nicola Sassi2, Mario Zoratti2,Erich Gulbins3 and Ildiko Szabo11Department of Biology, 2Department of BiomedicalSciences, CNR Institute of Neurosciences, Universityof Padova, 3Department of Molecular Biology Universityof Duisburg-Essen, Germany

Potassium channels are emerging therapeutic targets incancer. Kv1.3 has been shown to play an important regula-tory role during proliferation and apoptosis. In lymphocytesand other cells Kv1.3 is present in both the plasmamembraneand the inner mitochondrial membrane (mitoKv1.3). Inhibition

of mitoKv1.3 by pro-apoptotic Bax results in production ofreactive oxygen species and leads to cytochrome c releaseand death. Three distinct membrane-permeant inhibitors ofKv1.3 induce apoptosis in different cancer cell linesexpressing mitoKv1.3, while membrane-impermeant inhibi-tors do not induce cell death, suggesting that apoptosisinvolves inhibition of mitochondrial and not of plasma mem-brane Kv1.3. The effect of these drugs is abolished whenKv1.3 is absent or its expression is reduced by siRNA,demonstrating Kv1.3-specificity of the inhibitors. Mostimportantly, the drugs are able to induce death in two celllines lacking Bax and Bak. Our work thus identifies a novelpharmacological tool to induce apoptosis in mitoKv1.3-expressing cells and in vivo experiments are under way totest sensibility of tumors to these drugs.

New and notable

P-574Protein Dynamical Transition and Resolution Effectson Mean Square Displacement in Lysozyme SystemsA. Benedetto, S. Magazu, F. MigliardoDepartment of Physics, University of Messina, VialeD’Alcontres 31, P.O. Box 55, 98166 Messina University, Italy

In the present contribution a procedure for molecular motioncharacterization based on the evaluation of the Mean SquareDisplacement (MSD), through the Self-Distribution Function(SDF), is presented. It is shown how MSD, which representsan important observable for the characterization of dynamicalproperties, can be decomposed into different partial contri-butions associated to system dynamical processes within aspecific spatial scale. It is shown how the SDF procedureallows us to evaluate both total MSD and partial MSDsthrough total and partial SDFs. As a result, total MSD is theweighed sum of partial MSDs in which the weights areobtained by the fitting procedure of measured Elastic Inco-herent Neutron Scattering (EINS) intensity. We apply SDFprocedure to data collected, by IN13, IN10 and IN4 spec-trometers (Institute Laue Langevin), on aqueous mixtures oftwo homologous disaccharides (sucrose and trehalose) andon dry and hydrated (H2O and D2O) lysozyme with andwithout disaccharides. The nature of the dynamical transitionis highlighted and it is shown that it occurs when the systemrelaxation time becomes shorter than the instrumental energytime. Finally, the bioprotectants effect on protein dynamicsand the amplitude of vibrations in lysozyme are presented.

P-575DNA damage in breast cancer cells treatedwith combination of quercetin and anticancer drugsJoanna Bernasinska, Anna Pieniazek, Karolina Matczak,Aneta Koceva-ChylaDepartment of Thermobiology, Institute of Biophysics,University of Lodz, Lodz, Poland

Quercetin (Q) is a natural flavonol present at high levels innumerous of plants. It has been epidemiologically associatedwith bioactive protective effects, but has also been shown tobe mutagenic and toxic in in vitro cell systems.

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We evaluated Q for genotoxicity in MCF-7 breast cancer cellsin the presence or absence of doxorubicin (DOX), docetaxel(DTX) and paclitaxel (PTX), anticancer drugs commonlyused in chemotherapy of different solid tumors. Damage toDNA was determinated by comet assay. The cells, aftertreatment with investigated compounds, were washed upand cultured in fresh medium for 0, 24, 48, 72 and 96 hours.We have found that Q by itself caused significant DNAdamage. Moreover flavonol enhanced genotoxic effect ofanticancer drugs. The highest amount of DNA in the comettail was observed 48 h after treatment with combination of Qwith DOX. Similar changes were found in cells incubated withcombination of Q with taxanes – PTX or DTX. However,damage to DNA in this case was considerably lower thandamage caused by combination of Q with DTX.Our results confirmed anticancer and genotoxic activity ofquercetin, which makes it a promising candidate for apotential use as a modulator of cytotoxicity and anticanceractivity of anthracycline and taxane chemotherapeutics.

P-576Non-ionizing electromagnetic-own-signal-treatmentBokkon Istvan1, Till Attila2, Erdofi-Szabo Attila31Doctoral School of Pharmaceutical and PharmacologicalSciences, Semmelweis University, Budapest, Hungary,2National Institute for Medical Rehabilitation, Budapest,Hungary, 3Director of BioLabor, Budapest, HungaryE-mail: bokkoni@yahoo.com

Although the health effects of low-frequency and intensityelectromagnetic fields (LFI-EMFs) are controversial,increasing evidence suggests that LFI-EMFs are capable forinitiating various healing processes. Many (bio)physicalideas were suggested to explain the influence of LFI-EMFs inliving systems but the main effect of LFI-EMFs on cell func-tions remains vague. However, some effects of LFI-EMFsmay be explained by redox and membrane processes. Dur-ing diseases, cells not only demonstrate altered biochemicalprocesses but also produce altered non-linear bioelectro-magnetic complex patterns. Thus, it is reasonable to usenon-linear bioelectric and bioelectromagnetic signals fromcells of the body for potential therapeutic applications thatmay be more effective than the artificial LFI-EMFs signals.Our novel EMOST (Electromagnetic-Own-Signal-Treatment)method is based on the utilization of the non-linear, bio-electric and bioelectromagnetic signals of the patientswithout any electromagnetic wave modulation and inversionof recorded output signals of subjects. Here, we report oursome restorative results after EMOST application. We alsosuggest that the possible effects of the EMOST may beachieved via redox-related processes.

P-577Macroscopic properties of phospholipid vesicleswith domains in phase-separated membranesBojan Bozic and Janja MajhencInstitute of Biophysics, Faculty of Medicine, Universityof Ljubljana, Ljubljana, Slovenia

Ternary mixtures of a high-melting lipid, a low-melting lipid, andcholesterol are known to form domains of a liquid-ordered and

a liquid-disordered phase in bilayer membranes. Giant vesi-cles, prepared from a sphingomyelin/dioleoylphosphocholine/cholesterol mixture, are examined using fluorescence micros-copy. NBD-labeled lipid and BODIPY-labeled cholesterol areused to identify the phase domains of the membrane. A vesiclewith only two domains is chosen because of its simple geom-etry, for convenient comparison of the experimental results withthe theoretical predictions. The volume of the vesicles isgradually decreased and/or increased by changing the osmo-larity. The relevant energy terms of the membrane mechanicsare the bending energy and the Gaussian bending energy, andthe energy of the domain boundary. The energy of the domainboundary is proportional to its length. At the boundary betweenthe domains a contact angle is taken into consideration. Inorder to obtain the values of the lateral tension and the contactangle, the areas of the domains and the characteristicdimensions of the shape are determined for different volumes.The best fits were obtained for a line tension of 6 ± 3 pN and acontact angle of 1.4 ± 0.3 rad.

P-579Fractal avalanche ruptures in biological membranesIrep Gozen1, Paul Dommersnes1,2, Ilja Czolkos1, TatsianaLobovkina1,*, Aldo Jesorka1 and Owe Orwar11Chalmers University of Technology, Goteborg, Sweden,2Matieres et Systemes Complexes,Universite Paris Diderot,Paris, France, *Present address: Stanford University,Stanford, USA

Previously, membrane rupture dynamics has been studied inbilayer vesicles under tensile stress, which consistently pro-duces circular pores. We observed very different rupturemechanics in bilayer membranes spreading on solid supports:the ruptures proceeded in a series of rapid avalanches causingfractal membrane fragmentation. The intermittent character ofrupture evolution and the broad distribution in avalanche sizesis consistent with crackling-noise dynamics. Such noisydynamics appear in fracture of solid disordered materials, indislocation avalanches, in plastic deformations, and domainwall magnetization avalanches. We also observed similarfractal rupture mechanics in spreading cell membranes.

O-580Background K1 channel gating and cell excitabilityFlorian Lesage, Guillaume Sandoz, Sylvain Feliciangeli,Delphine Bichet, Maud Larroque and Franck ChatelainInstitut de Pharmacologie Moleculaire et Cellulaire, CNRS -Universite de Nice Sophia Antipolis, 06560 Sophia Antipolis,France

Background or leak potassium conductances are a majordeterminant of resting potential and input resistance, two keycomponents of cell excitability. These currents are not pas-sive but finely tuned and adapted to cell specific functions.K2P channels producing these currents are tightly regulatedby a variety of chemical and physical stimuli including tem-perature, membrane stretch, free fatty acids, pH, Ca2?,neurotransmitters and hormones as well as protein partners.These different stimuli converge on gating mechanisms thatshow remarkable conservation between intracellular K2P

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channels (TWIK1 channels) and K2P channels located at theplasma membrane (TREK1/2 channels).

P-581Living at the edge: volume 2 the conduction systemof interfacial forces into the alveolar type II cellN. Hobi1, A. Ravasio1and T. Haller11Dept. of Physiology and Medical Physics, Divisionof Physiology, 2 Dept. of Internal Medicine I, 3Dept.of Physiology and Medical Physics, Division of BiomedicalPhysics, Innsbruck Medical University, A–6020 Innsbruck,Austria

Many epithelia have contact with air-liquid interfaces (ALI).This applies particularly to the lung, where the surfactantsecreting alveolar type II cells (AT II) even project into the air.In previous investigations, using new microscopic approa-ches, we found that the presence of an ALI leads to aparadoxical situation: it is a potential threat that may causecell injury, but also a important stimulus: AT II cells respondpromptly, and show sustained Ca2?-signals that activateexocytosis. Exocytosed surfactant, in turn, clearly prolongedthe time to irreversible cell damage, and may be an adaptiveand evolutionary defense mechanism against the harmfulnature of surface forces. Recently we published that AT IIcells are sensing the ALI but how this stimulus is conductedand converted into the cell, is still obscure.Currently we are searching for potential calcium sources andit seems that the cells signal Ca2? by extracellular Ca2? entryprobably through mechanosensitive channels. Special-designed gene chips allowed a whole genome profiling of ALI

exposed single cells. These cells react with rapid changes onthe transcriptional level: cellular pathways that are involvedinclude e.g. defense response and lipid metabolism, and weidentified genes associated with several lung diseases andinjuries. We summarize, interface forces are strong, they areacting on the cells and triggering cellular events that areclosely related with classical concepts in mechanotransduc-tion, it is very plausible that those forces play a crucial role inthe lung surfactant homeostasis.

Abbreviations:Alveolar type II cell AT II cellAir-liquid-interface ALI

O-582Get a grip: engineering ultra-stable interactionsto proteins for biophysics and medicineMark Howarth, Bijan Zakeri, Claire Chivers, Lotta Holm,Estelle Crozat, David SherrattDepartment of Biochemistry, University of Oxford, SouthParks Road, Oxford, OX1 3QU, UK

Achieving strong and specific binding to proteins is importantfor many areas of biophysics and also for new therapeutics.The mammalian immune system usually addresses thischallenge within a few weeks, but the antibodies raised havemodest affinity. We have designed stronger interactions,through chemical and bioengineering approaches. For bind-ing endogenous proteins, we have generated electrophilicaffibodies, for proximity-induced covalent binding. For binding

peptide tags, we have designed genetically-encoded pep-tides based on the human pathogen Streptococcus pyogenesthat spontaneously form amide bonds to their protein partner.For binding to small-molecule ligands, we have modified thestreptavidin-biotin interaction to achieve even stronger biotinbinding and mechanical strength. These technologies havebeen applied to mammalian cell imaging, immunoassays, andfor testing the translocation mechanism of motor proteins in a‘‘molecular car-crash’’.

P-583DSC Characterization of protein aqueous solutionsP. Kamasa1, M. Bokor1, A. Gelencser2, P. Tompa2,K. Tompa21Research Institute for Solid State Physics and Optics,Hungarian Academy of Sciences, pob. 49, 1525Budapest,Hungary, 2Institute of Enzymology, BiologicalResearch Center, Hungarian Academy of Sciences, POB. 7,1518 Budapest, Hungary

Calorimetric method and instrumentation were worked outand applied for investigation aqueous solutions of proteins.Thermal effects were analyzed by own construction heat-flux-type DSC cell designed for temperature range from the boilingpoint of water down to 120 K. The achieved sensitivity of heatflow rate (HF) of the instrument is better than 50%W testedusing 1 ll of 150 mM aqueous solution of NaCl. From theintegral value of HF - the total enthalpy change DHtotal, theenthalpies of transitions were separated from the heatcapacities. Using the method, several types of proteins (BSA,ERD10, UBQ, a-, b-casein, and WT, A53T, a-synucleinmutants) were investigated in that temperature range. Theresults are shown in detail as an illustrative example. Poten-tial applications are outlined, which include (i) the distinctionbetween the solvent accessible surfaces of globular andintrinsically disordered proteins, (ii) the distinction betweenprotein mutants, and (iii) the identification of monomer andpolymer protein states. This method provides a possibility tostudy the polymerization process (amyloid formation) and toinvestigate in-situ the reason and circumstances of that.

P-584Finite element approach to understand self gravitationalbio in embryological compact massRajan Kashyap1, Sagarika Bhattacharjee2

and I.R.Bhattacharjee31Politecnico Di Torino, Italy, 2International Hospital, LotusTower, G.S.Road, Guwahati-781005, India, 3Institutefor Intrinsic Gravitation Biology (i3GB) 84, GMCH Road,Anandanagar, Dispur, Guwahati-781005, IndiaE-mail: rajankashyap6@gmail.com

Morphometry due to self gravity in living organism is anintegral part in understanding Self Gravitation Bio. Compu-tational studies of biological system, especially in ab-initioembryo as self gravitating mass, floating over amniotic fluid,as if maintaining anti – gravitation (extrinsic) mechanism,would be an interesting one to explore biomechanics ofintrinsic gravity. Since the work would be of exploratory nat-ure, both from the point of view of identifying appropriatestage of development of embryological morulla and available

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computational logics, it was contemplated to initiate functionalstudy with a few reported ultrasound evidential works on smallanimals like mice, grasshopper including compact humanembryo as mathematical structure that may allow the formaldefinition of concepts such as convergence, mapping andcontinuity. As many of the finite-dimensional function analysisin topological vector spaces are available, we initiated oursimulation and studies on concentrating locally compactbanach spaces. On following differential equation in 3-D withrequisite divergence, biparietal diameter and mentoverticaldiameter are parametrical values used to computationallysimulate and model a final compact cranial topological mass.

P-586Synthetic small molecules with cardiomyogenic effectson stem and progenitor cellsA.K. Mike1, X. Koenig1, S. Kimbacher1, H. Todt1, M. Koley2,M. Schnurch2, M.D. Mihovilovic2, G. Weitzer3 and K. Hilber11Center for Physiology and Pharmacology, MedicalUniversity of Vienna, Vienna, Austria, 2Institute of AppliedSynthetic Chemistry, Vienna University of Technology,Vienna, Austria, 3Max F Perutz Laboratories, MedicalUniversity of Vienna, Vienna, Austria

Cell transplantation into the injured heart is a new therapyafter myocardial infarction. Its success, however, is impededby the limited capacity of donor cells to differentiate intofunctional cardiomyocytes in the heart. A strategy to over-come this problem would be the induction of cardiomyogenicfunction in cells prior to transplantation. Recently, syntheticsmall molecules (SySMs) were identified, which exhibit aremarkable capacity to trans-differentiate cells into other celltypes. The aim of this study was to characterize SySMs withcardiomyogenic activity. Treatment of P19 embryonic carci-noma cells and C2C12 skeletal myoblasts with Cardiogenol-C (CgC) or MK142 significantly up-regulated cardiac markerexpression. Thereby, CgC was especially active on lineage-committed C2C12, whereas MK142 on uncommitted P19cells. Supporting this difference in target cell specificity, instudies on embryoid and cardiac body development fromvarious cell types, CgC produced more beating cardiomyo-cytes from committed A5 cardiovascular progenitor cells,while MK142 more from uncommitted W4 mouse embryonicstem cells. We conclude that MK142 promotes mesodermaldifferentiation of stem cells, whereas CgC drives cardiomy-ocyte maturation of already induced mesoderm. Supportedby AWS (Z090391).

P-587Elements and relations of a Life Theory (LT)Stephan OpielokFree University Berlin, Berlin, Germany

I propose a general theory about the genesis and nature oflife(optimum). It seems to be based as a rule on interactionbetween symmetry and asymmetry (* stability and flexibility)for equilibration. In that kind of multidisciplinary researchthese concept generalized and deepens previous trials toexplain the question what is life.

Finally the data of this exploration show that nature of life ispredictable by suitable methods for measurement like e.g.spectroscopy and probability.As a result, the way of life goes the optimal friction byessential level of efficiency energy use.

O-589Towards attosecond biophysics: what can ELI-ALPSoffer?Gabor SzaboDepartment of Optics & Quantum Electronics, Universityof Szeged, Szeged, Hungary

The Hungarian pillar of the ELI (Extreme Light Infrastruc-ture) project, ELI-ALPS (Attosecond Light Pulse Source),is designed to open new vistas in attosecond science. Itwill be a high power, high repetition rate XUV/x-ray pro-viding coherent pulses with attosecond and few-femtosecond time-structure allowing for practically all kindsof pump-probe experiments providing unprecedentedaccess to the ultrafast electronic dynamics that constitutesthe primary response of atomic and molecular systems toincident light. Especially important in understanding bio-molecules is the combination of attosecond and fewfemtosecond pulses that will make possible the directinvestigation of electron dynamics. Another key feature ofELI-ALPS that may find significant biological applicationsis its potential for sub-atomic 4D imaging. From a technicalpoint of view it is important to emphasize that the highphoton flux combined with extremely high (up to 100 kHz)repetition rate makes ELI-ALPS very user friendly inexperiments where complex samples, such as biologicalmaterial, are being studied.

O-590Hydrodynamic synchronisation of light drivenmicroscopic rotorsPal Ormos, Andras Buzas, Lorand Kelemen, Laszlo Oroszi,Gaszton Vizsnyicai, Roberto di LeonardoBiological Research Centre Szeged, Institute of Biophysics,Szeged, Hungary

Moving organelles of microscopic biological systems oftentend to synchronize. It has been proposed that the origin ofthe synchronization is hydrodynamic interactions. However,there has been yet no direct experimental demonstration ofthe effect at the corresponding microscale. We used afunctional model system to carry out experiments to dem-onstrate hydrodynamic synchronization.We have produced micrometer sized propellers by twophoton photopolymerization. They are held in optical twee-zers and are rotated by light. Such rotors were held androtated close to each other in optical traps produced byholographic optical tweezers. The distance of the rotors andthe rotating torque could be freely varied. Synchronization ofthe rotation was demonstrated. The effect was characterizedunder a broad range of parameters. Based on the compre-hensive data we built a model to describe the effect andtested it in detail.

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Membrane structure

O-591Single molecule biology—studying meetingswithin the plasma membraneGerhard J. SchutzInstitute of Applied Physics, Vienna University of Technology,Austria

In my lecture, I will show examples how to obtain insights intothe organization of the cellular Nanocosm by single moleculeexperiments. Our primary goal is to understand the molecularorganization of the plasma membrane and its impact onsignaling processes. For this, we apply single moleculetracking to resolve the plasma membrane structure at sub-diffraction-limited length-scales by employing the high pre-cision for localizing biomolecules of *15 nm. Brightness andsingle molecule colocalization analysis allows for studyingstable or transient molecular associations in vivo. In partic-ular, we developed a technique to detect molecular clusterformation directly in the live cell plasma membrane. With thismethodology, individual aggregates can be selectivelyimaged, and the load of each cluster can be determined. Weapplied this technique to investigate the association of afluorescent lipid analogues and lipid anchored proteins.Aggregates containing up to 4 probe lipids were observed todiffuse freely as stable platforms, shedding new light on thecurrent debate concerning the existence of ‘‘lipid rafts’’. Usingthe same technique we could further quantify the subunitstoichiometry of ion channels. Finally, single molecule toolsenable the direct observation of the random transitions abiomolecule experiences during its movements through theplasma membrane. For example, transient proximity due tointeractions with other proteins can be directly monitored andquantified. I will present results on the interaction betweenantigen-loaded MHC and the T cell receptor directly in theinterface region of a T cell with a mimicry of an antigen-presenting cell, using single molecule FRET.

O-592The native M2 proton channel structure from influenza AT.A. Cross1,2,3, M. Sharma1,2, M. Yi3,4, H. Dong3,4, H. Qin2,E. Peterson5, D.D. Busath5, H.-X. Zhou3,41National High Magnetic Field Lab, 2Departmentof Chemistry and Biochemistry, 3Institute of MolecularBiophysics, 4Dept. of Physics, Florida State University,Tallahassee, FL, 5Dept. of Physiology and DevelopmentalBiology, Brigham Young Univ. Provo, UT, USA

Anfinsen’s thermodynamic hypothesis states ‘‘that the nativeconformation (of a protein) is determined by the totality ofinter-atomic interactions and hence by the amino acidsequence in a given environment.’’ As in most Biochemistrytextbooks, too often these last four words are ignored. Formembrane proteins the complex and heterogeneous mem-brane environment can have a substantial impact on proteinstructure and dynamics as well as on our understanding ofprotein function. I will discuss a new structure of the M2proton channel from Influenza A virus that is a proven drugtarget. With solid state NMR we have characterized the full

conductance domain (residues 22-62) that has very similarelectrophysiology to that of the full length protein. Thisstructure has been characterized in a native-like liquid crys-talline lipid bilayer environment. Additional structures havebeen achieved by x-ray crystallography as well as solutionand solid state NMR, both with and without the antiviral drug,amantadine. Recently, resistance to the antiviral drugsamantadine and rimantadine has dominated the flu seasonsand the recent swine flu pandemic. The resistance resultedfrom the S31N mutation in the proton conducting pathway.Considerable debate has arisen from the various structuresof the M2 protein and their implications for both proton con-ductance and drug binding.The solid state NMR conductance domain represents ahighly constrained backbone structure with key sidechainrestraints. In restrained Molecular Dynamics and QM/MMcalculations a detailed model of the key HxxxW sequenceresponsible for acid activation, gating and conductance hasbeen achieved at neutral pH. Based on this model it hasbeen possible to develop a mechanism for proton conduc-tance showing in detail how the charge on the histidineresidues is stabilized and how protons are shuttled from theN-terminal pore to the C-terminal pore of this protein. Themechanistic details explain a broad range of chemical andphysiological data.

P-593Lipid composition is the dominant factor that determinesthe orientational states of membrane-boundantimicrobial peptidesSergii Afonin1, Ralf W. Glaser2, Carsten Sachse3, JesusSalgado4, Parvesh Wadhwani1, Katja Koch1, MarcoIeronimo1, and Anne S. Ulrich11Karlsruhe Institute of Technology (KIT), Karlsruhe,Germany, 2Friedrich-Schiller-Universitat, Jena, Germany,3EMBL, Heidelberg, Germany, 4Universitat de Valencia,Burjassot, Valencia, Spain

The perturbing activity of membrane-active peptides incellular membranes is attributed to the protein-lipid inter-actions. A systematic correlation of peptide behavior withthe bilayer properties should thus provide important cluesabout cytolytic mechanisms. Here, we have studied twostructurally unrelated antimicrobial peptides, the cyclic b-stranded gramicidin S, and the a-helical PGLa. Using solid-state 19F-NMR in oriented bilayers, we have previouslycharacterized different alignment states for them in lipidmembranes [JACS 130:16512; Top.Curr.Chem. 273:139].On this basis, we can now use the signal of a specific 19F-label as a fingerprint to monitor the re-alignment andmobility of these two peptides as a function of membranecomposition. An NMR temperature series passing throughthe relevant lipid phase transition allows to address thestate of the peptides in response to the lipid phase state.That way we have explored the effects of bilayer thick-ness, surface curvature, negative charge, and presence ofcholesterol. These data are then compared with the pep-tide behaviour in plasma membranes from erythrocytesand bacterial protoplasts. We discuss the results withrespect to the activities of these two peptides and evaluatethe meaningfulness of the various membrane models.

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O-596Perforin induces invaginations in model membranesGregor Anderluh1, Tilen Praper1, Andreas F.-P. Sonnen2,Mauro Dalla Serra3, Christopher J. Froelich4, Robert JCGilbert21Department of Biology, Biotechnical Faculty, Universityof Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia,2Division of Structural Biology, Welcome Trust Centrefor Human Genetics, University of Oxford, Roosevelt Drive,Oxford, OX3 7BN, UK, 3National Research Council - Instituteof Biophysics & Bruno Kessler Foundation, via alla Cascata56/C, 38123 Trento Povo, Italy, 4Department of Medicine,NorthShore University HealthSystem Research Institute,Evanston, IL, 60201, USA

Perforin (PFN) is an important pore forming protein from thehuman immune system, which enables the clearing of virus-infected or tumor cells. PFN pores exist with a range offunctional diameters. The larger pores allow the crossing ofgranzyme B across the membrane, but the direct passage ofthis probe appears to be rather slow process. We have alsoobserved that PFN can induce the formation of invaginationsand internal vesicles on giant unilamellar vesicles (GUVs)composed of pure lipids and when GUVs were formed fromred blood cell ghosts. Invaginations formation was dependenton the presence of Ca2? and functional PFN. The process ofinvaginations started rapidly and intraluminal vesicles werefilled with fluorescent dextrans from the surrounding medium,irrespective of the dextran size. Intraluminal vesicles werecomplete and in some cases separated from the originatingmembrane. PFN was not uniformly distributed over the wholeGUV surface but rather concentrated in the invaginations,according to the labeling by anti-PFN antibodies. Theobserved PFN activity may have implications for the transferof granzymes into the cytosol of the target cells.

P-597Phospholipid/cholesterol binary mixtures: polarityvariations with composition and temperatureDalila Arrais1, Jorge Martins1,21IBB-CBME, Faro, Portugal, 2DCBB - FCT, Universidadedo Algarve, Faro, Portugal

Phosphatidylcholine (PC), sphingomyelin (SM) and cholesterol(Chol) are considered the major constituents of the outer lipidicleaflet of cell plasma membranes. The binary mixtures of PCand SM with cholesterol gives rise to significant differences inlipid bilayers properties, such as water permeability, thicknessand molecular packing. Those variations will lead to someimportant changes in the fluidity and polarity gradients that canbe measured within model membranes.We studied the polarity variations of saturated (DPPC, egg-SM) and unsaturated (POPC, DOPC) phospholipids bilayers,and their respective mixtures with Chol at specific molarproportions, using the pyrene empirical polarity scale.For POPC and DOPC the polarity values tend to decrease,either increasing cholesterol concentration, as well as tem-perature. For DPPC and egg-SM, we verified the sametendency for lower Chol contents. For higher proportions,and higher temperatures, we observe an increase in theequivalent polarity, which is much more pronounced for egg-SM.

Funding from Fundacao para a Ciencia e a Tecnologia -Portugal, through a Ph.D. grant (SFRH/BD/41607/2007) toD.A., the project PTDC/QUI-BIO/112943/2009, and Centrode Biomedicina Molecular e Estrutural, IBB/CBME, LA,FEDER/POCI 2010, is acknowledged.

P-599Hard X-ray phase contrast imaging of black lipidmembranesAndre Beerlink1, Michael Mell2, Tim Salditt31Deutsches Elektronen-Synchrotron, HASYLAB, Hamburg,Germany, 2University of Madrid, Madrid, Spain, 3Institutefor x-ray physics, University of Gottingen, Germany

We report on hard x-ray phase contrast imaging of black lipidmembranes (BLMs), which are freely suspended over amicro machined aperture in an aqueous solution. This newway of membrane structure analysis allows investigating biomolecular and organic substances in aqueous environmentsby parallel and divergent beam propagation imaging, usingpartially coherent multi-keV x-ray radiation. The width of thethinning film is significantly smaller than the detector pixelsize, but can be resolved from quantitative analysis of theintensity fringes in the Fresnel diffraction regime down to itsnative thickness of about 5nm. To our knowledge this is thefirst time that such small features of a very weak phaseobject have been visualized by direct x-ray imaging tech-niques. We have put forward a simplified but extendablemodel, which enables the theoretical description of imageformation and characterization of membrane thickness andits decrease during the thinning process from a bulk to abimolecular film. On the basis of recent experiments, futureinvestigations will be performed to study the interactions ofmembranes, as they are for example known from synapticfusion, with high spatial resolution.

P-600Interaction between chorogenic acid and erythrocytemembraneDorota Bonarska-Kujawa, Hanna Pruchnik, HalinaKleszczynskaDepartment of Physics and Biophysics, Wroclaw Universityof Environmental and Life Science, C.K. Norwida 25/27,50-375 Wroclaw, Poland

Chlorogenic acid (CGA) is a compound of plant origin, beingpresent in large quantities in coffee beans and fruits andleaves of apple tree. Scientific studies have documented aprotective action of CGA on the human organism due to itsantioxidant, anticancer, fungicidal and bactericidal properties.However, the effect of the acid on the molecular and celllevels have not yet been studied. The aim of the researchpresented was to determine the effect of CGA on the eryth-rocyte membrane and liposomes formed of membrane lipids.The fluorimetric method was used to determine the antioxi-dant activity of CGA, and its location within membrane on thebasis of morphological changes of erythrocytes and fluo-rescence of some probes located in various regions of themembrane. The results indicate that CGA protects the redblood cell perfectly against oxidation. This research has

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shown that the acid incorporates mainly in the exterior part ofthe erythrocyte membrane, inducing creation of echinocytes.This suggests that it interacts predominantly with the outerpart of the lipid layer of erythrocytes and liposomes. It wasalso shown that the CGA decreases the packing order of thehydrophobic part of the membranes, without changing theanisotropic fluorescence of the hydrophobic part.This work was sponsored by the Ministry of Science andEducation, scientific project no. N N312 422340 and N N304173840.

P-601Giant unilamellar vesicle (GUV) as model systemin advanced 3d orientation determinationRichard Borner1, Nicky Ehrlich2, Christian G. Hubner11Institute of Physics, University of Lubeck, Ratzeburger Allee160, 23562 Lubeck, Germany, 2Bionanotechnologyand Nanomedicine Laboratory, University of Copenhagen,Universitetsparken 5, 2100 København, Denmark

One of the unique features of single molecule absorption/emission is their anisotropy due to the well-defined transi-tion dipoles for both processes allowing the determination ofthe molecule’s 3d orientation. Therefore, several techniqueshave been proposed in order to determine the full 3d ori-entation of dipole emitters on a single molecule level. Werecently demonstrated a technique that combines emissiondistribution and polarization detection [1,2,3]. As the methodis an intensity distribution technique and based on singlephoton detection in principle, one can extend the 3d ori-entation determination to fluorescence correlationspectroscopy (FCS) as well as dynamical anisotropy mea-surements. This allows for the determination of thedynamics in 3d orientation of single molecules down to ananosecond timescales. The 3d orientation is particularlyinteresting in non-isotropic environments. A lipid membraneis such a non-isotropic environment of enormous impor-tance in biological systems. We therefore use giantunilamellar vesicle (GUV) labeled with dyes like DiO as amodel system. Due to the defined curvature of such vesi-cles all possible dipole orientations can be achieved. Thisallows us to show the capabilities of our method on differenttimescales and to quantify the error in determination of 3dorientation dynamics in lipid membranes.

[1] J. Hohlbein & C. G. Hubner, APL, 86, 121104 (2005)[2] J. Hohlbein & C. G. Hubner, JCP, 129, 094703 (2008)[3] R. Borner et al., Proc. SPIE, 7905, 79050D (2011)

P-603Biological activity of strawberry leaf extractSylwia Cyboran1, Jan Oszmianski2, Halina Kleszczynska11Department of Physics and Biophysics, Wrocław Universityof Environmental and Life Sciences, Wroclaw, Poland,2Department of Fruit and Vegetable Technology, WrocławUniversity of Environmental and Life Sciences, Wroclaw,Poland

The aim of the studies was to determine the changes thatoccur in a biological membrane and the model lipid mem-brane as a result of interaction with strawberry leaf extract.

Numerous studies conducted all over the world have docu-mented a beneficial effect of polyphenolic compounds on thehuman organism. However, the mechanism of the interactionon the molecular and cell level is not yet known.In the work presented, the effect of strawberry leaf extract onthe erythrocyte and black lipid membranes has been inves-tigated. The applied methods - spectroscopic, fluorimetricand electric - allowed to determine the hemolytic and anti-oxidant activity, and the packing order in the erythrocytemembrane as well as the electric capacity of BLMs.The results obtained indicate that the extract is efficient inprotectingmembrane lipids against oxidation, does not inducehemolysis, increases osmotic resistance and decreasespacking order in the hydrophilic region of the erythrocytemembrane. Moreover, it increases stability and life-time of flatlipid membranes, without altering their specific capacity.The changes induced in erythrocyte membranes and BLMsindicate that strawberry leaf extract binds to the hydrophilicpart of membrane.This work was sponsored by the Ministry of Science andEducation, scientific project no N N304 173840.

P-604What is the difference between a supported and a freelipid bilayer? Insights from Molecular ModelingRoland FallerDept. of Chemical Engineering & Materials Science,UC Davis, Davis, USA

Supported Lipid Bilayers are an abundant research platform forunderstanding the behavior of cell membranes as they allowfor additional mechanical stability and enable characterizationtechniques not reachable otherwise. However, in computersimulations these systems have been studied only rarely up tonow. We present systematic studies on different length scalesof the changes that a support inflicts on a phospholipid bilayerusing molecular modeling. We characterize the density andpressure profiles as well as the density imbalance induced bythe support. It turns out that the changes in pressure profile arestrong enough that protein function should be impacted leadingto a previously neglected mechanism of transmembrane pro-tein malfunction in supported bilayers. We determine thediffusion coefficients and characterize the influence of corru-gation of the support. We also measure the free energy oftransfer of phospholipids between leaflets using the coarse-grained Martini model. It turns out that there is at equilibriumabout a 2-3% higher density in the proximal leaflet. Theseresults are in agreement with data obtained by very large scalemodeling using a water free model where flip-flop can beobserved directly. We are additionally characterizing theintermediate states which determine the barrier height andtherefore the rate of translocation. We also study the influenceof surface roughness and curvature on the behavior. Simula-tions in atomistic detail are performed for selected systems inorder to confirm the findings.

[1] C. Xing, R. Faller: Interactions of Lipid Bilayers withSupports: A coarse-grained Molecular Simulation Study JPhys Chem B 112(23) 7086-7094 (2008)[2] M.I. Hoopes, M. Deserno, M.L. Longo, R. Faller: Coarse-grained modeling of interactions of lipid bilayers with sup-ports J Chem Phys 129(17) 175102 (2008)

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[3] C. Xing, O.H.S. Ollila, I. Vattulainen, R. Faller: AsymmetricNature of Lateral Pressure Profiles in Supported LipidMembranes and Its Implications for Membrane ProteinFunctions Soft Matter 5(17) 3158-3161 (2009)[4] C. Xing, R. Faller: Density imbalances and free energy oflipid transfer in supported lipid bilayers J Chem Phys 131(17)175104 (2009)[5] E.I. Goksu, M.I. Hoopes, B.A. Nellis, C. Xing, R. Faller, C.W.Frank, S.H. Risbud, J.H. Satcher, M.L. Longo: Silica Xerogel/Aerogel-Supported Bilayers: Consequences of Surface Cor-rugation BBA Biomembranes 1798 (4) 719-729 (2010)

P-605Lipid-soluble hydroquinone modifications inducedon membranesSergio S. Funari1, Vivian Rebbin1, Liliana Marzorati2,Claudio Di Vitta21HASYLAB at DESY, Hamburg, Germany, 2Inst. Chemistry,Univ. Sao Paulo, Sao Paulo, Brazil

We synthesized new alkylthiohydroquinones (ATHs); in orderto investigate different aspects of lipid-soluble hydroquinonesinteractions with phospholipids normally found in cell mem-branes. They have the same long hydrophobic alky chainsfound in many lipids forming the cell membranes. In thesecompounds the tails should share the inner of the membrane,while the hydroquinone, as polar head group should remainon the surface. One or more alkylthio chains attached to thearomatic ring, modifies its hydrophobicity and should alter theelectron distribution. Moreover, the two OH groups becomechemically distinguishable, e.g. NMR spectroscopy and alsoshow different pKa values.We investigate the interaction of ATHs with lipid membranes,POPE and POPC and observe the formation of structureswith different morphologies, or curvature, of the lipid mem-brane, depending on temperature and pH. We attributed theirformation to changes in the balance charge/polarity inducedby the ATHs. Mixtures with POPE at pH=4 forms two cubicphases P4332 and Im3m that reach a maximum lattice sizewhile in basic conditions they only expand upon heating fromroom temperature. They coexist with lamellar or hexagonaland have been associated with inhomogeneous distributionof the ATHs molecules over the lipid matrix. The zwitterionicPOPC does not form cubic phases, but instead shows twolamellar structures up to a high temperature.In all mixtures of lipid/ATHs we observed the formation oflamellar and hexagonal phases, similar to the behaviour ofpure hydrated lipid e.g. POPC, while for POPE additionalcubic structures, depending on the environment conditions.

P-606The role of the I-BAR domain in the filopodia formationKinga Futo1, Laura M. Machesky2, Balazs Visegrady11Department of Biophysics, Faculty of Medicine, Universityof Pecs, Szigeti str. 12, Pecs H-7624, Hungary, 2BeatsonInstitute for Cancer Research, Garscube Estate, SwitchbackRoad, Bearsden, Glasgow G61 1BD, UK

The Inverse BAR (I-BAR) domain is part of the superfamily ofthe membrane-deforming protein is Bin-amphiphysin-Rvs(BAR) proteins which induce either positive or negative

membrane curvature both in vitro and in cells. Generation ofmembrane curvature by these membrane deforming proteinsoften works together with actin dynamics. I-BAR shares itsfunction between actin bundling and membrane binding but itis still obscured what molecular mechanisms are responsiblefor these functions. The aim of our project is to investigate thedetailed membrane binding properties of the I-BAR ofIRSp53 and its relations to the actin cytoskeleton. In vitroFRETexperiments and fluorescence quenching studies werecarried out between the I-BAR and liposomes made up fromdifferent lipid constructs. We have found that the I-BAR haspreference to bind to the negatively charged lipids however itcan also bind to the uncharged lipids. The fluorescencequenching studies reflected that the accessibility of theI-BAR surface was higher toward the negatively chargedlipids than for the uncharged ones. TNS fluorescence assayreflected that the I-BAR domain binds to the surface of themicells rather than penetrating into its core.

P-607Phase transitions and spatially-ordered counterionassociation in ionic-lipid bilayers: a statistical modelMario N. Tamashiro1, Renato Germano2, M. Teresa Lamy3,Vera B. Henriques31Instituto de Fısica ‘‘Gleb Wataghin’’, Universidade Estadualde Campinas, Campinas, Brazil, 2Departamento de Fısica,Universidade Federal do Piauı, Teresina, Brazil, 3Instituto deFısica, Universidade de Sao Paulo, Sao Paulo, SP, Brazil

Lipid bilayers present a well-known order-disorder chain tran-sition at ambient temperatures. This transition may becomeanomalous if the lipid head-group presents ionic dissociation atlow ionic strength, as detected by several experimental tech-niques: between the gel and the liquid phases an intermediatephase appears as a shoulder in the specific heat, a deep inturbulence or a maximum in conductivity. We propose a sta-tistical model which allows ionic dissociation of the polar groupon the membrane surface and thus introduces competitionbetween the hydrophobic interaction of hydrocarbonic chains,which favours the gel phase, at low temperatures, and theelectrostatic interaction of charged head-groups, which favoursthe fluid phase, at higher temperatures. Themodel presents anintermediate fluid phase with higher dissociation and chargeordering on the membrane surface, beyond a sharp gel-fluidtransition. The model presents increasing temperature of themain transition by addition of salt, as well as the shrinking of theanomalous region as chain length increases. Model thermo-dynamic behavior is compared to results for PGs,phospholipids with a glycerol head-group.

P-609Modulation of membrane fluidity by 2-hydroxylated fattyacid derivativesM. Ibarguren, D.J. Lopez and P.V. Escriba.Laboratory of Molecular and Cell Biomedicine, Universityof the Balearic Islands – Lipopharma Therapeutics, S.L.,Palma de Mallorca, Spain

Keywords: Membrane fluidity, Minerval, 2-hydroxylated fattyacids.

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Cellular functions are usually associated with the activity ofproteins and nucleic acids. Recent studies have shownthat lipids may alter the biophysical properties of lipidmembranes and modulate the localization and activity ofmembrane proteins, thus regulation the cellular processesin which they are involved. The present work describes theeffect of Minerval (2-hydroxyoleic acid), a potent antitu-moral drug, and other different 2-hydroxylated fatty acidderivatives on the fluidity of lipid membranes. The self-quenching method of NBD-phosphatidylethanolamineshowed that all the studied 2-hydroxylated fatty acidsinduced fluidification of domain-containing model mem-branes. However, phase separation was observed uponaddition of these synthetic molecules on vesicles com-posed of homogeneously distributed lipids. Results offluorescence confocal microscopy of giant unilamellarvesicles correlates with the disordering effects of the2-hydroxylated fatty acids on the membrane structure. Theability of changing the membrane biophysical propertiesmakes the 2-hydroxylated fatty acid derivatives candidatesto be modulators of the membrane associated proteinsactivities and the subsequent intracellular cell signalingprocesses.

P-610Transmembrane signalling through a heme/hemophorereceptor, HasRGisele Cardoso de Amorim1, Idir Malki1, Ada Prochnicka-Chalufour1, Julien Lefevre1, Catherine Simenel1, PhilippeDelepelaire2, Muriel Delepierre1 and Nadia Izadi-Pruneyre11Unite de RMN des Biomolecules, Institut Pasteur, CNRSURA 2185, Paris, France, 2Unite des membranesbacteriennes, Institut Pasteur, CNRS URA 2172, Paris,France

The outer membrane (OM) of Gram-negative bacteria actsas a selective permeation barrier. The nutrients that cannotpass through porins are internalised via OM specific trans-porters by an active process depending on the energy givenby an inner membrane complex. This is the case of ironsources, heme, vitamin B12, etc. We study on such anactive transport system through an OM transporter, HasR.HasR is a central component of a heme acquisition system‘‘Has’’ developed by several Gram-negative bacteria tosatisfy their need of iron. It functions in synergy with asecreted heme carrier protein, named hemophore or HasA.HasA extracts heme from host hemoproteins as hemoglobinand then shuttles it to HasR, wherein it is internalized. Theenergy for HasR functioning is brought by the inner mem-brane HasB complex. The HasR OM receptor binds hemeand HasA on its extracellular side and HasB on its peri-plasmic side. Using structural and biophysical approaches,we show that the binding of the substrate on the extracel-lular face of HasR modulates the interaction with HasB onthe periplasmic face. The transmitted signal depends on thenature of the substrate1,2,3,4.

1 Wolff, N et al., 2008 J. Mol. Biol.2 Krieg S et al 2009 PNAS,3 in prep.4 Lefevre J et al., 2008. J Mol Biol.

P-612Target-selective membrane fusion with endosomal-pH-responsivenessAyumi Kashiwada, Mana Tsuboi, and Kiyomi MatsudaDepartment of Applied Molecular Chemistry, Collegeof Industrial Technology, Nihon University, Narashino, Chiba275-8575, Japan

The well-programmed membrane fusion systems, operatingin a weakly acidic environment, have attracted attention inthe fields of biochemistry, biophysics, and pharmacybecause these acidic conditions are generally observed inendosomal membranes or tumor tissues. We have reporteda selective liposomal membrane fusion system toward asugar-like cyclic cis-diol structure on the target liposome.This system consists of a lapidated phenyl boronic acidderivative as membrane -bound fusogen and phosphatidyl-inositol as target. Here we report the preparation of a boronicacid / pH-responsive polypeptide conjugate as a novelmembrane fusion device and the development of a targetselective liposomal membrane fusion system with endo-somal pH-responsiveness.During the course of lipid-mixing, inner-leaflet lipid-mixing,and contents-mixing assays to characterize membranefusion behavior, we clearly observed a liposomal membranefusion phenomenon when the pH of the experimental systemwas changed from 7.4 (physiological) to 5.0 (endosomal).Our highly effective methods, which include a target selectiveliposomal membrane fusion, can be useful in the area ofnanomedicine such as hybridoma technology and liposome-based drug or gene delivery.

P-613Complete and reversible chemical denaturationof an a-helical membrane proteinJana Broecker, Sebastian Fiedler, Sandro KellerMolecular Biophysics, University of Kaiserslautern, Erwin-Schrodinger-Str. 13, 67663 Kaiserslautern, Germany

The question of how an unordered polypeptide chain assumesits native, biologically active conformation is one of thegreatest challenges in molecular biophysics and cell biology.This is particularly true for membrane proteins. Chemicaldenaturants such as urea have been used successfully for invitro un- and refolding studies of soluble proteins and b-barrelmembrane proteins. In stark contrast with these two proteinclasses, in vitro unfolding of a-helical membrane proteins byurea is often irreversible, and alternative denaturation assaysusing the harsh detergent sodium dodecyl sulphate sufferfrom a lack of a common reference state.Here we present the complete and reversible chemicaldenaturation of the bacterial a-helical membrane proteinMistic out of different micellar environments by urea. Weapplied multidimensional spectroscopy and techniques typi-cally used in b-barrel membrane protein unfolding. Misticunfolds reversibly following a two-state equilibrium thatexhibits the same unfolded reference state. This allows for adirect comparison of the folding energetics in different mem-brane-mimetic systems and contributes to our understandingof how a-helical membrane proteins fold as compared withboth b-barrel membrane proteins and water-soluble proteins.

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P-614Simple cell, complex envelope: modellingthe heterogeneous membrane of E. coliSyma Khalid, Thomas J Piggot and Daniel A Holdbrook.School of Chemistry, University of Southampton,Southampton SO17 1BJ, UK

Gram-negative bacteria such as E. coli are typically regardedas ‘‘simple’’ model organisms, yet their cell envelopes aresurprisingly complex. Recent experimental and theoreticalstudies have revealed that contrary to the traditional view ofthe cell membrane as a passive bystander in membraneprotein function, it plays a key role in protein folding, assembly,and function. To study the influence of the bacterial membraneon the dynamics of embedded proteins, we have performedatomistic molecular dynamics simulations of members of theTonB-dependent transporter family of proteins from E. coli. Inparticular, our aim has been to capture the details of lipidcomposition; we have modeled the outer membrane as anasymmetric bilayer containing an asymmetric distribution oflipopolysaccharide (LPS) and heterogeneous phospholipidmixtures. Our simulations enable us to explore the dynamicsof the extracellular loops with a level of detail that is not pos-sible using more traditional, simpler model membranes. Byextending the simulation timescales out to hundreds ofnanoseconds we are gaining new insights into the influence of‘‘realistic’’ membrane environments on the structure anddynamics of membrane proteins.

P-615Interaction between buckwheat extract and erythrocytemembraneAleksandra Włoch1, Jan Oszmianski2, Halina Kleszczynska11Department of Physics and Biophysics, Wrocław Universityof Environmental and Life Sciences, Norwida 25, 50-375Wrocław, Poland, 2Department of Fruit, Vegetable and GrainTechnology Technology, Wrocław Universityof Environmental and Life Sciences, Norwida 25, 50-375Wrocław, Poland

In recent years, buckwheat has been of great interest in theworld markets of healthy food, due to its high energy value,the content of unsaturated fatty acids, mineral constituentsand vitamins. Its seeds contain flavonoids which are natural,efficient antioxidants.The aim of the present studies was to investigate the effectof buckwheat extracts on the properties of biologicalmembrane, which is the main site of the interaction betweenthe substances buckwheat contains and the organism. Theresearch was conducted on red blood cells and their iso-lated membranes, using the spectrophotometric,microscopic and fluorimetric methods. From the resultsobtained it follows that the compounds contained in buck-wheat extracts increase the osmotic resistance oferythrocytes, making them less sensitive to the medium’sosmotic pressure, induce changes in cell shape, producingincreased number of echinocytes, and decrease the pack-ing order of the polar heads of membrane lipids. It can thusbe inferred that the compounds contained in the extractspenetrate the hydrophobic region of the erythrocyte mem-brane and alter its properties.

This work was sponsored by the Ministry of Science andEducation, scientific project no N N304 173840

P-616Re-alignment of the antimicrobial peptide gramicidin Sin lipid membranes: probing different sitesof the moleculeVladimir Kubyshkin1, Sergii Afonin1, Pavel K. Mykhailiuk2,Marina Berditsch1, Igor V. Komarov2, Anne S. Ulrich11Karlsruhe Institute of Technology (KIT), Karlsruhe,Germany, 2 National Taras Shevchenko University, Kyiv,Ukraine

Gramicidin S (GS) is a cyclic decapeptide (cyclo[Val-Orn-Leu-DPhe-Pro]).Due to its small size, symmetric structure,amphipathicity, proteolytic stability and testable mode ofactivity, the GS backbone is a convenient model system toexamine the structure-activity relationship of individual aminoacid substitutions.We have previously reported the structure analysis of two GSanalogues in which either the Val or Leu residues on thehydrophobic face of the molecule were substituted by thealiphatic 19F-labeled amino acid 4F-Phg. Using 19F-ssNMRin oriented lipid bilayers, we observed a re-alignment of thepeptide that is compatible with the formation of a putativepore [Top.Curr.Chem. 273:139]. Here, we present novelanalogs of GS with different 19F-prolines in the b-turn region,and with CF3-Bpg in place of Leu. Based on these 19F-ssNMR results and supported by CD, DSC and activity tests,we could demonstrate that all analogues are structurallyintact and antimicrobially active. We observe, however, dif-ferences in the re-alignment propensity when comparingthese GS analogues in DLPC and DMPC bilayers. Thesedifferences can be rationalized in terms of molecular shapebeing changed upon incorporation of unnatural amino acidsat various sites of the molecule.

P-618Viral membrane fusion inhibited by beta-propiolactonetreatmentPierre Bonnafous1, Marie Claire Nicolaı2, Michel Chevalier2,Jean-Christophe Taveau 1, Julie Morel2, Olivier Adam2,Frederic Ronzon2, Olivier Lambert11CBMN UMR-CNRS 5248, Universite Bordeaux, BordeauxFrance, 2Sanofi Pasteur Research, Lyon, France

The beta-propiolactone (BPL) is an inactivating reagentcommonly used to produce viral vaccine preparations (wholevirions or split-virions). Although BPL has been reported toinactivate nucleic acids, its mechanism of action on proteinsand the outcome on viral infection remains ill-defined. In thiswork, H3N2/Victoria/210/2009 influenza virus strain has beensubmitted to various BPL inactivation conditions (from 2lM to1 mM). Cell infection ability was progressively reduced andentirely abolished at 1 mM BPL. To clarify the BPL effect, wefocused on membrane fusion infection steps using kineticfluorescence molecule leakage from liposome and lipid FRETassays combined with cryo electron microscopy. Membranefusion measured at pH 5 on GM3 liposomes was reduced in adose-dependent manner. Interestingly the fusion activity was

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partially restored using the proton-ionophore monensin asconfirmed by cryoEM images. In addition, a decrease ofmolecule leakage irrespective to BPL concentration wasmeasured suggesting that the hemagglutinin affinity for GM3was slightly modified even at low BPL concentration. Alto-gether these results strongly suggest that BPL treatmentimpairs M2 protein activity likely by preventing proton trans-port and bring new light on the mechanism of action of BPL.

P-619Light scattering detects large vesicle deformationsalong the anomalous gel-fluid transition of DMPGThais A. Enoki, Daniela A. Nomura, Jozismar R. Alves, VeraB. Henriques, M. Teresa LamyInstituto de Fısica, Universidade de Sao Paulo, Sao Paulo,SP, Brazil

The saturated anionic lipid dimyristoyl phosphatidylglycerol(DMPG), at low ionic strength, exhibits a very peculiarthermo-structural behavior. Along a wide gel-fluid transitionregion, DMPG dispersions display several anomalous char-acteristics, like low turbidity, high electrical conductivity andviscosity. Here, static and dynamic light scattering (SLS andDLS) were used to characterize DMPG vesicles. SLS andDLS yielded similar dimensions for vesicles in the geland fluid phases. However, over the DMPG transition region,SLS indicated a threefold increase in vesicle radius of gyration,whereas the hydrodynamic radius, as obtained from DLS,increasedbyonly 30%.Over theDMPG transition region, largepositive values of the second virial coefficient clearly indicatedan increase in inter-vesicle repulsion, probably due to thesuggested increase in vesicle ionization. Despite the anoma-lous increase in the radius of gyration, DMPG lipid vesiclesmaintain isotropy, since no light depolarization was detected.Adding to results present in the literature, we propose aninterpretation in terms of large fluctuations of vesicle form andbilayer density along the DMPG transition region, associatedto head-group dissociation and formation of bilayer pores.Support: USP, FAPESP and CNPq.

P-620Synergy between membrane-curvature and liquidordered phase-state effectively sorts amphiphilicprotein-anchoring motifsJ. Larsen, M.B. Jensen, N. Hatzakis, V.K. Bhatiaand D. StamouBionanotechnology Laboratory, Department of Neuroscienceand Pharmacology, Nano-Science Center, LundbeckFoundation Center Biomembranes in Nanomedicin,University of Copenhagen, Universitetsparken 5, 2100 KBH Ø,E-mail: jannikl@sund.ku.dk

Cellular membranes have a heterogeneous lipid composi-tion, potentially forming nano-domains or membrane rafts,believed to be platforms of altered fluidity involved in proteinsorting and trafficking1. An alternative mechanism, potentiallyleading to protein sorting, has recently been proposed,suggesting that the curvature of membranes can alsoactively regulate protein localization2. Recently we showedthat a variety of protein anchoring motifs are membrane-

curvature sensors and thus up concentrate in regions of highmembrane curvature3. Furthermore the curvature sensingability of the anchoring motifs persisted independently of theirstructural characteristics. This leads us to speculate thatcurvature sensing might be an inherent property of anycurved membrane and as a consequence, the lipid compo-sition of the bilayer could potentially regulate this recruitmentby membrane curvature. Thus there might be an intimate, yetunrecognized, link between the way raft-like membranedomains and membrane-curvature promotes the localizationof membrane-anchored proteins.We examined how changing the lipid composition of lipo-somes influenced the recruitment by membrane curvature ofa model amphiphilic protein-anchoring motif. Employing oursingle liposome curvature assay, we tested lipid mixtureswith different ratios of DOPC, sphingomyelin and cholesterol,giving rise to liposome populations of different phase-states.We found an amplified recruitment by membrane curvaturefor all raft-like lo phase-state mixtures when compared to theld phase-state counterparts. Based on these findings wesuggest a synergetic effect when combining a raft-like lipidphase-state and high membrane curvature, resulting in ahighly potent mechanism for selective localization of mem-brane-anchored proteins.[1] D. Lingwood and K. Simons, ‘‘Lipid Rafts As a Membrane-Organizing Principle,’’ Science, vol. 327, pp. 46-50, January1, 2010 2010.[2] V. K. Bhatia, et al., ‘‘Amphipathic motifs in BAR domainsare essential for membrane curvature sensing,’’ EmboJournal, vol. 28, pp. 3303-3314, 2009.[3] N. S. Hatzakis, et al., ‘‘How curved membranes recruitamphipathic helices and protein anchoring motifs,’’ NatureChemical Biology, vol. 5, pp.835-841, 2009.

P-621Minerval and other 2-hydroxylated fatty acid derivativesinduce the formation of non-lamellar lipid structuresD.J. Lopez, M. Ibarguren and P.V. EscribaLaboratory of Molecular and Cell Biomedicine, Universityof the Balearic Islands – Lipopharma Therapeutics, S.L.,Palma de Mallorca, Spain

Keywords: Non-lamellar lipid structure, phase transition,Minerval, 2-hydroxylated fatty acid.Minerval (2-hydroxyoleic acid), a potent antitumoral drug, isknown tomodulate the lipid membrane structure by decreasingthe lamellar-to-non-lamellar phase transition temperature (TH).A series of 2-hydroxy fatty acid derivatives, varying in acylchain length and degree of unsaturation, have been analyzedin terms of their ability to stabilize the inverted hexagonal (HII)phase in palmitoyl-oleoyl-phosphatidylethanolamine mem-branes. Differential Scanning Calorimetry and 31P-NuclearMagnetic Resonance showed that mono- and polyunsatu-rated, but not saturated, 2-hydroxylated fatty acid moleculeswere able to decrease the TH. Lipid vesicles mimicking the lipidcomposition of a cell membrane were solubilized at 4�C in thepresence of Triton X-100. The results demonstrated that theamount of detergent-resistant membranes, which are related toliquid ordered (Lo) structures, decreased in the presence of2-hydroxylated fatty acids. The so-called Lipid MembraneTherapy focuses on the reversion of cell disfunction throughthe modulation of the membrane structure, thus altering the

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activity of membrane-associated proteins. The ability to modifythe biophysical properties of a lipid membrane makes thestudied 2-hydroxylated fatty acid molecules be prospectivecandidates for the use in the Lipid Membrane Therapy.

P-623Steady-state and time-resolved fluorescence studyof pyrenyl probes’ excimer formation and quenchingMiguel Manuel1, Jorge Martins1,21IBB-CBME, University of Algarve, Faro, Portugal, 2DCBB-FCT, University of Algarve, Faro, Portugal

Keywords: Pyrene excimer, lateral diffusion, 2D steady-statekinetics, lipid bilayers

Excimer formation of phospholipid-labeled pyrenyl probes b-py-C10-HPC and b-py-C6-HPC in POPC MLV vesicles wasexamined by combined steady-state and time-resolvedfluorescence. Our findings are in good agreement with thetheoretical predictions by a kinetic formalism specific forfluorescence quenching processes occurring in two-dimen-sional media [Razi Naqvi et al. (2000) J. Phys. Chem. B 104,12035]. However, a significant downward divergence occursabove 4 mol % of probe content, which might indicate devi-ations to ideal mixing in fluid phase. Results for b-py-C10-HPC, in mixtures of POPC with 10 and 20 mol % POPS wereindistinguishable from those obtained with pure POPC vesi-cles; however excimer formation in pure POPS bilayersappears to be appreciably higher.We also compared the excimer formation findings withquenching of the same probes by low concentrations ofDOXYL quencher groups labeled acyl phospholipid chain atthe same depth of the pyrenyl group. The results are alsoscrutinized by the same two-dimensional kinetic formalismand good correlation was also found.Funding from Fundacao para a Ciencia e a Tecnologia -Portugal, through a Ph.D. grant (SFRH/BD/41607/2007) toM.M. and Centro de Biomedicina Molecular e Estrutural, IBB/CBME, LA, FEDER/POCI 2010, is acknowledged.

O-624Biophysics of Membrane LipidomicsDerek MarshMax-Planck-Institut fur biophysikalische Chemie,37070 Gottingen, Germany

The amassing of comprehensive data on the lipid composi-tion of biological membranes by lipidomics initiativesprovides a potent challenge to the membrane biophysicistinterested in lipid structure. This resolves itself essentiallyinto two aspects. The first systematises the dependence ofmembrane biophysical parameters on lipid molecular struc-ture. Lipid volumes, membrane dimensions, chain-meltingtemperatures and enthalpies, nonlamellar phase formationand structure, critical micelle concentrations and thermody-namics of membrane formation, membrane-membraneinteractions and lipid transfer are amongst the properties ofcentral biophysical interest. The relevant structural parame-ters are lipid chain length, degree of unsaturation, chain

branching and headgroup configuration. The second, morecomplex and less well developed, aspect concerns the lipid-lipid interactions that determine the membrane properties oflipid mixtures. In part, these can be obtained from binaryphase diagrams, and the more limited number of ternaryphase diagrams - notably with cholesterol - that are available.Extrapolation to higher order mixtures lies in the future. I shallattempt to summarise some of the progress in these direc-tions. The immediate aim is a second edition of myHandbook of Lipid Bilayers, which, in addition to a vastlyexpanded database, will include interpretative features andwill be available in the early part of next year.

References:

Marsh,D., 2009. Cholesterol-induced fluid membranedomains, Biochim. Biophys. Acta 1788, 2114-2123.Marsh,D., 2009. Structural and thermodynamic determinantsof chain-melting transition temperatures, Biochim. Biophys.Acta 1798, 40-51.Marsh,D., 2010. Liquid-ordered phases induced by choles-terol, Biochim. Biophys. Acta 1798, 688-699.Marsh, D., 2010. Molecular volumes of phospholipids andglycolipids in membranes, Chem. Phys. Lipids 163, 667-677.Marsh, D., 2011. Pivotal surfaces in inverse hexagonal andcubic phases of phospholipids and glycolipids. Chem. Phys.Lipids 164, 177-183.

P-625Combined steady-state and time-resolved excimerformation analysis in POPC/cholesterol bilayersMonica Faria2, Miguel Manuel1, Jorge Martins1,21IBB - CBME, Faro, Portugal, 2DCBB - FCT, Universidadedo Algarve, Faro, Portugal

Lateral diffusion dynamics in phosphatidylcholine/cholesterolbilayers has been mostly accessed by means of EPR, NMRand FCS spectroscopic techniques. Reliable stady-stateflorescence quenching analysis of diffusion-controlled pro-cesses has been ampered by the lack of a self-consistentkinetic formalism for the two-dimensional (2D) counterpart ofthe classical Stern-Volmer analysis for three-dimensional(3D) solvents.We studied the excimer formation of phospholipid-labeledpyrenyl probes (proportion of 4 mol %) in mixed POPC/Cho-lestrol MLV liposomes by combined steady-state and time-resolved fluorescence The findings are in very good agree-ment with the theoretical predictions of the kinetic formalismspecific for fluorescence quenching processes occurring in2D media [Razi Naqvi et al. (2000) J. Phys. Chem. B 104,12035]. We analyzed POPC/Cholestrol mixtures putatively inthe liquid-ordered (35, 40 and 45 mol % Cholesterol) and inliquid-disordered (5 mol %) phases, and compare the out-comes with the results from pure POPC bilayers.Funding from Fundacao para a Ciencia e a Tecnologia -Portugal, through a Ph.D. grant (SFRH/BD/40671/2007) toM.M., the project PTDC/QUI-BIO/112943/2009, and Centrode Biomedicina Molecular e Estrutural, IBB/CBME, LA,FEDER/POCI 2010, is acknowledged.

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P-626The effects of 2N- and 3O-methylation of sphingosineon the membrane properties of ceramide in mixedbilayersTerhi Maula1, Mayuko Kurita2, Shou Yamaguchi2, TetsuyaYamamoto2, Shigeo Katsumura2, J. Peter Slotte11Biochemistry, Department of Biosciences, Abo AkademiUniversity, Turku, Finland, 2School of Science & Technology,Kwansei Gakuin University, Sanda City, Japan

The small hydrophobic head group, the closely packed acylchains, and the capability of interfacial hydrogen bondinghave been suggested to govern the characteristic membranebehavior of long chain saturated ceramides: the self-segre-gation, and the formation of hexagonal phases and highlyordered gel phases. While it has been shown that structuralalterations of the ceramide acyl chains induce positiondependent effects on their behavior, we wanted to study theeffect of interfacial properties, including hydrogen bonding, onceramide membrane properties. The H-bond donor functionsof 2NH and 3OH in the sphingosine backbone of palmitoyl-ceramide were disrupted either separately or simultaneouslyby replacing the hydrogen with a methyl-group. When thelateral phase behavior of mixed bilayers containing choles-terol/sphingomyelin-rich domains was studied in thepresence of the ceramide analogs, the 3O-methylated cera-mide appeared to form a thermally stable, sterol-excluding gelphase with sphingomyelin, whereas the 2O-methylated cer-amide failed in both thermal stabilization and steroldisplacement. The doubly methylated analog was the poorestceramide mimic. Together with the possible steric effectsinduced by the methylations, the lack of 2NH H-bond donorfunction impaired ceramide membrane behavior to a greaterdegree than the lack of 3OH H-bond donor function.

P-628Cytotoxicity and interactionsof x-(alkyldimethylamonium) alkylaldonamide bromideswith membranesE. Wozniak1, B. Ro _zycka-Roszak1, P. Misiak1, A. Czarny2,E. Zaczynska2, R. Skrzela3, K.A. Wilk31Department Physics and Biophysics, Wrocław Universityof Environmental and Life Sciences, Wrocław, Poland,2Institute of Immunology and Experimental Therapy, PolishAcademy of Sciences, Wrocław, Poland, 3Departmentof Chemistry, Wrocław University of Technology, Wrocław,Poland

Sugar-based surfactants are made from renewable resourcesusing the ‘‘green chemistry’’ methods, are easily biodegrad-able and used in washing agents, cosmetics, and drugcarriers. Besides, there are attempts to use them as nonviralvectors in gene therapy. We studied the influence of new x-(alkyldimethylamonium)alkylaldonamide bromides (CnGAB)with different chain lengths (n = 10, 12, 14, 16) on the ther-motropic phase behavior of DPPC, and DPPC/chol bilayersby means of differential scanning calorimetry. The surfactantswere added either to the water phase or directly to the lipidphase (a mixed film was formed). We analyzed the changesin the temperatures, enthalpies and shapes of the main phasetransitions as a function of concentration. Molecular modeling

methods were also used. Cytotoxicicity of the CnGABs wasdetermined in the cell line L929 and A549. For cytotoxicitytest, the cells were seeded in 96-well plates 1 ml of 2�106cells/ml in the culture medium Eagle’a or Dulbecco with 2%calf serum, penicillin and streptomycin was deposited intoeach plates. The cells were treated with various doses ofsurfactants and incubated. The minimal concentration whichwas toxic to approximately 50% of cells was taken asTCCD50. This work was supported by grant N N305 361739.

P-629Direct visualization of large and protein-free hemifusiondiaphragmsJoerg Nikolaus1, Jason M. Warner2, Ben O’Shaughnessy2,Andreas Herrmann11Humboldt University Berlin, Berlin, Germany, 2Departmentof Chemical Engineering, Columbia University, New York,New York, USA

Keywords: Membrane fusion, hemifusion, giant unilamellarvesicle, transmembrane domain

Membrane fusion is ubiquitous in life requiring remodeling oftwo phospholipid bilayers. As supported by many experimen-tal results and theoretical analyses, merging of membranesseems to proceed via similar sequential intermediates. Con-tacting membranes form a stalk between the proximal leafletswhich expand radially into a hemifusion diaphragm (HD) andsubsequently open to a fusion pore. Direct experimental veri-fication of the HD is difficult due to its transient nature. Usingconfocal fluorescence microscopy we have investigated thefusion of giant unilamellar vesicles (GUVs) containing fluo-rescent membrane protein anchors and fluorescent lipidanalogues in the presence of divalent cations. Time resolvedimaging revealed that fusionwas preceded by displacement ofpeptides and lipid analogues from the GUV-GUV contactregion being of several lm in size. A detailed analysis showedthat this structure is consistent with the formation of an HD. Aquantitativemodel of thehemifusionequilibriumandkinetics ofthe growing HD was developed. Bilayer tension could beshown to drive HD expansion and interleaflet tension wasfound to act as a counterforce, because the outer leaflets arecompressed uponHD growth. Themodel and its predictions fitnicely with observations above.

P-630Concentration effects of trehalose in the equivalentpolarity of fluid POPC bilayersC. Nobre2, D. Arrais1, J. Martins1,21IBB - CBME, Faro, Portugal, 2DCBB - FCT, Universidadedo Algarve, Faro, Portugal

Trehalose is an important disaccharide, formed by two unitsof glucose linked by a a-1,1 glicosidic bond. It is capable ofreplacing water molecules in the hydration shell of thephospholipid headgroups, in cases of extreme dehydration,by establishing hydrogen bonds with their –CO and –POgroups, preserving this way the membrane structure. Thepolarity gradient is a significant feature of lipid bilayers and isinfluenced by the amounts of water within this medium. It is

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therefore important to understand the effects of differentconcentrations of trehalose in simple model membranes.Using the pyrene empirical polarity scale, we monitorizedchanges in the polarity values when varying trehalose con-centration in the bounding aqueous phase. For lowerconcentrations (until 0.25 M), we observed a decrease inpolarity, comparing with POPC bilayers in pure water. Forhigher trehalose concentrations (above 0.5 M), the polarityvalues are indistinguishable from those POPC in water.Using the freeze and thaw technique we obtained the sameresults, except for the lower trehalose concentrations.Funding from Fundacao para a Ciencia e a Tecnologia -Portugal, through the project PTDC/QUI/64565/2006, andCentro de Biomedicina Molecular e Estrutural, IBB/CBME,LA, FEDER/POCI 2010, is acknowledged.

O-631Stereo-specific effect of the analgesic drug ketamineon lipid membranesGeorg Pabst1, Eva Sevcsik1, Michael Rappolt1,Thomas Stockner21Institute of Biophysics and Nanosystems Research,Austrian Academy of Sciences, Graz, Austria, 2Instituteof Pharmacology, Medical University of Vienna, Vienna, Austria

General anestheticsare indispensible toolsofdaily surgery.Yet,their molecular mode of action remains elusive. While oneschool favors specific (direct) interactions with proteins of thecentral nervous system, another school adheres to a nonspe-cificmodulation of biophysicalmembraneproperties.Oneof thestrongest arguments against lipid theories is the absence ofstereo-specific effects in model membranes, as opposed totheir detection by electrophysiological measurements on ion-channels. We have combined X-ray scattering and moleculardynamics simulations on palmitoyl-oleoyl-phosphatidylcholinebilayers with fluorescence microscopy on live cells to study theeffects of the stereoisomers of ketamine on membrane prop-erties. We find significant effects of both enantiomers on thedistribution of lateral pressures at clinically relevant concentra-tions, being more pronounced for S-(?)-ketamine. We furthercalculated the effect of the lateral pressure profile changes onthe opening probability of an ion-channel using crystallographicinformation. The observed channel inhibition comparesremarkably well with clinically observed effects of the enantio-mers. We thus provide first evidence for a stereo-specific, butindirect effect of general anesthetics on ion-channels.

O-632Dependence of gramicidin A channel lifetimeon membrane structure obtained from x-ray scatteringmeasurementsHoria I. PetracheDepartment of Physics, Indiana University Purdue UniversityIndianapolis, IN 46202, USA

The activity of ion channels, in particular the lifetime of theirconducting (open) state depends on the physical propertiesof lipid bilayers [1,2] which in turn depend on lipid headgroup

and acyl chain composition. In order to investigate thisdependence, we have performed measurements of gramici-din A (gA) channel lifetimes in three different lipid series. Ineach series, the lipid headgroups were phosphatidylcholine(PC), phosphatidylethanolamine (PE), and phosphatidylser-ine (PS), while the acyl chains consisted of symmetricmonounsaturated di(18:1), mixed (16:0)(18:1), and methyl-ated di(16:0-4ME). In order to minimize the effect ofheadgroup electrostatics, measurements where performed in1M KCl salts. We show how gA lifetimes depend on head-group and acyl chain composition and on structuralparameters determined by x-ray scattering. For the lipidsconsidered, gA lifetimes cover a range from 0.7 seconds inthe DOPE lipid to 18 seconds in DPhPS. In this range, wefind a Gaussian dependence of gA lifetime on bilayer thick-ness, consistent with hydrophobic matching models. Wediscuss different aspects of channel-lipid interactions and towhat extent measurements of gA lifetime in binary mixturesare consistent with measurements in pure lipid systems. [1]Rostovtseva et al., Biophys. J. 94, L23-25, 2008. [2] Lund-baek et al., PNAS, 107, 15427-15430, 2010.

P-633Interaction between chlorogenic acid and modelmembranesHanna Pruchnik, Dorota Bonarska-Kujawa, Sylwia Cyboran,Halina KleszczynskaDepartment of Physics and Biophysics, Universityof Environmental and Life Science, Norwida 25/27, 50-375Wrocław, Poland

The aim of the studies was to determine the effect of chlor-ogenic acid (CGA), which is the main constituent of plantextracts, on properties of the model membranes. Its effectwas studied on temperature of the main phase transition ofvarious lipids with and without presence of cholesterol, usingthe differential scanning calorimetry (DSC) method and thefluorimetric method. In particular, the degree of packing orderof the hydrophilic phase of liposomes was determined usingthe laurdan and prodan probes, and fluorescence anisotropyof the hydrophobic phase with the probes DPH and TMA-DPH. It had also been studied the effect of chlorogenic acidon the structure and capacity of black lipid membranes(BLMs), formed of egg lecithin and lipids extracted fromerythrocytes.The results obtained indicate that CGA lowers the mainphase transition temperature slightly, without changing thefluorescence anisotropy in the hydrophobic part of the bilyer,and causes a decease in the packing order of the hydrophilicphase. By monitoring the capacity during BLM formation wehave found that the presence of chlorogenic acid acceleratesthe process of lipid self-organization into a bilayer, andincreases stability and life of the BLMs. However, the was noeffect of CGA on specific capacity of the membranes, andthus on thickness of the liposome membrane hydrophobiclayer.This work was sponsored by the Ministry of Science andEducation, scientific project no. N N312 422340 and N N304173840.

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P-634Networking of sterols in lipid bilayersC. Millan-Pacheco1, H. Saint-Martin2, A.H. De Vries3,I. Ortega-Blake21Universidad Autonoma Metropolitana, Mexico City, Mexico,2Universidad Nacional Autonoma de Mexico, Cuernavaca,Morelos, Mexico, 3Rijksuniversiteit Groningen, Groningen,The Netherlands

Many examples have recently been found where biologicalprocesses in the lipid bilayer are affected by the changes inthe physicochemical properties of the membrane, e.g. thelocal curvature, the membrane tension and certainly themembrane structure. It has been shown that the activity ofpolyene antibiotics is strongly correlated to the phase dia-gram in a membrane composed of a mixture of POPC andergosterol or cholesterol (J Membrane Biol, 237:41-49,(2010)). It is known that polyene action is quite sensitive tothe type of sterol in the membrane, which enables its medicaluse, mainly as antifungals. It has been proposed that thisselectivity of the drug to fungi is related to structure modu-lation by the sterols (see for example, Biophys. J., 85, 2323,(2003)) and therefore the correlation found could be due tostructural differences between POPC/ergosterol and POPC/cholesterol along the corresponding phase diagrams. Toinvestigate this, Molecular Dynamics simulations of theabove mixtures along their phase diagrams were performed.It was found that there are indeed marked differences instructure along the phase diagrams, but for the sterol-steroldistribution function. An analysis of the behavior of thisobservable and the implications on polyene action isdiscussed.

P-635Acyl transfer from lipids without enzyme catalysis: a newparadigm for membrane protein ageing?John Sanderson, Catherine Pridmore, Jackie Mosely,Paul YeoDurham University, Department of Chemistry, Durham, UK

Membrane proteins are recycled in cellulo with half-livesranging from minutes to days. In other systems, such asenveloped viruses, proteins may equally remain membrane-bound for periods of days. It is therefore of interest toexamine the behaviour of proteins in model membranes overextended periods in order to determine the long-term stabilityof the mixed systems, both in kinetic terms (attainment ofequilibrium states) and chemical terms (reactivity). Thereactivity of proteins towards membranes has been exam-ined using the peptide melittin as a model for membraneproteins. Acyl transfer from phospholipids to the peptide wasfound to occur over a period of several days, in the absenceof any enzyme catalysis. Transfer was detectable after 2days and reached 50% conversion in 8 days. Using tandemmass spectrometry approaches, the sites of melittin modifi-cation were localised. These sites included the side chain oflysine, opening the possibility that this residue may bemodified in any membrane protein where this residue has anappropriate disposition. These observations challenge pre-conceptions concerning the membrane as an inert medium

and highlight potential new mechanisms for membrane pro-tein ageing.

P-636Interaction of poly(L-arginine) with negatively chargedbilayers studied by FT-IR spectroscopyChristian Schwieger, Alfred BlumeMartin-Luther-University, Halle-Wittenberg, Instituteof Chemistry, van-Dankelman-Platz 3, 06108 Halle / Saale,GermanyE-mail: christian.schwieger@chemie.uni-halle.de

Oligoarginine residues attached to macromolecules are knownto facilitate the transport through lipid membranes. Since themechanism of this transport is still unclear, the effect is oftencalled ‘‘arginine magic’’. We studied the interaction of poly(L-arginine) (PLA) of different molecular weight with negativelycharged lipid bilayers. We have shown by calorimetric andmonolayer techniques that the interaction is due to a combina-tion of electrostatic and hydrophobic forces. Nowwepresent anFT-IR spectroscopic study to reveal the effect of PLA binding onmembrane organisation and peptide conformation. We willshow that PLA binding reduces the lipid miscibility of negativelycharged (PG or PA) and zwitterionic (PC) lipids within thebilayer. From theshift of theC=Ostretchingvibrationwededucethat arginine side chains penetrate into the hydrophobic/hydrophilic interface and replace hydration water molecules.The binding reduces the rotational freedom of the lipid mole-cules, as could be shown by an analysis of the CH2-strechingvibrations. PLA binds in a b-sheet conformation to PGor PA gelphase membranes whereas its structure in bulk is random coil.The shift of the guanidyl vibration frequencies shows that alsohydrogen bonds contribute to the PLA –lipid interactions.

P-637Neutron scattering studies of model membraneas a function of hydration and temperatureFederica Sebastiani1,2, Alessandra Filabozzi1 and GiovannaFragneto21Dipartimento di Fisica, Universita degli Studi di Roma ‘‘TorVergata’’, Roma, Italy, 2Institut Laue-Langevin, Grenoble,France

Cell membranes carry out highly specialised functions inliving materials. The composition of bacterial membranes isessential to understand the mechanism of action of antimi-crobial peptides. In order to understand the role of thevarious components contributing to the overall behaviour, wehave reproduced the membrane of Bacillus Subtilis andcarried out neutron diffraction studies on D16 (smallmomentum-transfer diffractometer) and D17 (reflectometerused as a diffractometer), at ILL. An ordered and homoge-neous sample has been obtained by using the widely studiedDMPC. The measured d-spacing of DMPC as a function ofthe relative humidity (RH) is related to the physical andchemical conditions affecting the sample. Consequently thereliability of the humidity chamber, which has been previouslyupgraded, has been stated. Moreover, the most suitablepreparation technique has been set up.

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In order to investigate the component roles within BacillusSubtilis membrane, three samples of phospholipids wereprepared (with POPE, POPG and cardiolipin). Neutron dif-fraction measurements, performed at controlled RH andtemperature, suggested the presence of interesting phasetransitions or coexistence of phases.

P-640The rupture of membrane vesicles near solid surfacesAnnamaria Takats-Nyeste, Imre DerenyiDepartment of Biological Physics, Eotvos University, H-1117Budapest Pazmany P. stny. 1/A, Hungary

The behavior of lipid membranes near solid surfaces has agreat significance both in medicine and in technology. In spiteof the widespread use and study of such membrane phe-nomena, their theoretical analysis is rather scarce. Our maingoal here is to understand the process during which mem-brane vesicles first adhere to solid surfaces, then rupture (orgo through a series of transient ruptures) due to themechanical tension induced by the adhesion, and finallyspread along the surface forming a supported lipid bilayer. Inour theoretical description we simultaneously consider thedynamics of spontaneous pore opening and closing; volumeloss via leakage through the pores; and the advancement ofthe adhesion front. All these processes are supposed tofollow an overdamped dynamics and coupled to each otherthrough membrane tension.Our numerical simulations reveal that the rupture processconsists of three well distinguishable phases: a fast initialvolume loss; followed by a slow volume loss; ending with afinal burst and surface spreading. The second phase can beskipped if either the first phase advances far enough or thethird phase sets in early enough. The smaller the vesicle, thefurther the first phase can advance. The third phase can startearlier if either the surface is smooth enough, or the adhesionenergy is large enough, or the line tension is small enough.When the second phase is not skipped the time needed forthe rupture process can take very long with a large variance.In the realistic range of the material properties (line tension,bending rigidity) the process is qualitatively always the same,so the most decisive parameter remains the size of thevesicle: the smaller the vesicle the faster and easier itruptures.

P-641Liposomal delivery of antituberculoticsI. Voszka1, K. Herczeg1, K. Modos1, G. Csık1, K. Horvati2,Sz. B}osze21Department of Biophysics and Radiation Biology,Semmelweis University, Budapest, Hungary, 2ResearchGroup of Peptide Chemistry Hungarian Academyof Sciences, Eotvos L. University, Budapest, Hungary

Liposomes are applied to targeted drug delivery. Liposome-incorporated drugs have less side effects. Tuberculosis iscaused by Mycobacterium tuberculosis. The bacteriacan survive in macrophages. The aim was to develop lipo-somal drugs against TB. We made liposomes fromdiphalmitoil-phoshpatidylcholine (DPPC), or from dioleoyl-

phosphatidyletanolamine (DOPE), cholesteryl hemisuccinate(CHEMS) and polyethilene-glycol releated distearoylpho-sphatidyl-ethanolamine (DSPE-PEG). We measured the sizeof liposomes with dynamic light scattering on the formulationday, than 2 and 7 days later. Samples were stored at 4 and20 �C. We used isoniazid (INH) and peptide conjugated INHsas antituberculotics. We measured the drug encapsulationwith fluorescence anisotropy. The liposomal drugs’ in vitroactivity was determined on Mycobacterium tuberculosisH37Rv culture and on Mycobacterium tuberculosis H37Rvinfected MonoMac6 human monocyte culture. The DPPCliposomes showed significant aggregation both at 4 and 20�C. Neither the empty, nor the INH-containing DOPE:-CHEMS:DSPE-PEG liposomes aggregated in 7 days. TheINH and the peptide conjugated INH were encapsulated inboth type of liposomes. Both liposomal drugs were effectivein vitro on the bacterial culture and on the infected monocyteculture.

P-643Direct observation of the effect of plant polyphenolson lipid domains in giant unilamellar vesiclesOlga Wesołowska1, Krystyna Michalak1, Andrzej B.Hendrich21Department of Biophysics, Wroclaw Medical University,Wroclaw, Poland, 2Department of Biology and MedicalParasitology, Wroclaw Medical University, Wroclaw, Poland

Giant unilamellar vesicles (GUVs) produced by electrofor-mation possess an average diameter of 30 - 50 lm and canbe directly observed under fluorescence microscope ifformed from a lipid-fluorescent label mixture. In the presentstudy we used confocal microscopy technique to observe themorphology of lipid domains in GUVs prepared from ternarylipid mixture DOPC:cholestrol:sphingomyelin (1:1:1) andlabeled with carbocyanine fluorescent probe (DiIC12(3)). Wechose four plant-derived polyphenols (flavonoids and stilb-enes) of documented biological activity to study theirinfluence on lipid domain number, area, shape, and border-length. We found that resveratrol elevated the number ofdomains per vesicle, decreased their area and markedlyincreased the total length of domain border without affectingdomains’ circular shape. Surprisingly, no such effect wasobserved for piceatannol differing from resveratrol by onehydroxyl group only. Neither genistein nor 8-prenylnaringeninchanged the morphology of lipid domains significantly. Thepossible mechanism of resveratrol-induced effect on lipiddomains’ morphology could be its selective accumulation inthe interfacial regions between liquid ordered and liquid dis-ordered domains.

P-644Putative cholesterol recognition amino acid consensus(CRAC) motif in HIV coreceptors CXCR4 and CCR5Mikhail A. Zhukovsky, Albrecht OttBiological Experimental Physics Department, SaarlandUniversity, 66041 Saarbruecken, Germany

We identified a cholesterol recognition amino acid consen-sus (CRAC) motif in transmembrane domain 5 (TMD5) of

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two G protein-coupled receptors (GPCRs), human chemo-kine receptors CXCR4 and CCR5, coreceptors of humanimmunodeficiency virus (HIV). We suggest that residuesbelonging to this CRAC motif are involved in cholesterolbinding to CXCR4 and CCR5 that is responsible for cho-lesterol requirement for CXCR4 and CCR5 conformationand function and for the role that cell cholesterol plays in thecell entry of CXCR4-using and CCR5-using HIV strains.Putative CRAC sequences involve residues V214/L216-Y219-K225 in CXCR4 and L208/V209/V211-Y214-K219 in CCR5. InCXCR4, CRAC motif is highly conserved across Chordataspecies, whereas in CCR5, CRAC motif is less conserved.Moreover, we identified CRAC motifs in TMD5 in 16 of 20human chemokine receptors. In [Md. Jafurulla et al., Bio-chem. Biophys. Res. Commun. (2011), 404: 569] CRACmotifs in TMD5 of two other GPCRs were recently identified.CRAC motifs in these GPCRs, as putative CRAC motifs inCXCR4 and CCR5, include tyrosine residue that is highlyconserved among GPCRs. This tyrosine is located close tothe third intracellular loop, i. e., close to the membraneinterface, as can be expected for an effective associationwith cholesterol.

Conformational dynamics, folding and IDP

O-645Wide-line NMR and relaxation characterizationof interfacial water in protein solutionsM. Bokor1, P. Tompa2, K. Tompa1, K-H. Han31Research Institute for Solid State Physics and Optics,Hungarian Academy of Sciences, POB. 49, 1525 Budapest,Hungary, 2Institute of Enzymology, Biological ResearchCenter, Hungarian Academy of Sciences, POB. 7, 1518Budapest, Hungary, 3Korea Research Institute of Bioscience& Biotechnology, 111 Gwahangno, Yuseong-gu, Daejeon350-806, Korea

1H NMR FID, echoes, and relaxation times (T1 and T2) weremeasured at 82.55 MHz, at -70�C to ?40�C, for lyophilizedproteins, aqueous and buffered protein solutions. Examplesfor BSA, UBQ, ERD10, and WT a-synuclein and bird eyelenses are given. The melting of hydration water could bedetected well below 0�C and the quantity of mobile watermolecules (hydration, h vs. T) was measured. The h vs.Tcurves inform on the bonding character between the proteinsurfaces and H2O. The magnitudes and the slope of the h vs.T curve describe quantitatively the interfacial landscapearound the protein molecules and can be used for the dis-tinction between the globular and IDP states. The behavior ofthe T1 and T2 data showed that there are two reorientationtypes present for every protein solutions below 0�C, irre-spective for the nature of the protein or the solventcomposition. Local field fluctuation and the BPP models wereapplied, which failed for the buffered protein solutions and forthe IDPs dissolved in water. A main cause of the failure is thechanging h in the analyzed temperature range. This case isvalid for the solutions of IDPs and for buffered solutions ofboth protein types. Another cause can be the active

relaxation channels other than dipolar when ions of quadru-polar nuclei are present.

P-646Multinuclear NMR in buffered aqueous solutionsof a-synuclein mutantsP. Matus1, M. Bokor1, P. Tompa2, E. Hazy2, K. Tompa11Research Institute for Solid State Physics and Optics,Hungarian Academy of Sciences, POB. 49, 1525 Budapest,Hungary, 2Institute of Enzymology, Biological ResearchCenter, Hungarian Academy of Sciences, POB. 7, 1518Budapest, Hungary

1H, 23Na and 35Cl nuclear magnetic resonance (NMR) freeinduction decay (FID) signals have been investigated in buf-fered wild-type (WT) and A30P a-synuclein protein solutionsand in the solvent itself containing NaCl. The experiments havebeen carried out by Bruker SXP 100 and AVANCE III spec-trometers at Larmor frequencies 82.1 MHz (1H), 89.3 MHz(23Na) and 33.1 MHz (35Cl) in the temperature range of -150�Cand 30�C. Below 0�C, the melting of hydration is followed bythe broadening of the line width of all the three nuclei. Themotional narrowing of the NMR spectra of the quadrupolarnuclei (23Na, 35Cl) is the consequence of the molecular motionof water molecules and the interaction of protein surface withsodium and chloride ions on a molecular level.

P-647Calcium-induced structural, hydration and valencechanges of intrinsically disordered RTX proteinsAna Cristina Sotomayor Perez, Johanna C. Karst, MarilyneDavi, Daniel Ladant and Alexandre ChenalInstitut Pasteur, Paris, FranceE-mail: alexandre.chenal@pasteur.fr

Ligand-induced disorder-to-order transition plays a key rolein the biological functions of many proteins that containintrinsically disordered regions. This trait is exhibited by RTX(Repeat in ToXin) motifs found in more than 250 virulencefactors secreted by Gram-negative pathogenic bacteria. Weinvestigated several CyaA RTX polypeptides of differentlengths ranging from 111 to 706 residues. We showed thatthe RTX proteins exhibit the hallmarks of intrinsically disor-dered proteins in the absence of calcium: they adopt pre-molten globule conformations and exhibit a strong time-averaged apparent hydration, due in part to the internalelectrostatic repulsions between negatively charged resi-dues, as revealed by the high mean net charge. Calciumbinding triggers a strong reduction of the mean net charge,dehydration and compaction, folding and stabilization ofsecondary and tertiary structures of the RTX proteins. Wepropose that the intrinsically disordered character of the RTXproteins may facilitate the uptake and secretion of virulencefactors through the bacterial secretion machinery. Theseresults support the hypothesis that the folding reaction isachieved upon protein secretion and, in the case of proteinscontaining RTX motifs, could be finely regulated by the cal-cium gradient across bacterial cell wall.

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P-648C-terminal conformation changes of alpha-synucleinupon Al-induced oligomerizationManli Deng and Yves EngelborghsLaboratory of Biomolecular dynamics, Katholieke UniversiteitLeuven, Leuven, Belgium

Occupational exposure to heavy metals has been recog-nized to be a risk factor for Parkinson’s Disease via metal-triggered deposition of alpha-synuclein (aS)1,2. In thepresent work, Al3? induced conformational change andinstant oligomerization of aS have been studied usingFRET and FCS as main techniques. Donor and acceptorwere labeled in the C-terminal at positions A107C andA140C. The average lifetime of donor in the presence ofacceptor increases with the increase of Al3? concentration,indicating aS adopts a more extended conformation upon Al3?

binding. The intrinsic Tyr fluorescence rises sharply within themixing dead time, reflecting an enhanced hydrophobicity of theTyr environment and a fast conformational change of aS. Al3?

also induces an immediate oligomerization of aS as monitoredby FCS. The diffusion coefficient of aS changes from 85 ± 5lm2/s asmonomer state to 23 ± 5 lm2/s as oligomer state. Theoligomerization issupposed tobe inducedby the ligandbridgingof trivalent Al ions.

1. Rybicki, B. A.; Johnson, C. C.; Uman, J.; Gorell, J. M.Mov Disord 1993, 8, 87-92.

2. Uversky, V. N.; Li, J.; Fink, A. L. J.Biol.Chem. 2001, 276,44284-44296.

P-650Using neutron scattering to investigate unfoldedintermediates of ribonuclease AJennifer Fischer1, Ralf Biehl1, Bernd Hoffmann2 and DieterRichter11Forschungszentrum Julich, JCNS-1 and ICS-1, Julich,Germany, 2Forschungszentrum Julich, ICS-7, Julich,Germany

Ribonuclease A (RNase) is a well studied enzyme, catalyz-ing the degradation of RNA. As it provides a reversibletransition following a distinct unfolding pathway, RNase Aserves as a model system to investigate large scale thermalfluctuations inside the protein. This is one way to explore theenergy landscape at a given equilibrium state and will showus possible rearrangements of biological relevant confor-mations as well as local stable conformations. By changingenvironmental parameters such as temperature, pH andsolvent conditions, the level of energy can be varied.We are investigating bovine pancreatic RNase A under dif-ferent denaturation conditions using mainly small angleneutron scattering and neutron spin echo spectroscopy.Combining such techniques provides a unique tool to studythe structural arrangement of proteins in solution and theirdynamical properties. Large scale fluctuations of proteindomains can be accessed as these techniques are sensitiveto the structure and to collective dynamics. Using additionaltechniques, such as circular dichroism spectroscopy givesfurther insight into the secondary structure and how itresponds to changes in temperature.

O-651Fuzziness in protein-DNA interactions: beyond what canbe seenMonika FuxreiterInstitute of Enzymology, BRC, Hungarian Academyof Sciences, Hungary

Traditionally, specific DNA recognition is based on staticcontacts with the bases or phosphates. Residues far outsidethe binding context, however, can critically influence selec-tivity or binding affinity via transient, dynamic interactionswith the DNA binding interface. The corresponding regionsare usually intrinsically disordered (ID), which preserve theirconformational freedom even when bound to DNA. There arefour possible scenarios how distant ID segments impact DNAbinding. They modulate i) conformational preferences, ii)flexibility or iii) spacing of the DNA binding motif(s) or iv)serve as competitive partners. Despite their low sequencesimilarity, the ID regions are often conserved at the structurallevel in families of orthologous proteins. Fuzzy protein-DNAcomplexes offer a variety of regulatory pathways via protein–protein interactions, post-translational modifications or alter-native splicing in response to cellular cues.

P-652Understanding the plasticity of c-Src tyrosine kinasethrough very long molecular dynamics simulationsand experimentally validated free energy calculations.R. Boubeva1, A. Cristiani1, L. Pernot1, R. Perozzo1, L. Sutto2,L. Scapozza1, Francesco L. Gervasio2*1School of Pharmaceutical Sciences, University of Geneva,Quai Ernest-Ansermet 30, Geneve, CH-1211, Switzerland,2Spanish National Cancer Research Centre (CNIO), CalleMelchor Fernandez Almagro, 3, Madrid, E-29028, Spain,*E-mail: flgervasio@cnio.es

Nearly 2% of human genes encode protein-kinases (PK),enzymes involved in cellular signaling and several other vitalbiochemical functions, which transfer phosphate groups fromATP to specific target molecules, modifying their activity.[1]Deregulated PK have been linked to numerous diseasesincluding cancer and diabetes, making them attractive tar-gets for drug design.[2] Conformational transitions play acentral role in regulating the phosphorylation activity. PKadopt an on state that is maximally active and one or moreinactive states that shows minimal activity.[3] The similarity ofthe relatively rigid and largely conserved ATP binding sitemakes the design of selective inhibitors binding to the activestate very difficult. Indeed some of the best cancer therapiesavailable are based on inhibitors, as Imatinib, that bind toinactive states peculiar to a small subset of PK (Abl, c-Kit andPDGFR in the case on Imatinib). Thus, understanding theatomic details of the active to inactive transitions in kinaseshas a great importance. Here we study a particular active-to-inactive transition of c-Src, a fundamental proto-oncogeneinvolved in cancer and metastasis, by using multi-microsec-ond long fully solvated molecular dynamics simulations,metadynamics and PTmetaD calculations [4,5]. The results,validated by mutagenesis, x-ray crystallography and bindingkinetics, are suggestive of a functional role for the confor-mational transition. Moreover, we were able to single out the

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most important residues affecting the conformational transi-tion and to show that even a very conservative amino-acidsubstitution can have a dramatic effect on the conformationalfree energy landscape.

1. Manning, G., et al. Science 298, 1912 (2002)2. Druker, B., Lydon, N. J Clin Invest 105, 3 (2000)3. Huse, M. and Kuriyan J. Cell 109 275 (2002)4. Bussi, G., et al. J. Am. Chem. Soc. 128 13435 (2006)5. Laio, A., Gervasio, F.L. Rep Prog Phys 71 126601(2008)

O-653Folding rates studied by a combination of staticand time-resolved infrared spectroscopyC. Krejtschi and K. Hauser1Biophysical Chemistry, Department of Chemistry, Universityof Konstanz, 78457 Konstanz, GermanyE-mail: Karin.Hauser@uni-konstanz.de

The time-scales of protein folding events range over manyorders of magnitude. In order to understand the complexfolding mechanisms, peptides with well-defined secondarystructure are often used as model systems as they may beregarded as smallest folding units of proteins. The formationof secondary structure elements occur on the nanosecondto low microsecond time scale. Thus, stopped-flow tech-niques are too slow whereas pulsed laser techniques arecapable to trigger folding processes in nanoseconds and toanalyze faster folding events. We study ns-to-ls peptidedynamics by temperature-jump infrared spectroscopy. Afterinitiation of a nanosecond temperature jump, the spectralresponse is monitored at single wavelengths in the amide Iregion reflecting the dynamics of the peptide backbone.Relaxation rates are obtained. The helix-to-coil relaxation ofpolyglutamic acid is a multi-step process and requires morecomplex models than two-state kinetics. However, there arekinetic steps that are well described by single-exponentialbehavior and a two-state model. We demonstrate howequilibrium and time-resolved infrared spectroscopic datacan be combined to deduce folding rates.

P-654Protein unfolding and refolding by multidimensionalspectroscopySebastian Fiedler, Sandro KellerMolecular Biophysics, University of Kaiserslautern, Erwin-Schrodinger-Str. 13, 67663 Kaiserslautern, Germany

Unfolding and refolding studies using chemical denaturantshave contributed tremendously to our understanding of thethermodynamics and kinetics of protein folding and sta-bility. However, a major limitation of this approach lies inthe large uncertainty inherent in the extrapolation of thefree energy of unfolding in the absence of denaturant fromfree energy values measured at finite denaturantconcentrations.Here we show that this limitation can be overcome by com-bining multiple spectroscopic signals—including fluores-cence, circular dichroism, and absorbance—recorded in a

quasi-simultaneous and fully automated way at differentwavelengths. We have optimised the number of wavelengthvalues used, the integration time per data point, the incrementin the denaturant concentration, and the weighting schemeapplied for global data fitting. Compared with the traditionalapproach based on the use of a single or a few wavelengths,we could thus improve the precision of the free energy value byan order of magnitude. We exemplify and validate this novelapproach using representative, well-studied globular proteinsand explain how it can be exploited to quantify subtle changesin membrane-protein stability which have thus far remainedelusive.

P-655Kinetic effect of Hofmeister ions on the photocycleof photoactive yellow proteinPetro Khoroshyy, Andras Der and Laszlo ZimanyiInstitute of Biophysics, Biological Research Centreof the Hungarian Academy of Sciences, Szeged, Hungary

Photoactive yellow protein (PYP) is a water-soluble photo-sensor protein in purple photosynthetic bacteria with acovalently bound p-coumaric acid chromophore. Lightabsorption initiates a photocycle with chromophore isomeri-zation, transient protonation and conformational changes ofvarious magnitude. The formation and decay of the con-secutive photocycle intermediates of Halorhodospirahalophila PYP after an actinic laser pulse were followed withtime-resolved near UV – visible multichannel absorptionspectroscopy in the 100 nanosecond – seconds range inbuffers containing either NaF, NaCl or NaClO4 in 0.66 Mconcentration at pH 8.2. Hofmeister salts – originally classi-fied based on their salting-out or salting-in effects – influencethe conformational stability of proteins. We have analyzedthe spectrotemporal matrices obtained in different salts withthe method Singular Value Decomposition with ExponentialFit Assisted Self-Modeling (SVD-EFASM1) and investigatedthe correlation of the Hofmeister salt-dependence of variousrate constants of the PYP photocycle with the structuralmodel of the protein’s function.

[1] Zimanyi, L. 2004. J. Phys. Chem. B 108:4199-4209.

Supported by the Hungary-Romania Cross Border Cooper-ation Program of the EU, grant HURO/0901/219.

P-658A model with two elastically coupled reactioncoordinates for the internal friction of proteinsNorbert Orgovan, Anna A. Rauscher, Andras Malnasi-Csizmadia, Imre DerenyiDepartment of Biological Physics, Eotvos University,Budapest, Hungary

The rates of protein conformational changes are usually notonly limited by external but also internal friction, however,the origin and significance of this latter phenomenon ispoorly understood. It is often found experimentally that alinear fit to the reciprocal of the reaction rate as a functionof the viscosity of the external medium has a non-zero

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value at zero viscosity, signifying the presence of internalfriction. Furthermore, some of the experiments performed atdifferent temperatures indicate that the internal friction fol-lows an Arrhenius-like temperature dependence. To explainthese phenomena we suggest a simple model for proteinconformational changes in terms of two elastically coupledreaction coordinates, one of which being in contact with theexternal medium, and the other one experiencing the roughenergy landscape of the protein. Our analytical calculations,supplemented with numerical simulations demonstrate thatdepending on the coupling strength (which is related to theflexibility of the protein) the short-wavelength components ofthe energy landscape roughness can be observed as anincreased apparent internal friction with an apparent acti-vation energy.

O-659Probing cavities in SNase structure: a high pressureNMR studyRoche Julien1, Jose A. Caro2, Garcia E. Angel3, Garcia-Moreno Bertrand2, Royer A. Catherine1, RoumestandChristian11Centre de Biochimie Structurale, INSERM UMR 1054,CNRS UMR 5048, Universite de Montpellier, Montpellier,France, 2T. C. Jenkins Department of Biophysics, The JohnsHopkins University, Baltimore, Maryland, USA, 3Departmentof Physics, Applied Physics and Astronomy,Rensselear Polytechnic Institute, Troy, USAE-mail: roche@cbs.cnrs.fr; roumestand@cbs.cnrs.fr;royer@cbs.cnrs.fr; jcaro2@jhu.edu; bertrand@jhu.edu;angel@rpi.edu

Since Bridgman seminal experiments on high-pressuredenaturation of albumen in 19141, the physical basis ofpressure unfolding is still largely unknown. We report here aspecific study of cavities contributions to the volume differ-ence between unfolded and folded states (DVu), using foursingle point mutants of Staphylococcus nuclease (SNase).Each mutation is localised in a strategic position on theprotein structure and was designed to change a large buriedhydrophobic side chain into alanine, thus opening tunablecavities in the SNase 3D structure.Measuring HSQCs peaks intensities up to 2500 bar moni-tored the equilibrium high pressure unfolding and leads us toprecise estimations of DVu for more than two-thirds of the 143residues of each mutant. SO-FAST HMQC experiments2

were also performed to measure folding and unfolding ratesfrom 200 bar pressure jumps. High-pressure fluorescenceexperiments were done on six additional alanine mutants tocomplement the NMR study, allowing a more completeexploration of the local pressure sensitivity along the protein3D structure. All these highly reliable measurements shedlight on the real signification of the thermodynamic parameterDVu, and bring an unprecedented complex and heteroge-neous picture at a residue level of the apparent two-statefolding process of SNase.

1. Bridgman, P. W. J. Biol. Chem. 1914. 19, 511-5122. Shanda P., Kupce E. Brutscher B. J. Biomol. NMR. 2006.

33, 199-211

P-660Differential hydration, void volume: which factorprovides the main contribution to DVu?Roche Julien1, Rouget Jean-Baptiste1, Jose A. Caro2, AkselTural2, Barrick Doug2, Garcia E. Angel3, Garcia-MorenoBertrand2, Roumestand Christian1, Royer A. Catherine11Centre de Biochimie Structurale, INSERM UMR 1054,CNRS UMR 5048, Universite de Montpellier, Montpellier,France, 2T. C. Jenkins Department of Biophysics, The JohnsHopkins University, Baltimore, Maryland, USA, 3Departmentof Physics, Applied Physics and Astronomy, RensselearPolytechnic Institute, Troy, USA, E-mail: roche@cbs.cnrs.fr;rouget@cbs.cnrs.fr; roumestand@cbs.cnrs.fr;royer@cbs.cnrs.fr; tural@jhu.edu; jcaro2@jhu.edu;barrick@jhu.edu; bertrand@jhu.edu; angel@rpi.edu

Determination of contributing factors to the volume changemagnitude between unfolded and folded states (DVu) is a long-standing question in the high-pressure field. We provide herenew experimental and computational data using two well-characterized model proteins: Notch ankyrin repeat domain(Nank) and Staphylococcal nuclease (SNase). The repetitivenature of the Nank protein was used to study influence of theprotein size on DVu in a systematic way with a set of deletionmutants. High-pressure fluorescence data provided new evi-dences that neither peptide bonds hydration nor side chainsdifferential hydration could be considered as major contributorto the measured DVu value. Additional molecular dynamics(MD) simulations rather suggested that the heterogeneousdistribution of void volume in the folded states structures couldexplain the DVu variations among the Nank deletion mutants.The specific issue of the void volume contribution to DVu val-ues was studied using 10 cavity mutants in SNase, allowing alarge structural mapping of the alanine mutations on thisglobular protein. Combination of X-ray crystallography, high-pressure fluorescence, high-pressure NMR and MD simula-tions provided a first clear determination of the void volumecontribution to the DVu values. These results also bring anunprecedented complex and heterogeneous picture at a resi-due level of the apparent two-state folding process of SNase.

O-662Lactoferrin: dynamics of a flexible protein in solutionrevealed by neutron scatteringClemens Sill1, Ralf Biehl1, Bernd Hoffmann2, Dieter Richter11Julich Centre for Neutron Science JCNS (JCNS-1) &Institute for Complex Systems: Neutron Scattering (ICS-1),Forschungszentrum Julich, 52425 Julich, Germany, 2Institutefor Complex Systems: Biomechanics (ICS-7),Forschungszentrum Julich, 52425 Julich, Germany

The understanding of the functionality of proteins started witha rigid model, namely the Lock and Key analogy, in 1894.Meanwhile, a more dynamic and flexible picture of thesemacromolecules has evolved to explain protein function likecatalyzing biochemical reactions, transport, regulation, stor-age or defensive tasks. The importance of thermodynamicallydriven, internal motions for the functioning of proteins issubject of ongoing research.We will present recent investigations of protein dynamics onLactoferrin, a protein with antimicrobial activity which is partof the innate immune system. It consists of two binding sites,

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each is capable of binding and releasing one iron ion. Thecrystallographic structures show that the binding sites haveopen and closed conformations, assumedly depending onthe presence of iron (Andersen et al., Nature, 1990).We are analyzing the internal dynamics of different bindingstates to elucidate of the binding mechanism with neutronscattering. Our unique method includes large scale structuralcharacterization with small angle neutron scattering and theobservation of internal motions of subdomains with neutronspin echo spectroscopy on nanosecond scale. This way weare able to clarify the link between binding mechanism andconformational change.

P-663Comparison of the pressure- and temperature-dependent behavior of the ordered and disorderedregions of titinJ. Somkuti, Z. Martonfalvi, M.S.Z. Kellermayer, L. SmellerDept. Biophysics and Radiation Biology, SemmelweisUniversity, Budapest, Hungary

Titin is a giant protein responsible for striated-muscle elas-ticity. It contains a series of ordered domains and a largedisordered segment called the PEVK domain. The ordereddomains belong to the immunoglobulin (Ig) type C2 andfibronectin (FN) type III superfamilies. The disordered PEVKdomain is named after the residues proline, glutamate, valineand lysine which provide the majority of its constituents.We expressed an Ig domain (I27) and a 170-residue-longfragment of the PEVK domain in order to investigate theeffect of temperature and pressure on their conformation.FTIR spectroscopy is a useful method for investigating thesecondary structure of proteins. We analyzed the amide Iband to obtain information on protein structure. Fluorescencelabeling was also used in some experiments. To generatehigh pressures, a diamond anvil cell was employed.The FTIR and fluorescence spectra of the protein fragmentswere recorded across the pressure and temperature rangesof 0-1 GPa and 0-100 �C, respectively. Moderate changeswere observed in the conformation of the PEVK fragments inthe explored range of the T-p plane, suggesting that thedomain is a highly flexible, random-coil across the entirestudied T-p range. By contrast, the I27 domain showed quitestable secondary structure.

P-664Impact of variable DMSO additives on the catalyticand calorimetric performance of a-chymotrypsinTatyana Tretyakova, Mikheil Shushanyan, MayaMakharadze, Tamar Partskhaladze, Dimitri E. Khoshtariyaand Rudi van EldikLEPL Life Science Research Centre, 0160, Tbilisi, Georgia,Institute for Biophysics and Bionanosciences, Departmentof Physics, I. Javakhisvili Tbilisi State University, 0128,Tbilisi,Georgia, Department of Chemistry and Pharmacy,University of Erlangen-Nurnberg, 91058 Erlangen,Germany

New insights in understanding of fundamental links betweenproteins’ stability, conformational flexibility and function can

be gained through altering protein’s thermodynamic anddynamic properties. In the present work an impact of non-specific moderate denaturant, dimethylsulphoxide, DMSO,on the kinetic (functional) and thermodynamic (thermal sta-bility) patterns of a hydrolytic enzyme, a-chymotrypsin (a-CT)has been investigated. For the a-CT-catalysed process ofATEE hydrolysis, within the DMSO concentration range of0-25% V/V we observed increase of the Michaelis constant(KM) whereas the catalytic constant (kcat) remainedunchanged. These observations indicate on a preservation ofthe protein’s active conformation accompanied by increaseof the conformational flexibility of its active centre. In thepresence of similar DMSO concentrations microcalorimetricdata reflecting the a-CT thermal denaturation clearly point tothe stability increase of the protein’s compact part. Sub-sequent increase of the DMSO concentration leads to theprotein’s global destabilization, however the enzymatichydrolysis process still obeys the Michaelis-Menten pattern,indicating about the involvement of the molten-globule-likestate of a-CT.

O-665How scarce sequence elements control the functionof single ß-thymosin/WH2 domains in actin assemblyDominique Didry2, Francois-Xavier Cantrelle1, ClotildeHusson2, Pierre Roblin2,3, Anna M. Eswara Moorthy2, JavierPerez3, Christophe Le Clainche2, Maud Hertzog2, EricGuittet1, Marie-France Carlier2, Louis Renault2 and Carinevan Heijenoort11Laboratoire de Chimie et Biologie Structurales, FRC 3115ICSN-CNRS, 91198 Gif-sur-Yvette, France, 2CytoskeletonDynamics and Motility, FRC 3115 LEBS-CNRS, 91198 Gif-sur-Yvette, France, 3Synchrotron SOLEIL, BP 48 SaintAubin, 91192 Gif-sur-Yvette, France

b-thymosin (ßT) and WH2 domains are widespread, intrin-sically disordered, proteins that fold upon binding actin. Theydisplay significant sequence variability associated to versa-tile regulations of actin assembly in motile processes. Herewe reveal the structural basis by which, in their basic 1:1stoichiometric complexes with actin, they either inhibitassembly by sequestering actin monomers (Thymosin-ß4),or enhance motility by directing polarized filament assembly(Ciboulot ßT or WASP/WAVE WH2 domains).We combined mutational, functional, and structural analysisby X-ray crystallography, SAXS and NMR on Thymosin-ß4,Ciboulot and the WH2 domain of WASP-interacting protein(WIP). Functionally different ßT/WH2 domains do not tar-get alternative actin binding sites but rather differ by alternativedynamics of their C-terminal half interactions with G-actinpointed face. The interaction dynamics largely depends on thestrength of electrostatic interactions of a single residue alongtheir sequence. The results open perspectives for elucidatingthe functions of ßT/WH2domains in othermodular proteins andenlighten how intrinsic structural disorder can lead to a novelmode of functional versatility.

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P-666Interfacial water in b-casein molecular surfaces: wide-lineNMR, relaxation and DSC characterizationT. Verebelyi1, M. Bokor1, P. Kamasa1, P. Tompa2, K. Tompa11Research Institute for Solid State Physics and Optics,Hungarian Academy of Sciences, POB. 49, 1525Budapest,Hungary, 2Institute of Enzymology, BiologicalResearch Center, Hungarian Academy of Sciences, POB. 7,1518 Budapest, Hungary

Wide-line proton NMR FID, echoes, spin-lattice and spin-spinrelaxation times were measured at 82.55 MHz frequency inthe -70�C to ?40�C temperature range, in lyophilized b-casein and aqueous and buffered solutions, and DSCmethod were also applied. The motivation for the selection ofb-casein is the uncertainty of structural order/disorder. Nat-urally, the NMR and thermal characteristics were alsoevaluated. The melting of hydration water could be detectedwell below 0�C and the quantity of mobile water molecules(hydration) was measured. The hydration vs. melting tem-perature curve has informed us on the bonding characterbetween the protein surfaces and water molecules. Thegenerally used local field fluctuation model and the BPPtheory were applied in the interpretation, and the limits of themodels were concluded.

O-667Promiscuous liaisons: functional interactionsof intrinsically disordered proteins in biologicalsignalingPeter E. WrightDepartment of Molecular Biology, The Scripps ResearchInstitute, La Jolla, CA, USA

Intrinsically disordered proteins participate in importantregulatory functions in the cell, including regulation oftranscription, translation, the cell cycle, and numerous sig-nal transduction events. Disordered proteins often undergocoupled folding and binding transitions upon interactionwith their cellular targets. The lack of stable globularstructure can confer numerous functional advantages,including, paradoxically, both binding promiscuity and highspecificity in target interactions. NMR is unique in beingable to provide detailed insights into the intrinsic confor-mational preferences and dynamics of unfolded and partlyfolded proteins, and into the mechanism of coupled foldingand binding. The function of intrinsically disordered proteindomains in transcriptional regulation and signaling will bedescribed, with particular reference to the general tran-scriptional coactivators CBP and p300, the tumorsuppressor p53, and the adenovirus E1A oncoprotein. Theglobular domains of CBP/p300 are targets for coupledfolding and binding of disordered transactivation motifs ofnumerous transcription factors and viral oncogenes, whichcompete for binding to limiting amounts of CBP/p300. Manyintrinsically disordered proteins contain multipartite inter-action motifs that perform an essential function in theintegration of complex signaling networks. The role ofmultipartite binding motifs and post translational modifica-tions in regulation of p53-mediated signaling pathways willbe discussed.

Systemic and collective behavioural aspectsin biology

O-668Vasculogenesis and collective movement of endothelialcellsAndras CzirokDept of Biological Physics, Eotvos University, Budapest,Hungary, Dept of Anatomy & Cell Biology, Universityof Kansas Medical Center, Kansas City, USA

The early vascular network is one of the simplest functioningorgans in the embryo. Its formation involves only one cell typeand it can be readily observed and manipulated in avianembryos or in vitro explants. The early vascular network ofwarm-blooded vertebrates self-organizes by the collectivemotility of cell streams, or multicellular ‘‘sprouts’’. The elon-gation of these future vascular network segments depends ona continuous supply of cells, moving along the sprout towardsits tip. To understand the observed self-organization process,we investigate computational models containing interactionsbetween adherent, polarized and self-propelled cells. Bycomparing the simulations with data from in vivo or simplisticin vitro experiments, we explore the role of active migration,leader cells, invasion of the ECM, and cell guidance by mi-cromechanical properties of adjacent cell surfaces.

P-669Isotopic dilution mass spectrometric transmembranartransport study of amino acids in human erythrocytesE. Horj1, S. Neamtu2, M. Culea11Babes-Bolyai University, 1 Kogalniceanu St., Cluj-Napoca,400084, Romania, 2National Institute for Researchand Development of Isotopic and Molecular Technologies,71-103 Donath St., 400293, Cluj-Napoca, Romania

A study of amino acid transmembranar transport in human redcells is presented. An isotopic dilution GC/MS technique wasdeveloped for in vitro transport study of leucine and glycine inhuman erythrocytes. 15N-labeled glycine and 15N-leucinewere used as internal standards. The amino acids valueswere calculated by using two different methods, by usingregression curves or the least squares method. The methodswere validated and good values were obtained for parametersas linearity, precision and accuracy. Small values for bothamino acids transport were measured. Influences of param-eters such as time, temperature and electromagnetic field ontransmembranar transport of amino acids were studied.

P-670Logic estimation of the optimum source neutron energyfor BNCT of brain tumorsM.A. Dorrah, M.A. Kotb2, F.A. Gaber3, M.M. Mohammed2

and M.A. Abd Elwahab1Nuclear Engineering; National Center of Nuclear Safetyand Radiation Control; Egypt Atomic Energy Authority,2Bio-Medical Physics Dept., Medical Research Institute;Alexandria University, Alexandria, Egypt

Boron Neutron Capture Therapy (BNCT) is a promisingmethod for treating the highly fatal brain tumor; glioblastoma

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multiform. It is a binary modality; in which use is made of twocomponents simultaneously; viz. thermal neutrons andBoron-10.The biophysics of BNCT is very complicated; primarily due tothe complexity of element composition of the brain. Moreover;numerous components contributes to the over all radiationdose both to normal brain and to tumor. Simple algebraicsummation cannot be applied to these dose components,since each component should at first be weighed by its rela-tive biological effectiveness (RBE) value. Unfortunately, thereis no worldwide agreement on these RBE values.Thermal neutrons were formerly employed for BNCT, but theyfailed to prove therapeutic efficacy. Later on; epithermal neu-trons were suggested proposing that they would be enoughthermalized while transporting in the brain tissues. However;debate aroused regarding the optimum source neutronsenergy for treating brain tumors located at different depths inbrain. Insufficient knowledge regarding the RBE values of dif-ferent BNCT dose components was a major obstacle.A new concept was adopted for estimating the optimum sourceneutrons energy appropriate for different circumstances ofBNCT. Four postulations on the optimum source neutronsenergy were worked out, almost entirely independent of theRBE values of the different dose components. Four corre-sponding condition on the optimum source neutrons energywere deduced. An energy escalation study was carried outinvestigating 65 different source neutron energies, between0.01 eV and 13.2 MeV. MCNP4B Monte_Carlo neutron trans-port code was utilized to study the behavior of these neutronsin the brain. The deduced four conditions were applied to theresults. A source neutron energy range of few electron volts(eV) to about 30 keV was estimated to be optimum for BNCTofbrain tumors located at different depths in brain.

P-671Simulation of mutation induction by inhaled radonprogenies in the bronchial epitheliumBalazs G. Madas, Arpad Farkas and Imre BalashazyHungarian Academy of Sciences KFKI Atomic EnergyResearch Institute, Konkoly-Thege Miklos ut 29-33.,Budapest, H-1121, Hungary

Radon is considered as the second most important cause oflung cancer after smoking. To understand the mechanismsleading from radon exposure to cancer formation is of crucialimportance. This study focuses on the description of muta-tion induction by radon progenies in the bronchial epithelium.Computational fluid and particle dynamics approach wasapplied to determine the radio-aerosol deposition distributionin the central airways. A numerical replica of a small fragmentof the bronchial epithelium was prepared based on experi-mental data. Microdosimetric computations were performedto quantify the cellular radiation burdens at the very site ofdeposition accumulation. A mutagenesis model was appliedsupposing that radiation induces DNA damages andenhances the cell turnover rate.The results show that both considered mutagenic effects ofdensely ionising radiation contribute significantly to mutationinduction and mutation rate depends non-linearly on expo-sure rate. Furthermore, simulations suggest that the localmaintenance capacity of the bronchial epithelium can be

exhausted by chronic exposure to radon progenies withactivity concentration characteristic of some uranium mines.The present work demonstrates possible applications ofnumerical modelling in radon related carcinogenesis studies.

P-672On Future approaches to biophysicsDaniele Martin1, Michael Ch. Michailov1, Eva Neu1, UrsulaWelscher1, Joachim Milbradt1,2, Jakob Stiglmayr1,2, Klara-Stephen-S. Molnar1, Erich Gornik3, Germain Weber41Inst. Umweltmedizin c/o Int. Council Sci. Dev./Int. Acad.Sci., Austria, Bulgaria, Germany, Slovakia & Univ. Bratislava,Univ. Innsbruck, Univ. Erl.-Nurnberg. ICSD e.V. Postfach(POB) 340316, D-80100 Munchen, Germany, 2 BotanicalGarden, Univ. Erl.-Nurnberg, Germany, 3Fac. Physics, TUVienna, Austria, 4Fac. Psychology (dean), Univ. Luxembourg& Vienna, Austria

Introduction: Globalization needs new organizational mod-els also for biophysics. Reports on necessity of int. institutesfor biophysics (IIB) c/o int. universities (proposed by BritishNobel Laureate B.Russell) are given (Neu et al. Basic&Clin.Pharm.&Tox. 17/S1,489-2010; J.Physiol.Sci. 59/S1,447/8-2009; Eur.Biophys.J. 34/6,819-2005).Conception: Proposals for EBSA-discussion: 1. Enlarge-ment of Executive Committee by a. honorary & presidents(permanent 1-3: moral support & 1-3 fixed term), b. Inter-disciplinary commission: Scientists from biology, medicine,physics, etc. (FEPS/IUPS, IUPHAR, IUPAB, etc). 2. Impli-cation of interdisciplinary topics to ESBA/IUPAB congress-programmes, 3. also for biophysical journals. 4. Organizationof common interdisciplinary sessions not only to biophysical,but also to other congresses. 5. Co-operation betweenESBA/IUPAB with int. interdisciplinary organisations (WAAS,ICSD/IAS, Eur. academies) for creation of IIB by network ofnational ones: Successive common personnel, possibility forwhole life work, etc.Conclusion: Realization of proposals 1.-5. could increasescientific/political authority of EBSA/IUPAB, leading to modelfor renewal of scientific organizations, supporting UNO-Agenda21 for better health, education, ecology, economy inall countries.

O-673Collective migration of neural crest cells: a balancebetween repulsion and attractionRoberto MayorUniversity College London, UK

The Neural Crest is a group of cells found in all vertebrateembryos. It forms in the neural folds at the border of the neuralplateandgives rise toahugevarietyof cells, tissuesandorgans.One of the astonishing characteristic of neural crest cells is thatthey are able to migrate very long distances in the embryo. Theneural crest has been called the ‘‘explorer of the embryo’’ as it isone of the embryonic cell types that migrate most during devel-opment, eventually colonizing almost every tissue.In this talk I will discuss our recent finding about neural crestmigration. We have shown that neural crest cells, classicallydescribed as mesenchymal cells, migrate in large clusters

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and that interactions with neighbor cells are essential tocontrol directional migration. One of these interactions iscontact inhibition of locomotion [Nature. 2008. 456, 957] andI will show evidence of a new cell interaction behavior that wehave recently discovered. I will present our cellular andmolecular data that identify collective cell migration as afeature of neural crest and the molecules responsible forsuch behavior [Dev Cell. 2010. 19, 39]. Finally, I will discusssome mathematical modeling that integrates different kindsof cell interactions to explain collective neural crest migration,and tumor metastasis.

O-674Pattern formation by collective cell migration-drivensegregationEl}od Mehes, Enys Mones, Valeria Nemeth, Tamas VicsekDepartment of Biological Physics, Eotvos Lorand University,Budapest, Hungary

Pattern formation by cell sorting is an important process invarious biological events including embryonic development.To investigate the impact of collective cell migration on cellsorting we used fluorescent time-lapse videomicroscopy intwo-dimensional mixed cell cultures made of various pairs ofcell types with different motility characteristics. We demon-strate that more correlated cell migration, characterized bylonger directional persistence length of migrating cells,results in more extensive segregation into homogeneouscell clusters, monitored by various statistical physicalmethods.

O-675Signaling cascade dynamics after a hyper-osmoticshock in the yeast Saccharomyces cerevisiaeAgnes Miermont, Samuel Bottani, Pascal HersenMatiere et Systemes Complexes Laboratory, 10 rue AliceDomon et Leonie Duquet, 75013 Paris, France

Eukaryotic cells use signaling pathways to detect stimuli fromtheir environment and convey this information to the nucleuswhere transcriptional adaptation takes place. Mitogen Acti-vated Protein Kinases (MAPK) pathways are involved inseveral cellular responses such as stress reaction or prolif-eration controlFADDIN EN.CITE . The High-OsmolarityGlycerol (HOG) pathway in the yeast Saccharomyces cere-visiae is in charge of the osmo-regulation and its activationleads to the nuclear translocation of the protein Hog1p. Thedynamics of this cascade to mild osmotic shock has beenrecently investigated by several groups both experimentallyand theoretically. Here, we combine a microfludic device withfluorescent microscopy to study the HOG signal transductiondynamics in response to increasing osmotic shock. We showthat the nuclear translocation dynamics of Hog1p is sloweddown when increasing the osmotic shock intensity. Surpris-ingly, the cascade is still operational, since Hog1pphosphorylation is not altered. Moreover, we show that thisslow down is universal since other signaling pathways arealso severely slowed down when activated in a hyperosmotic environment. Our results suggest that the nucleo-cytoplasmic transport is altered by hyper osmotic shocks.

O-676Polar actin cortex mechanics and cell shape stabilityduring cytokinesisJakub Sedzinski1,2, Mate Biro1,2, Annelie Oswald1, Jean-Yves Tinevez1,2,3, Guillaume Salbreux4 and Ewa Paluch1,21Max Planck Institute of Molecular Cell Biology and Genetics,Dresden, Germany, 2International Institute of Molecularand Cell Biology, Warsaw, Poland, 3Present address:Pasteur Institute, Imagopole, Paris, France, 4Max PlanckInstitute for the Physics of Complex Systems, Dresden,Germany

Cytokinesis relies on tight regulation of the mechanicalproperties of the cell cortex, a thin acto-myosin network lyingunder the plasma membrane. Although most studies ofcytokinetic mechanics focus on force generation at theequatorial acto-myosin ring, a contractile cortex remains atthe poles of dividing cells throughout cytokinesis. Whetherpolar forces influence cytokinetic cell shape is poorlyunderstood. Combining cell biology and biophysics, wedemonstrate that the polar cortex makes cytokinesis inher-ently unstable and that any imbalance in contractile forcesbetween the poles compromises furrow positioning. We showthat limited asymmetric polar contractions occur during nor-mal cytokinesis, and that perturbing the polar cortex leads tocell shape oscillations and division failure. A theoretical modelbased on a competition between cortex turnover and con-traction dynamics accurately accounts for the oscillations. Wefurther propose that blebs, membrane protrusions that com-monly form at the poles of dividing cells, stabilise the positionof the cleavage furrow by acting as valves releasing corticalcontractility. Taken together, our findings show that thephysical properties of the entire cell are integrated into a fine-tuned mechanical system ensuring successful cytokinesis.

O-677Transition to collective motion in bacterial coloniesFernando Peruani1, Joern Starruss2, Vladimir Jakovljevic3,Lotte Soogard-Andersen3, Andreas Deutsch3,Markus Baer41Max Planck Institute for the Physics of Complex Systems,Dresden, Germany, 2TU-Dresden, Dresden, Germany, 3MaxPlanck Institute for Terrestrial Microbiology, Marburg,Germany, 4Physikalisch-Technische Bundesanstalt, Berlin,Germany

Collective motion of individual cells marks the onset of thetransition to multicellularity in many microorganisms. Thistransition is often mediated by intercellular communicationsignals between cells. Here, we show, in contrast, that thetransition fromsingle cell to collectivemotion in anensemble ofgliding bacterial cells can be understood as a dynamical self-assembly process of self-propelled rods. Experiments werecarried out with a mutant of the bacterium Myxococcus xan-thus moving by means of the A-motility system only andwithout undergoing reversals. The collective motion phase isconfined to a monolayer and is characterized by the organi-zation of cells into larger moving clusters. A transition tocollectivemotion is detected in experiments by imageanalysis,that reveals a qualitative change of the cluster-size distributionat a critical cell packing fraction around 17%. This transition ischaracterized by a scale-free power-law cluster size

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distribution with an exponent 0.88. We provide a theoreticalmodel for cluster formation of self-propelled rods that repro-duces the experimental findings for the cluster sizedistribution. Our findings suggest that the interplay of self-propulsion of bacteria and volume exclusion effects of the rod-shapedcell bodies is sufficient to explain the onset of collectivemotion and the related changes in the cluster statistics.

Single molecule biophysics

O-680Millisecond-piconewton force steps reveal the kineticsof DNA overstretchingPasquale Bianco1, Lorenzo Bongini2, Luca Melli1, MarioDolfi1and Vincenzo Lombardi11Laboratorio di Fisiologia, Dipartimento di BiologiaEvoluzionistica, Universita degli Studi di Firenze, Via G.Sansone 1, I-50019 Sesto Fiorentino, Italy, 2Departamentode Fisica Fundamental, Universitat de Barcelona, CarrerMarti i Franques, 1 E-08028 Barcelona, Spain

Until now the kinetics and energetics of the transition fromthe basic conformation of ds-DNA (B state) to the 1.7 timeslonger and partially unwound conformation (S state) have notbeen defined. The force-extension relation of the ds-DNA ofk-phage is measured here with unprecedented resolutionusing a dual laser optical tweezers that can impose milli-second force steps of 0.5- 2 pN via bead attached to oppositestrands of the molecule (temperature 25 �C). This approachreveals the transition kinetics of ds-DNA elongation and theirload-dependence. We show that (1) the elongation (DL) fol-lowing the force step imposed on the molecule in the regionof overstretching transition has an exponential time coursewith a rate constant (r) that has a U-shaped dependency onthe force attained by the step (F); (2) the r-F relation isunaffected by the reduction of the force step size from 2 to0.5 pN; r is related to the extent of elongation DL0 through apower equation that, once linearized by double log transfor-mation shows a slope of *0.6. These results are interpretedwith a two-state reaction model that provides structuralinformation on the transition state and an estimate of the sizeof the elementary reaction step of 25 bp. Supported by EnteCassa di Risparmio di Firenze.

P-681Single molecule microscopy: from nanodiamondsto nanomanipulationBenedikt Kraemer1, Felix Koberling1, Marcelle Koenig1,Volker Buschmann1, Michael Wahl1, Uwe Ortmann1,Olaf Schulz2, Robert Ros2 and Rainer Erdmann11PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin,Germany, info@picoquant.com, 2Arizona State University,Tempe, AZ 85287-1504, USAE-mail: robert.ros@asu.edu

Single molecule based techniques made their way fromstudies of biological dynamics and conformations, towardsDNA sequencing and ultra high resolution imaging. From thevery beginning, confocal microscopy was the workhorse due

to its versatility and straight forward multiparameter detectioncapability.Photon coincidence measurements (antibunching) started asthe ultimate proof for the existence of a single emitter butnowadays also decipher the true number of immobilisedemitters in the sub-micron observation volume. We use thistechnique to characterise a new and promising class aluminescent labels, NV defect centers in single nanodia-monds, where knowledge about the number of independentemitters in a single nanodiamond is the prerequisite tounderstand the complex fluorescence decay behaviour.The combination of atomic force microscopy (AFM) with single-molecule-sensitive confocal fluorescencemicroscopyenablesafascinating insight into the structure, dynamics and interactionsof single biomolecules and their assemblies. Sub ensemblesingle fluorophore counting becomes possible as well as theobservation of sub-diffraction imaging features. Adding an AFMtip to the confocal observation volume allows to complement theoptically acquired information with topographic imaging. Inaddition, nanophotonic effects, such as fluorescence quenchingor enhancement due to the AFM tip, are used to increasethe optical resolution beyond the diffraction limit, thus allowingto identify different fluorescence labels within e.g. a macromo-lecular complex. Silicon tip induced single molecule quenchingcould be demonstrated on individual organic fluorophores [1].

References:

[1] O. Schulz, F. Koberling, D. Walters, M. Koenig, J. Viani,R. Ros; Simultaneous single molecule atomic force andfluorescence lifetime imaging, Proc. of SPIE 7571,757109 (2010)

P-682Simulation of chain extension of folded and unfoldedDNA molecules in nanochannelsPeter Cifra, Zuzana Benkova, Tomas BlehaPolymer Institute, Slovak Academy of Sciences, 84541Bratislava, SlovakiaE-mail: bleha@savba.sk

Molecular simulations combined with the concepts of poly-mer physics expand our understanding of single moleculeexperiments. Stretching and confining in nanoscale chan-nels and capillaries is used e.g. for sorting fragments ofdsDNA of different length. We have employed Monte Carlosimulations based on a discrete worm-like chain model tocompute the chain extension R of DNA as a function ofchannel dimension D (J. Phys. Chem. B, 2009,113,1843).The chain extension profiles R(D) computed for a rectan-gular channel, tube and slit displayed three sections, inqualitative agreement with nanofluidic measurements ofDNA at high salt concentrations. DNA behavior in channelsof intermediate widths was explained by the statistics ofideal chain blobs and the scaling relation with the exponentof about x=-1. The transition to regime in narrow channelsoccurs when the diameter D is about equal to the DNApersistence length. Simulations revealed a considerableamount of folded structures of DNA molecules. Since hair-pins are much shorter than the straight forms, they tend to

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significantly reduce the equilibrium chain extension R. Theback-folding of DNA chains is manifested especially atintermediate channel widths. The individual folding-unfoldingevents can be identified in MC traces.

P-683NanoTracker: force-sensing optical tweezersfor quantitative single-molecule nanomanipulationJoost van Mameren, Helge Eggert, Gerd Behme, TorstenJaehnke, Thomas NeickeJPK Instruments AG (JPK), Bouchestrasse 12, 12435 Berlin,Germany

In the past decade, experiments involving the manipulationand observation of nanostructures with light using opticaltweezers methodology have developed from proof-of-prin-ciple experiments to an established quantitative techniquein fields ranging from (bio)physics to cell biology. Withoptical tweezers, microscopically small objects can be heldand manipulated. At the same time, the forces exerted onthe trapped objects can be accurately measured. JPK hasdeveloped a quantitative optical tweezers platform, theNanoTracker. This platform allows the controlled trappingand accurate tracking of nanoparticles, suspended either ina microfluidic multichannel flow chamber or even in atemperature-controlled open Petri dish. With its 3D detec-tion system, particle displacements in the trap can berecorded with nanometer precision. Moreover, dynamicforces acting on the particle (e.g. exerted by motor pro-teins) can be measured with more than piconewtonresolution on a sub-millisecond time-scale. Several suc-cessful biophysical applications will be demonstrated. Inparticular, we show how one of the hallmarks of single-molecule biophysics, the overstretching transition of DNA,can be studied in a versatile manner and used for protein-DNA interaction mechanics.

O-684Magnetic tweezers studies of AddAB: a molecular motorfor repairing broken DNACarolina Carrasco1, Joseph T. Yeeles2, Mark S. Dillingham2,Fernando Moreno-Herrero11Centro Nacional de Biotecnologıa, CSIC, Campus UAM,Darwin 3, 28049, Cantoblanco, Madrid, Spain, 2Dept.of Biochemistry, School of Medical Sciences, Universityof Bristol, University Walk, Bristol, BS8 1TD, UK

In Bacillus subtilis, broken DNA ends are first processed to a30-ssDNA overhang terminated at a recombination hotspot(Chi) sequence. This reaction is catalysed by the AddAB he-licase-nuclease that unwinds the DNA duplex and degradesthe nascent single-strands in a Chi-regulated manner (Yeelesand Dillingham, 2007). We have recently shown that recom-binational hotspots regulate AddAB function by preventing

reannealing of nascent single strands via formation of a DNAloop (Yeeles et al., 2011). Here, we have used MagneticTweezers to measure the real-time dynamics of AddAB at thesingle molecule level. We havemeasured AddAB translocationrate for Chi-containing and Chi-free DNA molecules whichshowed a complex appearance with the presence of constant-velocity segments of 300 bp/s at RT punctuated by stochasticpauses. Pauses duration followed a single exponential distri-bution with a decay time of 0.6 s. Interestingly, specific nickingin the AddAB translocating strand in both Chi-containing andChi-free substrates favored pausing of AddAB at this nickedposition, but did not prevent further AddAB translocation.Moreover, we observed that the presence of Chi sequencesincreases the frequency of AddAB pausing and reduces theAddAB translocation rate. The molecular basis for theseobservations is currently under evaluation.

Yeeles, J. T., and Dillingham, M. S. (2007). A dual-nucleasemechanism for DNA break processing by AddAB-type heli-case-nucleases. J Mol Biol 371, 66-78.Yeeles, J. T., van Aelst, K., Dillingham, M. S., and Moreno-Herrero, F. (2011). Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation toDNA unwinding by the AddAB helicase-nuclease. MolecularCell (In press).

O-685Direct observation of the interconversion of normaland pathogenic forms of a-synucleinNunilo Cremades1,*, Samuel I. A. Cohen1, Allen Y. Chen1,Angel Orte1,2, Massimo Sandal1, Richard W. Clarke1, PaulDunne1, Francesco A. Aprile1,3, Carlos W. Bertoncini1,4,Tuomas P. J. Knowles1, Christopher M. Dobson1,* and DavidKlenerman1,*1Department of Chemistry, University of Cambridge,Cambridge, United Kingdom, 2Department of PhysicalChemistry, Faculty of Pharmacy, University of Granada,Granada, Spain, 3Department of Biotechnologyand Bioscience, University of Milano-Bicocca, Milan, Italy,4Institute for Research in Biomedicine, Barcelona, Spain,*Corresponding authors

Despite much speculation on the existence of structurally dis-tinct oligomeric species associated with the conversion ofcertain monomeric proteins into amyloid fibrils, it has not pre-viously been possible to observe them directly or to relate themto any key mechanistic steps involved in the interconversionprocess. We have developed a novel application of single-molecule intermolecular FRET to investigate in unprecedenteddetail the aggregation and disaggregation of alpha-synuclein,the protein whose pathogenic deposition as intracellular Lewybodies is a characteristic feature of Parkinson’s disease. Ourstudy reveals that a range of oligomers of different size andstructure are formed, even at physiologically relevant con-centrations. Interestingly, the resistance to degradation of theaggregated state of alpha-synuclein, which is a well-

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established generic characteristic of amyloid fibrils, resultsfrom a specific conformational transition that we have beenable to observe directly. Moreover, we show that this transitionconverts disordered into ordered soluble oligomers and isremarkably slow, and indeed is likely to be the molecular originof the disruption to the normal clearance mechanisms of thecell that is associated with protein deposition.

O-686Single-molecule torque spectroscopy for biophysicalinvestigationsJ. Lipfert, F. Pedaci, M. Wiggin, Z. Huang,J.W.J. Kerssemakers, G.M. Skinner, S. Barland,T. Jager, M. van Oene, N.H. DekkerDepartment of Bionanoscience, Kavli Instituteof Nanoscience, Delft University of Technology, Delft,The Netherlands

The torsional properties of DNA play an important role incellular processes such as transcription, replication, andrepair. To access these properties, a number of single-mol-ecule techniques such as magnetic tweezers have beendeveloped to apply torque to DNA and coil it. I will briefly referto investigations of DNA-protein interactions using thesetechniques, and describe what has been learnt.I will then focus on the development of novel magnetic tech-niques that go beyond standard magnetic tweezers, such asthe magnetic torque tweezers1 and the freely-orbiting mag-netic tweezers2. These approaches allow one to quantifyconjugate variables such as twist and torque. For example,the magnetic torque tweezers rely on high-resolution trackingof the position and rotation angle of magnetic particles in a lowstiffness angular clamp. We demonstrate the experimentalimplementation of this technique and the resolution of theangular tracking. Subsequently, we employ this technique tomeasure the torsional stiffness C of both dsDNA moleculesand RecA heteroduplex filaments.Lastly, I will describe novel applications of the optical torquewrench3,4. The optical torque wrench is a laser trapping tech-nique developed at Cornell capable of applying and directlymeasuring torque on microscopic birefringent particles via spinmomentum transfer.Wehave focusedon the angular dynamicsof the trapped birefringent particle4, demonstrating its excit-ability in the vicinity of a critical point. This links theoptical torquewrench to non-linear dynamical systems such as neuronal andcardiovascular tissues, non-linear optics and chemical reac-tions, which all display an excitable binary (‘all-or-none’)response to input perturbations. Based on this dynamical fea-ture,wedeviseaconceptually novel sensing techniquecapableof detecting single perturbation events with high signal-to-noiseratio and continuously adjustable sensitivity.

References:

1. J. Lipfert, J.W.J. Kerssemakers, T. Jager, and N.H.Dekker, Nature Methods (2010).

2. J. Lipfert, M. Wiggin, J.W.J. Kerssemakers, F. Pedaci,and N.H. Dekker (2011).

3. A. La Porta and M. D. Wang, Phys. Rev. Lett. 92(19),190801 (2004).

4. F. Pedaci, Z. Huang, M. van Oene, and N. H. Dekker,Nature Physics (2010).

P-687Towards a single-molecule study of the AddAB helicase-nuclease with Optical TweezersBenjamin Gollnick1, Mark S. Dillingham2 and FernandoMoreno-Herrero11Department of Macromolecular Structures, Centro Nacionalde Biotecnologıa, CSIC, c/ Darwin 3, Campus deCantoblanco, 28049 Madrid, Spain, 2DNA: proteinInteractions Unit, School of Biochemistry, Medical SciencesBuilding, University of Bristol, University Walk, Bristol BS81TD, UK

Double-strandedDNAbreaksarea frequent formofnucleicaciddamage that can lead to cell death, premature ageing or cancer.In bacteria, recombinational DNA break repair is initiated by aclass of enzymes called helicase-nucleases. The prototypicalmember of this class in the model organism Bacillus subtilis isthe protein complex AddAB. It processes a double-strandedend to a 3’-terminated single-stranded DNA overhang, thuscreating a substrate for the subsequent repair steps [1, 2].To study the role of force in the complex process of DNArepair initiation, we have set up and characterized a custom-built single-beam Optical Tweezers adapted from a publisheddesign [3]. Positions of trapped microspheres are measuredby high-speed video microscopy as well as detection ofbackscattered laser light. Our novel instrumentation willenable us to investigate the real-time dynamics of repairprocesses catalyzed by the molecular motor AddAB. So far,proof-of-principle DNA force-extension measurements havebeen carried out. At present, we are endeavouring to deter-mine the stalling force of AddAB, which should be larger than4 pN, based upon observations from a complementaryMagnetic Tweezers study (unpublished data).

[1] Chedin, F., Ehrlich, S.D. and Kowalczykowski, S.C.(2000) J. Mol. Biol. 298, 7-20.

[2] Yeeles, J.T.P. and Dillingham, M.S. (2007) J. Mol. Biol.371, 66-78.

[3] Keyser, U.F., Van der Does, C., Dekker, C. and Dekker,N.H. (2006) Rev. Sci. Instrum. 77, 105105.

O-688Protein dynamics and stability: universality vs.specificityRony GranekThe Stella and Avram Goren-Goldstein Departmentof Biotechnology Engineering, the Ilse Katz Institute for Mesoand Nanoscale Science and Technology, and the ReimundStadler Minerva Center for Mesoscale MacromolecularEngineering, Ben-Gurion University of The Negev, BeerSheva 84105, Israel

Two seemingly conflicting properties of native proteins, suchas enzymes and antibodies, are known to coexist. Whileproteins need to keep their specific native fold structurethermally stable, the native fold displays the ability to performlarge amplitude motions that allow proper function. Thisconflict cannot be bridged by compact objects which arecharacterized by small amplitude vibrations and by a Debyedensity of low frequency modes. Recently, however, itbecame clear that proteins can be described as fractals;

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namely, geometrical objects that possess self similarity.Adopting the fractal point of view to proteins makes it pos-sible to describe within the same framework essentialinformation regarding topology and dynamics using threeparameters: the number of amino acids along the proteinbackbone N, the spectral dimension ds and the fractaldimension df . The fractal character implies large amplitudevibrations of the protein that could have led to unfolding. Weshowed that by selecting a thermodynamic state that is‘‘close’’ to the edge of stability against unfolding, nature hassolved the thermostability conflict. Starting off from a thermalmarginal stability criterion we reached a universal equationdescribing the relation between the spectral and fractaldimensions of a protein and the number of amino acids.Using structural data from the protein data bank (PDB) andthe Gaussian network model (GNM), we computed df and ds.for about 5,000 proteins (!) and demonstrated that theequation of state is well obeyed.Proteins have been shown to exhibit anomalous dynamics.The anomalous behavior may, in principle, stem from variousfactors affecting the energy landscape under which a proteinvibrates. We focused on the structure-dynamics interplayand showed how the fractal-like properties of proteins lead tosuch anomalous dynamics. We used diffusion, a methodsensitive to the structural features of the protein fold andthem alone, in order to probe protein structure. Conducting alarge scale study of diffusion on over 500 PDB structures wefound it to be anomalous, an indication of a fractal-likestructure. Taking advantage of known and newly derivedrelations between vibrational dynamics and diffusion, wedemonstrated the equivalence of our findings to the exis-tence of structurally originated anomalies in the vibrationaldynamics of proteins. More specifically, the time dependentvibrational mean square displacement (MSD) of an aminoacid is predicted to be subdiffusive. The thermal variance inthe instantaneous distance between amino acids is shown togrow as a power law of the equilibrium distance. The auto-correlation function in time of the instantaneous distancebetween amino acids is shown to decay anomalously. Ouranalysis offers a practical tool that may aid in the identifica-tion of amino acid pairs involved in large conformationalchanges.More recently, we studied the effect of the hydrodynamicinteraction between amino acids using a Zimm-type model.We computed the time-dependent MSD of an amino acid andthe time-dependent autocorrelation function of the distancebetween two amino acids, and showed that these dynamicquantities evolve anomalously, similar to the Rouse-typebehavior, yet with modified dynamic exponents.We also studied the dynamic structure factor S(k,t) of proteinsat large wavenumbers k, kRg[[1, with Rg the gyration radius,that are sensitive to the protein internal dynamics. We showedthat the decay of S(k,t) is dominated by the spatially averagedMSD of an amino acid. As a result, S(k,t) effectively decays asa stretched exponential. We compared our theory with recentneutron spin-echo studies of myoglobin and hemoglobin forthe Rouse and Zimm models of hydrodynamic friction.In addition, I will mention two other projects currently under-way: (i) A new elastic network model that accounts for thetensorial aspects of protein elasticity and is a combination ofstretch-compress springs and bond-bending energies. (ii) Theunfolding of a protein under the exertion of a large pulling force.

P-689A discrete number of activity states constitutean efficient mechanism to regulate enzymatic activityNikos S Hatzakis, Li Wei, Sune K Jorgensen, AndreasKunding, Per Hedegard, Dimitrios G StamouUniversity of Copenhagen, Department of neuroscienceand pharmacology, Nano-science Center, Copenhangen,Denmark

Allosteric regulation of enzymatic activity is crucial for con-trolling a multitude of fundamental cellular processes. Yet themolecular level details underlying regulation often remainpoorly understood. Here we employed single moleculeactivity studies to dissect the mechanistic origin of enzymaticactivity regulation. As a model system we employed a lipaseand measured its activity as a function of accessibility tosurface tethered liposomes (1), which are known regulatorsof its activity. Our results surprisingly revealed that the lipaseoscillates between 2 states of different activity. We accuratelyquantified for the first time both the interconversion ratesbetween activity states and the inherent activity of thesestates. Based on these we calculated the energetic land-scape of the entire reaction pathway and identified thatregulatory interactions redistributed the probability to resideon preexisting enzymatic activity states but did not alter theactivity of these states. Our findings provide the missing linkbetween conformational and activity substates supportingand represent the first direct validation of the textbookhypothesis of conformational selection for regulation ofenzymatic activity

1. N. S. Hatzakis et al., Nat. Chem. Biol., 5, 835 (2009).2. Nikos S Hatzakis, et al., submitted

P-690Proteomic identification of nucleoside diphosphatekinase as a novel cGMP-binding protein in ArabidopsisK.V. Kolesneva (Kaliasniova), D.L. Sodel, L.V. Dubovskaya,I.D. VolotovskiInstitute of Biophysics and Cell Engineering of NASof Belarus, 220072 Minsk, Academicheskaya, 27, Belarus

To identify the potential targets of cGMP in Arabidopsisplants we adopted a proteomic approach to isolate possiblecGMP-binding proteins. Purification of soluble cGMP-bindingproteins was performed using cGMP-agarose-based affinitychromatography procedure. Next eluted proteins were ana-lyzed by SDS-PAGE which revealed ten bands. We focusedthe subsequent analysis on low-molecular peptides of 15, 16and18 kDa which were bound cGMP more intensively. After2D-IEF-PAGE of the proteins isolated by cGMP-agarose-affinity chromatography eight most abundant protein spots inthe low-molecular area were visualized. These spots ofinterest were excised from the gel and in gel digested bytrypsin. Then tryptic peptides were analyzed by MALDI-TOFmass spectrometry and identified as isoforms of nucleosidediphosphate kinase (NDPK) from Arabidopsis. Thus, ourdata suggest that NDPK is a potential target of cGMP sig-naling in Arabidopsis.This research was supported by Belarusian RepublicanFoundation for Fundamental Research (grant B11M-191).

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P-691Surface enhanced Raman spectroscopy for in vivostudies of erythrocytesVenera V. Khabatova1, Nadezda A. Brazhe1, Alexey R.Brazhe1, Anna A., Semenova2, Eugeniya Y. Parshina1,Eugene A. Goodilin2,3, Olga V., Sosnovtseva4, Georgy V.Maksimov11Biology Faculty, Moscow State University, 119991,Leninskie gory, Moscow, Russia, 2Faculty of MaterialsScience, Moscow State University, 119991, Leninskie gory,Moscow, Russia, 3Chemistry Faculty, Moscow StateUniversity, 119991, Leninskie gory, Moscow, Russia,4Department of Biomedical Sciences, Faculty of HealthSciences, Copenhagen University, Copenhagen, Denmark,E-mail: venera_khabatova@mail.ru

For the first time we report a comparative approach based onsurface enhanced Raman spectroscopy (SERS) and Ramanspectroscopy to study different types of haemoglobin mole-cules in living erythrocytes. In erythrocytes there are twofractions of haemoglobin: cytosolic (Hbc) and membrane-bound (Hbm). The concentration of Hbm is less then 0,5%and therefore it is impossible to study Hbm with traditionaloptical techniques. Modifications of cellular membrane canaffect conformation of Hbm. Therefore, it can be used as asensitive marker of pathologies.Firstly, we investigated enhancement of SERS signal of Hbmdepending on Ag nanoparticles’ size. We found that theintensity of SERS spectra of Hbm and enhancement factorincrease with the decrease in Ag nanoparticles’ size. Sec-ondly, we investigated the dependence of haemoporphyrinconformation in both Hbc and Hbm ion pH values. Weobserved different sensitivity of Hbc and Hbm to the pH andfound that conformational movements of haemoporphyrin(vibrations of pyrrol rings and side radicals) in Hbm are ste-rically hampered comparably with Hbc.Our observation is an evidence of a benefit of application ofsurface enhanced Raman spectroscopy to investigate proper-ties of the Hbm in erythrocytes and provide new informationabout conformational changesand functionalpropertiesofHbm.

P-692Single-molecule FRET study of RNA free energylandscape: cooperative effects of nucleotidemodifications and cationsAndrei Yu. Kobitski1, Martin Hengesbach4a, KirstenDammertz4b, Salifu Seidu-Larry4a, Christine S. Chow4c,Arthur van Aerschot4d, Mark Helm2, G. UlrichNienhaus1,31Institute of Applied Physics and Center for FunctionalNanostructures (CFN), Karlsruhe Institute of Technology(KIT), Karlsruhe, Germany, 2Institute of Pharmacyand Biochemistry, University of Mainz, Mainz Germany,3Department of Physics, University of Illinois at Urbana-Champaign, Urbana, USA, 4aHeidelberg University,Heidelberg, Germany, 4bUniversity of Ulm, Ulm, Germany,4cWayne State University, Detroit, USA, 4dKatholiekeUniversiteit Leuven, Leuven, Belgium

RNA nanotechnology is an emerging field with high potentialfor nanomedicine applications. However, the prediction of

RNA three-dimensional nanostructure assembly is still achallenging task that requires a thorough understanding therules that govern molecular folding on a rough energy land-scape. In thiswork,wepresent a comprehensive analysis of thefree energy landscape of the human mitochondrial tRNALys,which possesses two different folded states in addition to theunfolded one. We have quantitatively analyzed the degree ofRNA tertiary structure stabilization, firstly, for different types ofcations,1 and, secondly, for several naturally-occurring nucleo-tide modifications in the structural core of the tRNALys.2,3 Thus,notable variations in the RNA binding specificity was observedfor the divalent ions of Mg2?, Ca2? and Mn2?, that can beattributed to their sizes and coordination properties to specificligands. Furthermore, we observed that the presence ofm2G10modification together with the principal stabilizing m1A9 modi-fication facilitates theRNA folding into the biologically functionalcloverleaf shape to a larger extent than the sum of individualcontributions of these modifications.

1K. Dammertz et al., Biochemistry (2011), 50, 3107-31152 A. Kobitski et al., Angew Chem Int Ed (2008), 47, 4326-43303A. Kobitski et al., Chem & Biol (2011), accepted

P-693Brownian motions of peptide/MHC complex definethe activation of T cellsHaruo Kozono1, Naoki Ogawa2, Yuko Kozono1, KentaroHoshizaki3, Hiroshi Sekiguchi3, Osami Kanagawa3,Yuji Sasaki31Research Institute for Biological Science, Tokyo Universityof Science,2669 Yamazaki, Noda, Chiba 278-0022, Japan,2Department of Physics, College of Humanitiesand Sciences, Nihon University, 3-25-40Sakurajosui,Setagaya-ku,Tokyo 156-8550, Japan,3Department of Advanced Materials Science, GraduateSchool of Frontier Sciences, The University of Tokyo, 5-1-5Kashiwanoha, Kashiwa, Chiba 277-8561, Japan

TCR should recognize conformations of peptide/MHC withrigid form. Recently, however, Unanue et al. reported thatcertain T cells (Type B) only recognize MHC with looselybound peptide. In order to elucidate the mechanism of therecognition, we used diffracted X-ray tracking method (DXT)that monitors real-time movements of individual proteins insolution at the single-molecule level. We found that peptidesmove distinctly from I-Ak, and the rotational motions of pep-tides correlate with the type B T cell activation. In the case ofdiabetogenic I-Ag7, immediately after peptide exchange, allthe peptides moves magnificently but the motion ceased in aweek, then new ordered motion appears; the rotationalmotion of peptides correlate to T cell activation, which isanalogous to the peptide in I-Ak. The rotational motion ofpeptides may create transient conformation of peptide/MHCthat recognized by a population of Tcells. DXT measurementof peptide/MHC complex well correlated to other biologicalphenomenon too. Our finding is the first observation thatfluctuations at the level of Brownian motion affect to thefunctions of proteins.

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P-694Quaternary structure of SecA in solution and boundto SecYEG probed at the single molecule levelIlja Kusters1, Geert van den Bogaart2,#, Alexej Kedrov1,Victor Krasnikov3, Faizah Fulyani1,2, Bert Poolman2

and Arnold J.M. Driessen1,*1Department of Microbiology, Groningen BiomolecularSciences and Biotechnology Institute, and Zernike Institutefor Advanced Materials, University of Groningen, Kerklaan 30,9751 NN Haren, The Netherlands, 2Departmentof Biochemistry, Groningen Biomolecular Sciencesand Biotechnology Institute, Netherlands Proteomics Centreand Zernike Institute for Advanced Materials, Universityof Groningen, Nijenborgh 4, 9747 AG Groningen, TheNetherlands, 3Department of Optical Condensed MatterPhysics, University of Groningen, Nijenborgh 4, 9747 AGGroningen, The Netherlands, *Corresponding author:Tel.?31-50-3632164; Fax.?31-50-3632154; #Current address:Max Planck Institute for Biophysical Chemistry, Departmentof Neurobiology, AmFassberg 11,Gottingen,D-37077GermanyE-mail: a.j.m.driessen@rug.nl

Dual-color fluorescence-burst analysis (DCFBA)wasapplied tomeasure thequaternary structure andhighaffinity bindingof thebacterial motor protein SecA to the protein-conducting channelSecYEG reconstituted into lipid vesicles. DCFBA is an equilib-rium technique that enables the direct observation andquantification of protein-protein interactions at the single mole-cule level. SecA binds to SecYEG as a dimer with a nucleotide-and preprotein-dependent dissociation constant. One of theSecAprotomersbindsSecYEG inasalt-resistantmanner,whilebinding of the second protomer is salt-sensitive. Since proteintranslocation is salt-sensitivewe conclude that the dimeric stateof SecA is required for protein translocation. A structural modelfor the dimeric assembly of SecA while bound to SecYEG isproposed based on the crystal structures of the Thermatogamaritima SecA-SecYEG and the Escherichia coli SecA dimer.

Highlights

• DCFBA is a flurorescence based single molecule tech-nique that allows assessment of the stoichiometry ofligands bound to membrane receptors

• Dimeric SecA binds asymmetrically to the protein-con-ducting membrane channel SecYEG

• Monomeric SecA binds SecYEG but dimeric SecA isrequired for protein translocation

• Protein translocation depends on receptor cycling of thedimeric SecA

P-695Helix specific electrostatic effects in DNA braidingand supercoilingR. Cortini1, A.A. Kornyshev1, D. J. Lee1, S. Leikin21Department of Chemistry, Imperial College London, SW72AZ, London, UK, 2Section of Physical Biochemistry, EuniceKennedy Shriver National Institute of Child Healthand Human Development, National Institutes of Health,Bethesda, Maryland 20892, USA

If the DNA charge is sufficiently neutralized by counter-ions,electrostatic interactions between helical charge patterns can

cause attraction [1]. Helix specific interactions also cause tilt,in one direction, between two DNA fragments [1]. In braidsand supercoils, this impetus to tilt breaks positive-negativesupercoil symmetry. We show that these effects may causespontaneous braiding of two molecules, lowering the DNApairing energy [2]. The pairing is more energetically favour-able for homologues (same base pair text) than fornonhomologous pairs. This might explain pairing betweenonly homologues observed in NaCl solution [3]. Also, weconstruct a simple model for a closed loop supercoil,including chiral electrostatic interactions. There are veryinteresting effects, for sufficient charge neutralization andgroove localization of counter-ions. i.) Positive super-coilsare more energetically favourable than negative ones. ii.) Atransition between loosely and tightly wound supercoils asone moves from negative to positive values of the super-coiling density. iii.) In positive super-coils the chiral interactionunderwinds DNA.

[1] A. A. Kornyshev et Al, Rev. Mod. Phys.,79, 943[2] R. Cortini et Al, submitted to Biophysical Journal[3] C. Danilowicz et Al, PNAS USA, 106, 19824

P-696Binding and conformational changes of VWFunder shearSvenja Lippok1, Hanna Engelke1,2, Joachim Radler11Center for NanoScience, Ludwig-Maximilians-Universitat,Geschwister-Scholl-Platz 1, 2University of California,Berkeley, CA 94720, USA

Von Willebrand Factor (VWF) is a large multimeric proteinthat is crucial for the force sensing cascade triggering pri-mary hemostasis. It mediates binding of activatedthrombocytes to injured epithelial tissue and serves as atransporter for coagulation factor VIII. While it was shownthat the hemostatic activity of VWF is affected by shearstress [1], the exact impact that shear forces have on theinflammatory cascade remains unclear. It is assumed thathydrodynamic forces lead to partial unfolding of VWF, whichin consequence exposes more binding sites.In order to observe shear-induced changes of the protein’sfunctionality, we measure conformational changes of VWFunder flow with fluorescence correlation spectroscopy (FCS).We aim to measure the degree of uncoiling of VWF multi-mers under various buffer conditions, e.g. in the presence ofcolloids, vesicles or platelets. As only large multimers showsignificant hemostatic activity we intend to monitor themolecular weight distribution of VWF. Shifts in this distribu-tion indicate various pathological conditions making ourmultimer analysis a fast diagnostical tool for VWF-relateddiseases. This will serve as a basis for studies of VWFbinding to collagen, FVIII, GPIb, vesicles and membrane-coated nanoparticles under shear flow.

[1] Schneider SW, Nuschele S, Wixforth A, Gorzelanny C,Alexander-Katz A, Netz RR, Schneider MF: Shear-induced unfolding triggers adhesion of von Willebrandfactor fibers. PNAS (2007) 104: 7899-7903

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P-697Nanomechanics of neural junction—stretching FNIIIdomains of human contactinsK. Mikulska, J. Strzelecki, A. Balter, W. NowakTheoretical Molecular Biophysics Group, Institute of Physics,N. Copernicus University, Torun, Poland

Data on mechanical properties of medically important pro-teins located in neural junctions are very limited. Contactins(CNTN) and paranodin proteins, located in extracellular partof Ranvier nodes, are important for proper brain wiring. Herewe study a new series of FNIII modules from human CNTN-1and -4 using a single molecule AFM force spectroscopy andadvanced, all-atom steered molecular dynamics (SMD)computer simulations. Mutations in CNTNs are responsiblefor numerous brain disorders including autism or pathologicaldevelopment of odor maps. Perhaps mechanical propertiesof individual FNIII mutated protein modules are compro-mised, thus we address this problem. A comparison of ourAFM force spectra with those of reference proteins will bepresented [1-2], and the molecular level interpretation FNIIInanomechanics, based on our SMD data will be given. Webelieve that these data should help to understand a role ofCNTN in regulation of sodium ion channels in both normaland autistic subjects.Supported in part by Polish Ministry of Education and Sci-ence, grant no. N202 262038, the Computational CenterTASK in Gdansk and license for Accelrys software.

[1] J. Strzelecki et al., Acta Phys Pol A S156, 116 (2009)[2] K. Mikulska et al., J Mol Mod, DOI: 10.1007/s00894-

011-1010-y (2011)

P-699Building and visualizing DNA-motorsusing single-molecule fluorescence spectroscopyEyal NirBen-Gurion University of the Negev, Departmentof Chemistry, Beer-Sheva, Israel

Recent achievements in rational DNA-motors engineeringdemonstrate the possibility to design nano-motors and nano-robots capable of performing externally controlled or pro-grammed tasks. A major obstacle in developing such acomplex molecular machine is the difficulty in characterizingthe intermediates, the final products and their activity. Typi-cally, non in-situ Gel and AFM and in-situ bulk fluorescencemethods are used. I will present two DNA-motors recentlydeveloped and studied using in-situ single-molecule fluo-rescence resonance energy transfer (smFRET), alternatinglaser excitation (ALEX) and total internal reflection fluores-cence spectroscopy (TIRF), and will demonstrate that thesemethods can improve the way we design, construct, measureand understand highly complex DNA-based machines.A motor made of bipedal DNA-walker, which walks on a DNAtrack embedded on a DNA-origami, capable of long walkingdistance and maintaining structural stability, will be pre-sented. The motor is non-autonomous; it receives ss-DNAfuel/anti-fuel commands from outside (as in Shin & Pierce,JACS, 2004). The motors assembly stages and single-motor’s walking steps are monitored using smFRET.

The second motor is based on published bipedal autonomousDNA-motor (Seeman, Science 2009). It is characterized bycoordinated activity between the different motor domainsleading to processive, linear and synchronized movementalong a directionally polar track. To prove that the motorindeed walks, the authors chemically froze the motor at eachstep and use a complicated radioactive Gel assay. I willdemonstrate that using single-molecule approach, we areable to directly and in-situ measure single-motor’s move-ments in few simple experimental steps, and measure itsstructural dynamics and kinetics.

P-700Translation by a single eukaryotic ribosomeAntoine Le Gall1, Nicolas Fiszman1, Nathalie Westbrook1,Karen Perronet1, Helene Chommy2, Matthieu Saguy2

and Olivier Namy21Laboratoire Charles Fabry de l’Institut d’Optique, UMR8501, CNRS, Universite Paris-Sud, 2 avenue Fresnel, 91127Palaiseau cedex, FRANCE, 2Institut de Genetique etMicrobiologie, UMR 8621, Universite Paris Sud, Bat 410,91400 Orsay, France

Using single molecule total internal reflection fluorescencemicroscopy, we observed translation of a short messengerRNA (mRNA) strand by single eukaryotic ribosomes. Theribosome-mRNAs complexes are fixed to a microscope cov-erslip through the mRNA, and mRNAs are located throughfluorescently labelled oligonucleotides hybridized to it down-stream start codon. Because of the ribosome helicase activity,thedouble strand formedby theoligonucleotideand themRNAis opened while the ribosome translates this region of themRNA. Thus, the loss of the fluorescence signal allows us tomeasure the distribution of translation speed of single ribo-somes. Careful attention was given to photobleaching for thedata analysis. This experiment opens the door to the study ofeukaryotic translation at the single molecule level.

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P-701Single molecule cut and paste for protein basedfunctional assemblyDiana A. Pippig, Stephan F. Heucke, Kamila Klamecka,Stefan K. Kufer, Philip M. Severin, Stefan W. Stahl, MathiasStrackharn and Hermann E. GaubCenter for Nanoscience & Physics Department, Ludwig-Maximilians-Universitat, Amalienstr. 54, 80799 Munich,Germany

Single-molecule cut-and-paste surface assembly (SMCP) hasformerly been employed in the controlled deposition of individ-ual fluorophores in well-defined nanometer sized patterns [1].The technique allows for the creation of patterns of arbitraryshapeandwitharbitrarynumbersofsinglemoleculesconsistingof multiple species. The accuracy has been shown to be ±10nmwith thegivenspacer andDNAsequence lengths [2]. SMCPhasbeenalsoused tobuildupaBiotinscaffold thatStreptavidin-coated nanoparticles could bind to [3].Utilizing specific molecular interactions, for example betweenDNA-binding proteins andDNA or antibodies and antigens, thistechnique is capable of providing a scaffold for the controlledself-assemblyof functional complexes.Furthermore, thisallowsfor the introduction of SMCP into protein science.We aim to employ DNA-binding Zinc-finger variants andGFP-binding nanobodies as shuttle-tags fused to the pro-teins of interest. Thus a fully expressible system that can beused for the step-wise assembly of individual building blocksto form, for example, large enzyme complexes or proteinnetworks, is provided.

[1] Kufer, S. K., Puchner, E. M., Gumpp, H., Liedl, T. &Gaub, H. E., Single-Molecule Cut-and-Paste SurfaceAssembly, Science 319, 594-596 (2008)

[2] Kufer S.K., Strackharn M, Stahl S.W., Gumpp H.,Puchner E.M. & Gaub H.E., Optically monitoring themechanical assembly of single molecules, NatureNanotechnology, Vol. 4, (2009)

[3] Puchner, E. M., Kufer, S. K., Strackharn, M., Stahl, S. W.& Gaub, H. E., Nanoparticle Self-Assembly on a DNA-Scaffold written by Single-Molecule Cut-and-Paste,Nano Letters, Vol. 8, No. 11 (2008)

P-703Force spectroscopy characterization of fibrinogen-erythrocyte binding and its conditioning by agingFilomena A. Carvalho, Sofia de Oliveira, Teresa Freitas,Sonia Goncalves, Nuno C. SantosInstituto de Medicina Molecular, Faculdade de Medicina daUniversidade de Lisboa, Lisbon, Portugal

Erythrocyte hyperaggregation, a cardiovascular risk factor,has been associated to high plasma concentrations offibrinogen. Using atomic force microscopy (AFM)-basedforce spectroscopy measurements, we have recently identi-fied the erythrocyte membrane receptor for fibrinogen, anintegrin with a a3 or a3-like subunit [1]. After this, we exten-ded the study to the influence of erythrocyte aging onfibrinogen binding [2]. Force spectroscopy measurementsshowed that upon erythrocyte aging, there is a decrease ofthe binding to fibrinogen by decreasing the frequency of itsoccurrence (from 18.6% to 4.6%) but not its force. This

observation is reinforced by zeta-potential and fluorescencespectroscopy measurements. Knowing that younger eryth-rocytes bind more to fibrinogen, we could presume that thispopulation is the main contributor to the cardiovascular dis-eases associated with increased fibrinogen blood content,which disturbs the blood flow. Our data also show that sialicacid residues on the erythrocyte membrane contribute for theinteraction with fibrinogen, possibly by facilitating the bindingto its receptor.

[1] Carvalho et al. (2010) ACS Nano, 4:4609-20.[2] Carvalho et al. (2011) PLoS ONE, 6: e18167.

P-704Structure-function investigation of a novel dendrimericand lipidated antimicrobial peptideM.A. Scorciapino1,2, J. Buerck3, M. Casu1, A. Giuliani4,G. Pirri4, A.S. Ulrich3, A.V. Vargiu1,2, A.C. Rinaldi11University of Cagliari, Monserrato (CA), Italy, 2CNR-INFMSLACS – Sardinian LAboratory for Computational materialsScience, Monserrato, Italy, 3Karlsruher Institut furTechnologie, Karlsruhe, Germany, 4R&D unit of SpiderBiotech s.r.l., Colleretto Giacosa (TO), Italy

Antimicrobial peptides are usually polycationic and amphiphilicwith high affinity for bacterial membranes. In order to charac-terize their therapeutic potential it is crucial to disclose whichproperties of the peptide/lipids are important for target selec-tivity, and to examine the peptide structure and its associationwith lipid bilayers. In this work, first experiments have beencarried out on a promising peptide called SB056, which mightrepresent the basis for developing a novel class of antibiotics.With the goal of enhancing the activity of a new semi-syntheticsequence, two identical peptides (WKKIRVRLSA) wereassembled via a lysine-linker, carrying also an octanoyl-lipidanchor. A highly active compound was obtained, but its struc-ture and mode-of-action remain unexplored. This dendrimericpeptide and its linear deca-peptide counterpart are being stud-ied in parallel to highlight the relevant properties and differencesbetween dendrimeric structure and the sequence. Monolayerintercalation is investigated with microtensiometry, Fluores-cence spectroscopy is applied to study thermodynamics andkinetics of the binding process. Circular dichroism, NMR andMDsimulations are employedwith the aim of elucidating the 3Dstructure in the membrane-bound state.

P-705Coronavirus nsp7-nsp8 complex formationHenning Seidel1, Yibei Xiao2, Rolf Hilgenfeld2, Christian G.Hubner11Institute of Physics, University of Lubeck, Ratzeburger Allee160, 23538 Lubeck, Germany, 2Institute of Biochemistry,University of Lubeck, Ratzeburger Allee 160, 23538 Lubeck,GermanyEmail: seidel@physik.uni-luebeck.de

The capability of proteins to build structures via self-organi-zation is fascinating biophysicists since decades. With the

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advent of single-molecule methods, namely fluorescencecorrelation spectroscopy (FCS) and fluorescence resonanceenergy transfer (FRET), the process of complex formation isbecoming accessible to direct observation.Coronaviruses (CoV) are enveloped positive-stranded RNAviruses. For SARS-CoV, it was shown that coronavirusesencode a RNA-dependent RNA-polymerase (RdRp) buildfrom non-structural protein 7 (nsp7) and non-structuralprotein 8 (nsp8). This hexadecameric nsp7-nsp8 complexis a hollow, cylinder-like structure assembled from eightcopies of nsp8 and held together by eight nsp7 molecules[1,2].We are aiming at understanding the assembly process andconformational changes of the complex for the relatedFeline Coronavirus. First results implicate that nsp8 aloneforms a dimer, where interchain FRET is more efficient thanintrachain FRET. For the complex the results indicate thatnsp7-nsp8 form a heterodimer which is different fromSARS-CoV.Our experiments highlight the potential of single-moleculeFRET for the study of protein complex formation.

References

[1] Imbert I. et al., The EMBO Journal 2006, Vol 25,4933-4942

[2] Zhai Y. et al., Nature Structural and Molecular Biology2005, Vol 12, No 11, 980-986

P-706Torsional motion analysis of group II chaperonin usingdiffracted X-ray trackingHiroshi Sekiguchi1,2, Ayumi Nakagawa3, Kazuki Moriya3,Masafumi Yohda3, Kouhei Ichiyanagi1,2 and Yuji C. Sasaki1,21JST Crest Sasaki-team, 2The University of Tokyo, Kashiwacity, Japan, 3Tokyo University of Agriculture and Technology,Koganei city, Japan

Diffracted X-ray Tracking (DXT) has been considered as apowerful technique for detecting subtle dynamic motion ofthe target protein at single molecular level. In DXT, thedynamics of a single protein can be monitored through tra-jectory of the laue spot from the nanocrystal which waslabeled on the objective protein.In this study, DXT was applied to the group II chaperonin, aprotein machinery that captures an unfolded protein andrefolds it to the correct conformation in an ATP dependentmanner. A mutant group II chaperonin from Thermococcusstrain KS-1 with a Cys residue at the tip of the helical pro-trusion was immobilized on the gold substrate surface andwas labeled with a gold nanocrystal. We monitored diffractedspots from the nanocrystal as dynamic motion of the chap-eronin, and found that the torsional motion of the chaperoninin the presence of ATP condition was 10 times larger thanthat in the absence of ATP condition. And UV-light triggeredDXT study using caged ATP revealed that the chaperonintwisted counterclockwisely (from the top view of chaperonin)when the chaperonin closed its chamber, and the angularvelocity from open to closed state was 10 % faster than thatfrom closed to open state.

P-707Single molecule fluorescence measurements of solubletau oligomersSarah Shammas1, Satish Kumar2, Priyanka Narayan1, EvaMandelkow2, Eckhard Mandelkow2, David Klenerman11University of Cambridge, Cambridge, UK, 2Max-Planck-Unitfor Struct. Mol. Biol., DESY, Hamburg, Germany

Peptides or proteins may convert (under some conditions)from their soluble forms into highly ordered fibrillar aggre-gates. In vivo such transitions can lead to neurodegenerativedisorders such as Alzheimer’s Disease. Alzheimer’s Diseaseis characterised by the extracellular deposition of Abetapeptide in amyloid plaques, and the intracellular formation ofneurofibrillary tangle (NFT) deposits within neurons, the lattercorrelating well with disease severity. The major constituentof NFT deposits are paired helical filaments (PHF) composedof a microtubule-associated protein known as tau. Studyingthe process by which tau forms these large aggregates maybe an essential step in understanding the molecular basis ofAlzheimer’s Disease and other Tauopathies. We haveapplied a two-colour single molecule fluorescence technique,and single molecule intermolecular FRET measurements tostudy the soluble oligomers of tau which are formed duringthe aggregation and disaggregation of PHF’s.

P-708Histone fold modifications control nucleosomeunwrapping and disassemblyMarek Simon1, Justin A. North1, John Shimko2. RobertForties1, Michelle Ferdinand2, Mridula Manohar2, MengZhang3, Richard Fishel1,3,4, Jennifer J. Ottesen2 and MichaelG. Poirier1,2,3,41The Department of Physics, 2The Departmentof Biochemistry and Ohio State Biochemistry Program, 3TheOhio State Biophysics Graduate Program, 4The Departmentof Molecular Virology, Immunology, and Medical Genetics,The Ohio State University and The Ohio State UniversityMedical Center, Columbus, USA

The nucleosome structure compacts the genome by wrap-ping 147 base pairs of DNA 1.65 times around a histoneprotein octamer, while alterations of this structure such aspartial DNA unwrapping and nucleosome disassembly reg-ulate gene expression, replication, and repair. We examinedthe regulation of these structural changes by histone post-translational modifications (PTMs) in key regions of histone-DNA interface. Precise histone PTMs were introduced intonucleosomes by preparing semi and fully synthetic histonesby expressed protein ligation and native chemical ligation.Single molecule mechanical measurements of nucleosomearrays determined that only PTMs within the nucleosomedyad region increase the nucleosome disassembly rate. Incontrast, Forster resonance energy transfer (FRET) analysisand enzyme kinetic experiments with mononucleosomesshow 2-to-3 fold increase in DNA unwrapping only for PTMslocated within the entry/exit region. Our studies suggest thatthe nucleosome regions that control DNA unwrapping andnucleosome disassembly are decoupled, where DNA histonecontacts near the dyad regulate disassembly and the DNAentry-exit region regulates DNA unwrapping.

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P-709Nanomechanical manipulation of Mason-Pfizer monkeyretroviral RNA fragment with optical tweezersMelinda Simon1, Zsolt Martonfalvi1, Pasquale Bianco1,Beata Vertessy2, Miklos Kellermayer11Dept. Biophysics and Radiation Biology, SemmelweisUniversity, Budapest, Hungary, 2Institute of Enzymology,Budapest, Hungary

Mason-Pfizer monkey virus (MPMV) is an excellent model forthe analysis of retrovirus assembly and maturation. However,neither the structure of the viral RNA, nor its modulation bycapsid-protein binding are exactly known. To explore thestructure of the MPMV genome, here we manipulated indi-vidual molecules of its packaging signal sequence withoptical tweezers.The 207-base-long segment of MPMV RNA corresponding tothe packaging signal, extended on each side with1200-base-long indifferent gene segments for use asmolecular handles, was cloned into a pET22b vector. RNAwas synthesized in an in vitro transcription system. RNA/DNA handles were obtained by hybridization in a PCR withcomplementary DNA initiated with primers labeled with eitherdigoxigenin or biotin. The complex was manipulated inrepetitive stretch and relaxation cycles across a force rangeof 0-70 pN. During stretch, transition occurred whichincreased the RNA chain length and likely corresponds tounfolding. The length gain associated with the unfoldingsteps distributed across three main peaks at *13, 20, 32nm, corresponding to *35, 57, 85 bases, respectively. Oftenreverse transitions were observed during mechanical relax-ation, indicating that refolding against force proceeds in aquasi-equilibrium process.

P-710Structural investigation of GPCR transmembranesignaling by use of nanobodiesJan Steyaert1,21Structural Biology Brussels, Vrije Universiteit Brussel,Pleinlaan 2, 1050 Brussel, Belgium, 2Departmentof Structural Biology, VIB, Pleinlaan 2, 1050 Brussel, Belgium

In 1993, scientists at the Vrije Universiteit Brussel discoveredthe occurence of bona fide antibodies devoid of light chainsin Camelidae. The small and rigid recombinant antigenbinding fragments (15kD) of these heavy chain only anti-bodies – known as VHHs or Nanobodies – proved to beunique research tools in structural biology.By rigidifying flexible regions and obscuring aggregativesurfaces, nanobody complexes warrant conformationallyuniform samples that are key to protein structure determi-nation by X-ray crystallography:

• Nanobodies bind cryptic epitopes and lock proteins inunique native conformations

• Nbs increase the stability of soluble proteins and solubi-lized membrane proteins

• Nbs reduce the conformational complexity of solubleproteins and solubilized membrane proteins

• Nbs increase the polar surface enabling the growth ofdiffracting crystals

• Nbs allow to affinity-trap active protein

I will focus my talk on the use of Nbs for the structuralinvestigation of GPCR transmembrane signaling to illustratethe power of the Nanobody platform for generating diffractingquality crystals of the most challenging targets includingGPCRs and their complexes with downstream signalingpartners.

P-713Dynamics of the Type I interferon receptor assemblyin the plasma membraneStephan Wilmes, Sara Lochte, Oliver Beutel, ChangjiangYou, Christian Paolo Richter and Jacob PiehlerUniversity of Osnabruck, Division of Biophysics,Barbarastrasse 11, 49076 Osnabruck, Germany

Type I Interferons (IFN) are key cytokines in the innateimmune response and play a critical role in host defense. AllIFNs bind to a shared cell surface receptor comprised of twosubunits, IFNAR1 and IFNAR2. Detailed structure-functionanalysis of IFNs has established that the IFN-receptor inter-action dynamics plays a critical role for signalling specificities.Here we have explored the dynamics of receptor diffusion andIFN assembly in living cells. By using highly specific orthog-onal posttranslational labelling approaches combined withTIRF-microscopy we probed the spatio-temporal dynamics ofreceptor diffusion and interaction in the plasma membrane oflive cells on the single molecule level. For this purpose, weemployed posttranslational labelling with photostable organicfluorophores. This allowed us to map diffusion and lateraldistribution of IFNAR1 and IFNAR2 with very high spatial andtemporal resolution by using single particle tracking (SPT)and single molecule localization imaging. Observed events of‘‘transient confinement’’ and co-localization with the mem-brane-proximal actin-meshwork suggest partitioning ofIFNAR1/2 in specialized microcompartments. This will beinvestigated in terms influence on receptor assembly andrecruitment of cytoplasmic effector proteins.

O-714Cytoplasmic dynein moves through uncoordinatedaction of the AAA1 ring domainsAhmet YildizDepartment of Physics, and Department of Molecularand Cell Biology, University of California, Berkeley, CA 94720USA

Cytoplasmic dynein is a homodimeric AAA? motor thatmoves processively toward the microtubule minus end. Themechanism by which the two catalytic head domains interactand move relative to each other remains unresolved. Bytracking the positions of both heads at nanometer resolution,we found that the heads remain widely separated and moveindependently along the microtubule, a mechanism differentfrom that of kinesin and myosin. The direction and size ofsteps vary as a function of interhead separation. Dyneinprocessivity is maintained with only one active head, whichdrags its inactive partner head forward. These results chal-lenge established views of motor processivity and show thatdynein is a unique motor that moves without strictly coordi-nating the mechanochemical cycles of its two heads.

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O-715Self-controlled monofunctionalization of quantum dotsand their applicaitons in studying protein-proteininteraction in live cellsChangjiang You, Stephan Wilmes, Sara Loechte, OliverBeutel, Domenik Lisse, Christian Paolo Richter,and Jacob PiehlerUniversitat Osnabruck, Fachbereich Biologie,Barbarastrasse 11, 49076 Osnabruck, Germany

Individual proteins labeled with semiconductor nanocrystals(quantum dots, QD) greatly facilitate studying protein-proteininteractions with ultrahigh spatial and temporal resolution.Multiplex single molecule tracking and imaging require mono-valent quantum dots (mvQD) capable of orthogonally labelingproteins with high yield. For this purpose, we prepared mono-valance QD-trisNTA by a chemical conjugation method. Ourresults indicated that monovalent QD-trisNTA was obtained inhigh yield by restricting the coupling by means of electrostaticrepulsion. Monovalent functionalization of the QD-trisNTA wasconfirmed by assays in vitro and in vivo. Two-color QD trackingof interferon receptors IFNAR1 and IFNAR2 based on mvQD-trisNTA were realized on live cell [1].To broaden the multiplextoolbox of mvQDs, we extended the electrostatic-repulsioninduced self-control concept for mono-functionalizing quantumdots with different affinity moieties. As a first instance, we usednegatively-charged biotin peptide to produce QD with biotinmono-functionalization. We confirmed our approach was ageneral method to rend QD monovalent by single moleculeassays based on stepwise photobleaching. These mvQDsfacilitate obtaining spatiotemporal information of IFNARs’organization in livecells. Byorthogonal labelingU5Acells stablyexpressing IFNAR2 at low level with biotin mvQD and mvQD-trisNTA-IFN, we verified colocalization and colocomotion ofindividual IFN and IFNAR2 at minute scale. Combined withsuper-resolution imaging of IFNARs’ cytosolic effecter STAT2,we observed the dynamic coming-and-going contact betweenthe microcompartments of IFNAR2 and STAT2.

Reference:

[1] You C, Wilmes S, Beutel O, Lochte S, Podoplelowa Y,Roder F, Richter C, Seine T, Schaible D, Uze G, ClarkeS, Pinaud F, Dahan M, Piehler J. Angew Chem Int EdEngl. 2010 49 (24), 4108-12;

P-716Establishing the composition of alpha-synucleinoligomers using single-molecule photobleachingNiels Zijlstra, Christian Blum, Ine Segers-Nolten, MireilleClaessens, Vinod SubramaniamNanobiophysics, MESA? Institute for Nanotechnology,University of Twente, PO Box 217, 7500 AE Enschede,The Netherlands

The neuronal protein alpha-synuclein is considered to play acritical role in the onset and progression of Parkinson’s

disease. Fibrillar aggregates of alpha-synuclein are the mainconstituents of the Lewy bodies that are found in the brains ofParkinson patients. However, there is growing evidencesuggesting that oligomeric aggregates are significantly moretoxic to cells than fibrillar aggregates. Very little is knownabout the structure and composition of these oligomericaggregates.We present results using single-molecule photobleachingapproaches to determine the number of monomeric sub-units constituting the oligomers. Our results show that theoligomers have a narrow size distribution, consisting of*13-20 monomers per oligomer. Fluorescence correlationspectroscopy data confirm the narrow size distribution andadditionally indicate a very loose packing of the oligomers.In combination with bulk fluorescence spectroscopyresults of tryptophan containing mutants of alpha-synuclein,we present a structural model for the alpha-synucleinoligomer.

Micro and nanotechnology

P-717Directly assessment of drug release dynamics from goldnanoparticlesV. Antonini1, L. Minati2, B. Rossi2,3, G. Speranza2,M. Dalla Serra11Istituto di Biofisica, Consiglio Nazionale delleRicerche,Trento, Italy, 2Fondazione Bruno Kessler TrentoItaly, 3Dip. Fisica Universita di Trento, Trento, Italy

Gold colloids are widely used for in vitro and in vivo imaging.Compared to the traditional optical tags SERS-coded nano-particles show a narrow emission bandwidth with structuredspectra typical of the molecule used, a wider excitationbandwidth, higher emission intensity, a better photo-stability,and a lower toxicity.This is why in cancer therapy, besides being consideredgood tools for the delivery of anti-tumor drugs, AuNP can bealso good optical tags for the analyses of both NP localiza-tion by laser scanning microscopy and the process of drugrelease inside the cells by Raman.In our work we used 10 nm diameter AuNP loaded withRhodamine 6G, a molecule with a high Raman and fluores-cence efficiency, and with a chemical structure similar toDoxorubicin, the antitumoral drug used in our system. Thedata showed that AuNP are internalized by cells and SERScan be performed.10 nm and 60 nm diameter AuNP loaded with Doxorubicinwere incubated at different time points with A549 cell line(human adenocarcinomic alveolar basal epithelial cells).Only 60 nm AuNP showed intense Raman emission typicalof the doxorubicin phonon transitions.AcknowledgmentProvincia Autonoma di Trento for supporting this research(project Nanosmart).

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P-720Effect of non-modified and Fenton-modifiednanodiamond powder on human endothelial cellsK. Solarska1, A. Gajewska1, J. Skolimowski2, K. Mitura3,A. Boudier4, P. Leroy4, G. Bartosz11Department of Molecular Biophysics, University of Lodz,Pomorska 141/143, 90-237 Lodz, 2Department of OrganicChemistry, University of Lodz, Tamka 12, 91-403 Lodz,Poland, 3Koszalin University of Technology, Racławicka15/17, 75-620 Koszalin, Poland, 4EA 3452 Ciblestherapeutiques, formulation et expertise preclinique dumedicament, Faculty of Pharmacy, Nancy-University, BP80403, 54001 Nancy Cedex, France

In recent years biomedical applications of diamond nanoparti-cles have become of significant interest, which rises questionsof their biocompatibility and mechanisms of interactions withcells. The aim of this study was to compare the effect of non-modified diamond nanoparticles (DNPs) andDNPsmodifiedbythe Fenton reaction on human endothelial cells.DNPs (\10 nm particle size, SIGMA) were modified by theFenton reaction introducing surface –OH groups. Immortal-ized human endothelial cells (HUVEC-ST) were incubatedwith 2-100 lg/ml DNPs in the OPTIMEM medium.Diamond nanoparticles modified by the Fenton reaction hadsmaller hydrodynamic diameter estimated by dynamic lightscattering and the surface potential (zeta potential) mea-sured using laser-Doppler electrophoresis. They were morecytotoxic as evaluated by the MTT reduction assay.DNPs augmented generation of reactive oxygen species inthe cells, estimated by oxidation of 2’,7’-dichlorofluorescin,the effect being higher for the Fenton-modified DNPs after48-h incubation. Cellular production of nitric oxide, estimatedwith DAF-FM, was also affected by DNPs; after 72h, Fenton-modified OH, in contrast to non-modified diamond,decreased NO production.Diamond nanoparticles affected also the cellular level ofglutathione and activities of main antioxidant enzymes(superoxide dismutase, catalase, glutathione peroxidase,glutathione reductase and glutathione S-transferase).

P-721Time-resolved FTIR difference spectroscopyof bacteriorhodopsin under vibrational controlC. Bauer1,3*, M. Gensch2 and J. Heberle31Institute of Radiation Physics, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany, 2 Institute of RadiationPhysics/Institute of Ion Beam Physics and MaterialsResearch, Helmholtz-Zentrum Dresden-Rossendorf,Dresden, Germany, 3 Experimental Molecular Biophysics,Department of Physics, Freie Universitaet Berlin, GermanyE-Mail: c.bauer@hzdr.de

We aim at investigating how photoreactions of proteins canbe controlled by means of intense THz radiation tuned inresonance to specific vibrational modes, much in analogy tocoherent control experiments conducted by fs NIR laserpulses [1]. For this we will combine a time-resolved IR dif-ference spectroscopic setup with uniquely intense, tunablenarrow bandwidth THz radiation (3 – 280 lm) at the ps

beamline of the THz free electron laser FELBE. Theseexperiments will be performed on bacteriorhodopsin (bR)which is the sole protein of the purple membrane of thearchaebacterium Halobacterium salinarum [2]. Upon illumi-nation, the chromophore retinal isomerizes around theC13-C14 double bond [3] and bR pumps a proton from thecytoplasmic to the extracellular side. This proton gradient isused by the bacterium to drive photosynthetic ATP produc-tion under low oxygen tension [4]. In our experiment, thephotoreaction is initiated by a visible laser pulse as in stan-dard experiments, but then the sample will be irradiated by aTHz pulse from the free electron laser tuned into resonancewith low-energy vibrational modes which is supposed toinfluence the photoreaction [1]. Such vibrational control willbe monitored by time-resolved FTIR spectroscopy using thestep-scan technique [5].

References

[1] Prokhorenko V.I., Nagy A.M., Waschuk S.A., BrownL.S., Birge R.R., Miller R.J.D., (2006), Science 313,1257-1261

[2] Oesterhelt D., Stoeckenius W., (1973), Proc. Natl. Acad.Sci. U S A 70, 2853-2857

[3] Stoeckenius W., Lozier R.H., Niederberger W., (1977),Biophys. Struct. Mech. 3, 65-68

[4] Lozier R.H., Bogomolni R.A., (1975), Biophys. J. 15,4462-4466

[5] Radu I., Schleeger M., Bolwien C., Heberle J. (2009),Photochem. Photobiol. Sci. 8, 1517-1528

P-722Structural stability of surface-adsorbed liposomesTamas Bozo, Ricardo H. J. Pires, Nora Kaszas, Pal Grof,Miklos S. Z. KellermayerSemmelweis University, Department of Biophysicsand Radiation Biology, Budapest, Hungary

Liposomes are increasingly studied as nanoscale drugdelivery systems and biomembrane models. However theexact structure dynamics and mechanical behavior of lipo-somes is little known. Atomic force microscopy (AFM) is apowerful tool to characterize nanoscale morphology andenables the mechanical manipulation of submicron-sizedvesicles. A drawback of AFM, however, is that liposomesmay flatten and rupture on substrates to form patches orsupported planar bilayers (SPB).Our aim was to obtain better understanding of factors affectingliposomes on substrates and find experimental conditions atwhich liposomes preserve their structural integrity.In the presence of divalent cations DPPC liposomes formedSPB on mica. Vesicles sedimented subsequently preservedtheir integrity and showed stronger attachment to SPB. Inaddition to cross-bridging lipid head groups, divalent cationsinfluence the surface charge of liposomes, thereby modu-lating liposome-substrate and liposome-liposome interfacialinteractions. Preserved vesicles stabilized by divalent cationsmay provide a unique experimental system for studyingmembrane-protein interactions.

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P-724Micro-viscosimeter generated and manipulated by lightAndras Buzas1, Laszlo Oroszi1, Lorand Kelemen1,Pal Ormos1, Antonio Sasso21Biological Research Centre of the Hungarian Academyof Sciences, Institute of Biophysics, Temesvari krt. 62,Szeged H-6726, Hungary, 2Universita di NapoliFederico II – Dipartimento di Scienze Fisiche,Complesso Universitario Monte S. Angelo,Via Cinthia, I-80126 Napoli, Italy

A micron sized viscometer was fabricated using the Couettetype geometry that is capable of measuring the complexviscosity of fluids. The viscometer was produced by twophoton polymerization of SU8 photopolymer using a femto-second laser system, a high NA objective and a piezotranslator stage. The viscometer was manipulated by holo-graphic optical tweezers and operated in the 0.005-1 Hzfrequency range. Video analysis algorithm was used toevaluate our measurements. We tested the viscometer withwater-glycerol solutions.

P-725Shaping substrates at the nanoscale for single cellcharacterizationStefania Corvaglia1, Luca Ianeselli2, Daniela Cesselli4,Antonio P. Beltrami4, Giacinto Scoles234, Denis Scaini1,Loredana Casalis31University of Trieste, P. Europa 1, 34127 Trieste, Italy,2SISSA, Scuola Internazionale Superiore di Studi Avanzati,via Bonomea, 265, 34136 Trieste, Italy, 3ELETTRA,Sincrotrone Trieste, S.S.14 Km 163.5, 34012 Basovizza,Trieste, Italy, 4Centro Interdipartimentale MedicinaRigenerativa, University of Udine, 33100 Udine, Italy

One of the main reasons for lack of reliability in proteinanalysis for disease diagnostics or monitoring is a lack of testsensitivity. This is because, for many tests, to be reliable,they need to be performed on a homogeneous, and thereforevery small, sample. Current in-vitro techniques fail in accu-rately identifying small differences in protein content, functionand interactions starting from samples constituted of few oreven single cells. A nanotechnology approach may over-come the current limits in low abundance protein detection.We aim at designing a microwell device for the trapping (innative environment) and the parallel characterization of rarecells (e.g. adult stem cells). Such versatile device, based onsoft and nanolithography, will promote cell adhesion andviability on differently functionalized bio-compatible materi-als, allowing for the morphological characterization of thecells, at a single cell level. In parallel, by facing our microwelldevice with a protein nanoarray, produced via atomic forcemicroscopy nanolithography, we can run proteomic studies ata single/few cells level.Moreover, we could foresee the possibility to deliver differentstimuli to each cell, correlating the changes in chemistry/morphology with the protein profile at a single cell level.

P-726Mechanical characterisation of hollow siliconmicroprobes fabricated by Deep Reactive Ion EtchingZ. Fekete1,2, A. Pongracz1, G. Marton1, P. Furjes11Research Institute for Technical Physics and MaterialsScience - MFA, H-1525 Budapest P.O. Box 49, Hungary,2Budapest University of Technology and Economics,Budafoki str. 8., H-1111 Budapest, Hungary, Correspondingauthor: Zoltan Fekete, H-1525 Budapest P.O. Box 49,HungaryE-mail: fekete@mfa.kfki.hu

Summary: Hollow in-plane microprobes were fabricated insilicon by deep reactive ion etching. Sealed microchannels inthe probeshaft were formed by a modified Buried ChannelTechnology (BCT). Besides the improvement in the fabrica-tion process, the influence of channel design on themechanical stability of the probes was also characterized bythe Stress-Strain Module of COMSOL Multiphysics simula-tion code. An in-vivo test was carried out in order to revealthe robustness of the device.

O-727SSM-based electrophysiology: transport mechanismand pH-regulation of the Na1/H1 antiporter NhaAfrom E. coliThomas Mager1, Abraham Rimon2, Etana Padan2,Klaus Fendler11Max-Planck-Institut fur Biophysik, 60438 Frankfurt/Main,Germany, 2Institute of Life Sciences, Hebrew Universityof Jerusalem, 91904 Jerusalem, Israel

Using an electrophysiological assay the activity of NhaA wastested in a wide pH range from pH 5.0 to 9.5. Forward andreverse transport directions were investigated at zero mem-brane potential using preparations with inside out and rightside out oriented transporters with Na? or H? gradients asthe driving force. Under symmetrical pH conditions with aNa? gradient for activation, both the wt and the pH-shiftedG338S variant exhibit highly symmetrical transport activitywith bell shaped pH dependencies, but the optimal pH wasshifted 1.8 pH units to the acidic range in the variant. In bothstrains the pH dependence was associated with a systematicincrease of the Km for Na? at acidic pH. Under symmetricalNa? concentration with a pH gradient for NhaA activation anunexpected novel characteristic of the antiporter wasrevealed; rather than being down regulated it remainedactive even at pH as low as 5.These data allowed to advance a transport mechanismbased on competing Na? and H? binding to a commontransport site and to develop a kinetic model quantitativelyexplaining the experimental results. In support of theseresults both alkaline pH and Na? induce the conformationalchange of NhaA associated with NhaA cation translocationas demonstrated here by trypsin digestion. Furthermore,Na? translocation was found to be associated with thedisplacement of a negative charge. In conclusion, theelectrophysiological assay allowed to reveal the mecha-nism of NhaA antiport and sheds new light on the conceptof NhaA pH regulation.

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O-728Swimming motility of bacteria near solid surfacesPeter Galajda, Orsolya HajaInstitute of Biophysics, Biological Reserach Centerof the Hungarian Academy of Sciences, Hungary

Swimming motility is widespread among bacteria. However,in confined or structured habitats bacteria often come incontact with solid surfaces which has an effect on theswimming characteristics.We used microfabrication technology to quantitatively studythe interaction of swimming cells with solid boundaries. Wetracked bacteria near surfaces with various engineeredtopologies, including flat and curved shapes. We were ableto study several surface related phenomena such as hydro-dynamic trapping and correlated motion.We think that our results may help to understand howphysical effects play a role in surface related biological pro-cesses involving bacteria such as biofilm formation.

P-729Cell labeling efficiency of oppositely chargedmagnetic iron oxide nanoparticles—a comparativestudyRaimo Hartmann1, Christoph Schweiger2, Feng Zhang1,Wolfgang. J. Parak1, Thomas Kissel2,#, Pilar Rivera_Gil1,#1Biophotonics, Institute of Physics, Philipps Universityof Marburg, 2Pharmaceutical Technology, Instituteof Pharmacy, Philipps University of MarburgE-mail: kissel@staff.uni-marburg.de;pilar.riveragil@physik.uni-marburg.de

The interaction of nanomaterials with cells is a key factor whenconsidering their translocation into clinical applications. Espe-cially an effective accumulation of nanoparticles inside certaintissues is beneficial for a great number of applications. Pre-dominantly size, shape and surface charge of nanoparticlesinfluence their cellular internalization and distribution. To inves-tigate this, twoseriesofmaghemite (c-Fe2O3)nanoparticlesweresynthesized either via aqueous coprecipitation or via thermaldecomposition of organometallic precursor molecules. Size andthe spherical shape of both nanoparticle types were kept con-stant whereas the chargewas changedbymodifying the surfaceof the nanoparticles with polymers of opposite charge, in detailpoly(ethylene imine) (PEI) and apolymaleic anhydridederivative(PMA). The positively and negatively charged c-Fe2O3 nano-particles were characterized with respect to size, zeta potential,colloidal stability and magnetic properties. Furthermore, theuptake rate and localization of both formulations into A549 car-cinoma cells after fluorescent labeling of the carriers as well asthe resulting alteration in MR-relaxation times were evaluated.

O-730Surface-Enhanced InfraRed Absorption Spectroscopy(SEIRAS) of membrane protein monolayersKenichi Ataka, Ionela Radu, Melanie Nack, HenningKrassen, Joachim HeberleFreie Universitaet, Department of Physics, Arnimallee 14,14195 Berlin, Germany

Membrane proteins are the target of more than 50% of alldrugs and are encoded by about 30% of the human

genome. Electrophysiological techniques, like patch-clamp,unravelled many functional aspects of membrane proteinsbut usually suffer from poor structural sensitivity. We havedeveloped Surface Enhanced Infrared Difference Absorp-tion Spectroscopy (SEIDAS)1,2 to probe potential-inducedstructural changes of a protein on the level of a monolayer.A novel concept is introduced to incorporate membraneproteins into solid supported lipid bilayers in an orientatedmanner via the affinity of the His-tag to the Ni-NTA termi-nated gold surface3. Full functionality of surface-tetheredcytochrome c oxidase is demonstrated by cyclic voltam-metry after binding of the natural electron donorcytochrome c. General applicability of the methodologicalapproach is shown by tethering photosystem II to the goldsurface4. In conjunction with hydrogenase, the basis is settowards a biomimetic system for H2-production. Recently,we succeeded to record IR difference spectra of a mono-layer of sensory rhodopsin II under voltage-clampconditions5. This approach opens an avenue towardsmechanistic studies of voltage-gated ion channels withunprecedented structural and temporal sensitivity. Initialvibrational studies on the novel light-gated channelrho-dopsin-2 will be presented6.

1. Ataka, K., & Heberle, J. (2003), J. Am. Chem. Soc. 125,4986-4987

2. Ataka, K., & Heberle, J. (2007), Anal. Bioanal. Chem.388, 47–54

3. Ataka, K., Giess, F., Knoll, W., Naumann, R., Haber-Pohlmeier, S., Richter, B., & Heberle, J. (2004), J. Am.Chem. Soc. 126, 16199-16206

4. Badura, A., Esper, B., Ataka, K., Grunwald, C., Kuhl-mann, J., Heberle, J., & Rogner, M. (2006), Photochem.Photobiol. 82, 1385–1390

5. Jiang, X., Zaitseva, E., Schmidt, M., Siebert,F., Engel-hard, M., Schlesinger, R., Ataka, K., Vogel, R., &Heberle, J. (2008), Proc. Natl. Acad. Sci. USA 105,12113-12117

6. Radu, I., Bamann, C., Nack,M., Nagel, G., Bamberg, E., &Heberle, J. (2009), J. Am. Chem. Soc. 131, 7313-7319

P-731Probing biomass-chromatographic bead interactionsby AFM force spectroscopyGesa Helms, Marcelo Fernandez-Lahore, Rami ReddyVennapusa, and Jurgen FritzSchool of Engineering and Science, Jacobs UniversityBremen, 28759 Bremen, GermanyE-mail: g.helms@jacobs-university.de

In expanded bed adsorption (EBA), bioproducts are purifiedfrom an unclarified fermentation broth by their adsorption onchromatographic beads in a fluidized bed. The unspecificdeposition of biomass onto the adsorbent matrix canseverely affect the process performance, leading to a poorsystem hydrodynamics which then decreases the success ofthis unit operation.To quantify the bead-biomass interactions different chro-matographic beads are attached to AFM cantilevers, andforce spectroscopy experiments are performed with thesecolloidal probes on model surfaces and cells in solution. Theexperiments are conducted under varying conditions to study

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the influence of e.g. pH and ionic strength on various chro-matographic bead - biomass combinations. To analyze theforce curves, possible elastic contributions, e.g. fromdeforming cellular membranes, have to be decoupled fromthe interaction forces. Then, bead-biomass interactions willbe modeled using (extended) DLVO theory and resultingdata can also be compared to real-life EBA processes. Theproject aims for a better understanding of the interactionforces in chromatography and might help to improve theprocess quality of EBA.

O-732Multifunctional magnetic nanoparticlesfor cell imagingDaniel Horak1,4, Michal Babic1,4, Pavla Jendelova2,4,Vıt Herynek3,41Institute of Macromolecular Chemistry AS CR, Prague,Czech Republic, 2Institute of Experimental Medicine, AS CR,Prague, Czech Republic, 3Institute for Clinicaland Experimental Medicine, Prague, Czech Republic,4Center for Cell Therapy and Tissue Repair, CharlesUniversity, Prague, Czech Republic

Long-term non-invasive in vivo monitoring of the survival,migration, homing and fate of transplanted cells is of keyimportance for the success of cell therapy and regenerativemedicine. Tools for in vivomagnetic resonance (MR) imaging oflabeled cells are therefore being developed.We have preparedsuperparamagnetic iron oxide nanoparticles by the coprecipi-tation of Fe(II) and Fe(III) salts and oxidation. To stabilize theparticles and to facilitate their internalization by the cells, thenanoparticles were coated with several novel low- and high-molecular weight compounds including D-mannose, poly(L-lysin), poly(N,N-dimethylacrylamide) and dopamine-hyaluro-nate conjugate. The surface-modified magnetic nanoparticleswere thoroughly characterized by a range of physico-chemicalmethods, which proved the presence of the coating on theparticles. The particles were then investigated in stem cellexperiments in terms of real time cell proliferation analysis,viability, labeling efficiency and differentiation. The iron oxideconcentration of the labeled cells was assessed using MR re-laxometry. The advantages/disadvantages of particular ironoxide coatings will be discussed and the optimal coating sug-gested. Excellent contrast was achieved by labeling the cellswith dopamine-hyaluronate-coated nanoparticles.Support of the AS CR (No. KAN401220801) is acknowledged.

P-733Selective bio-functionalization of electrodes inside microand nano fluidic channelsLuca Ianeselli1, Loredana Casalis2, Morena Silvestrini3,Gianluca Grenci4, Massimo Tormen4, Giacinto Scoles21SISSA, via Bonomea 265, 34136 Trieste, ITALY,2Sincrotrone Trieste S.C.p.A., Strada Statale 14 - km 163,5,34149 Basovizza, Trieste, ITALY, 3Universita di Venezia,Dorsoduro2137 -30121Venezia, ITALY, 4LaboratorioNazionaleTASC, Basovizza S.S. 14 Km 163.5, 34149 Trieste, Italy

In the last decades the interest towards the fabrication ofinnovative bio-sensors with improved sensitivity and reli-ability for medical-diagnostics applications has been

constantly risen. Among the different techniques, microfluidicsystems are playing a major role. In order to detect extremelylow concentrations of biomolecules (pM and fM), attentionshould be placed on the controlled, selective functionaliza-tion of micro- and nano-channels.In this work we propose a new approach to functionalize goldpatches inside fluidic channels. We start from self-assembledmonolayers (SAMs) of thiolated molecules on a gold electrodedeposited inside the channel. Then, by using an electrochem-ical approach [1,2] we remove molecules from the SAM atselected locations, by applying a negative voltage to the elec-trode.Thenewlyexposedgold surfacecanbe re-functionalizedby using a thiolated biomolecule (i.e an antibody) capable tobind specific proteins flowing inside the channel. The cycle canbe applied to other electrodes in the microfluidic system, cre-ating a multiplexing device which, as we will show, candifferentially measure ionic current flows in different channels.

[1] Widrig et al.; J. Electroanal. Chem., 310 (1991)[2] Ghaly et al.; Langmuir, 26(3) (2010)

P-734Microtools made by two-photon polymerizationfor optical tweezers systems used in biologicalmanipulation experimentsLorand Kelemen, Badri A. L. Rao, Andras Buzas, Pal OrmosBiological Research Centre of the Hungarian Academyof Sciences, Institute of Biophysics, Szeged, Hungary

Optically actuated micromanipulation and micro-probing ofbiological samples are increasingly important methods in thetoday’s laboratory. Microbeads as probes are the most com-monly used tools in this field, although the only manipulativemotion they allow is translation. We present polymerized 3Dmicrostructures which can also be used for optical microma-nipulation with more degree of freedom than microbeads.The two-photon polymerization (TPP), based on focused fslaser beam into appropriate photopolymers is a powerfulmethod to build structures of arbitrary complexity with sub-micrometer resolution. The presented tools have theadvantage of being capable of twisting and rotationalmanipulative motion, and also that the position of biologicalmanipulation and optical trapping is spatially separated. Dif-ferent manipulative interfaces, the positioning stability andsurface activation of the manipulators will be discussed.

P-735Sequential multiplexed quantum dot labeling to profilepathway-associated protein expression of advancedhuman prostate cancerHaiyen E. Zhau1, Peizhen Hu1, Ruoxiang Wang1, YuzhuoWang1 and Leland W. K. Chung11Uro-Oncology Research Program, Department of Medicine,Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center; Los Angeles, CA, USA; 2BC CancerAgency, University of British Columbia, 3Molecular PathologyLab of the Vancouver Prostate Center at Vancouver GeneralHospital, Vancouver, B.C., Canada

The potential application of multiplexed quantum dot label-ing, MQDL, in clinical detection, prognosis and monitoring

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therapeutic response has attracted high interests from bio-engineers, pathologists and cancer biologists. MQDL issuperior comparing with conventional organic dye staining inits narrow emission bandwidths, wide signal dynamic ranges,high detection sensitivity, and low noise to signal ratios.However, the majority of the MQDL application has beenlimited to identification of specific cell type or cancer subtypeand the improvement in labeling methodology. In this study,we focused on simultaneous detection and analysis of 5proteins in the c-METactivation pathway, i.e. RANKL, VEGF,NRPLN-1, p-c-MET and Mcl-1, which are known to beassociated with human prostate cancer progression andmetastasis. Two experimental systems were analyzed: 1)fixed xenograft tissues from an established LTL313 castra-tion resistance human prostate cancer or CRPC model; and2) clinical prostate tissue specimens from localized cancerand bone metastasis. In the presentation we will report ourexperience in 1) the MQDL protocol optimization for thesequential reactions of individual primary antibody, the bio-tinylated secondary antibody and streptavidin-coated QDconjugate with nuclear DAPI staining; and 2) the multiplexedimage catching, image unmixing, and subsequent per cellbase quantification. For future multi-specimen analyses andvalidation, we will introduce a high throughput Vectra ImageAnalysis System.

P-736Self assembled lipid nanostructures in aligned carbonnanotubesCatharina Paukner1, Krzysztof Koziol1 and ChandrashekharV. Kulkarni21Department of Materials Science and Metallurgy, Universityof Cambridge, UK, 2Department of Chemistry, Universityof Graz, Heinrichstrasse 28, Austria

Carbon nanotubes (CNTs)1 are already quite popularamong many scientific and technological disciplines2. Inrecent years they have been targeted for biotechnologicaland medical applications. In this work we have investigatedthe nanostructural self assemblies of biological lipid mole-cules in presence of CNTs. Advantage of using highlyaligned CNTs3 for this purpose being the possibility ofstudying the interactions of lipid molecules on the macro-molecular surface as well as in the confinement of alignedCNTs. We have observed various lyotropic nanostructuresthat are found for corresponding lipids in the bulk under dryand hydrated conditions.4 Nanostructural studies weremainly performed using Small and wide angle X-ray scat-tering techniques. This work is crucial for designing thenano-micro-fluidic architectures and supported modelmembranes where both – functionalization of CNTs andnanostructural assembling of lipids, could be employedsimultaneously.

1. Ijima, S. Nature 1991: p. 354: 562. Endo, M., et al. Philos Transact A Math Phys Eng Sci,

2004. 362(1823): p. 2223-38.3. Ducati, C., et al. Journal of Physics: Conference Series,

2006. 26(1): p. 199.4. Kulkarni, C.V., et al. Physical Chemistry Chemical

Physics (PCCP), 2011 Advance Article.

P-738Neural signal recordings with a novel multisite siliconprobeGergely Marton1, Anita Pongracz1, Laszlo Grand2,3, EvaVazsonyi1, Istvan Ulbert2,3, Gyorgy Karmos2,3, ZoltanFekete1, Gabor Battistig11Research Institute for Technical Physics and MaterialsScience, Hungarian Academy of Sciences, H-1525, P.O. Box49, Budapest, Hungary, 2 Peter Pazmany Catholic University,Faculty of Information Technology, H-1083, 50/A Prater St.,Budapest, Hungary, 3Institute for Psychologyof the Hungarian Academy of Sciences, H-1068, 83-85Szondi St., Budapest, Hungary

Uncovering physiological processes at the cellular level isessential in order to study complex brain mechanisms. Usingmultisite signal recording techniques in the extracellularspace, functional connectivity between different brain areascan be revealed. A novel microfabrication process flow, basedon the combination of wet chemical etching methods wasdeveloped, which yields highly reproducible andmechanicallyrobust silicon-based multielectrode devices. The fabricatedshaft of the probe is 280 lm wide, 80 lm thick, has roundededges and ends in a yacht-bow like, sharp tip. Its unique shapeprovides decreased invasivity. The sensor contains 24 plati-num recording sites at precisely defined locations. Murine invivo experiments showed that the probes could easily pene-trate the meninges. High quality signals, providing local fieldpotential, multi- and single unit activities, were recorded. Theinterfaces between the tissue and the platinum contacts werefurther improved by electrochemical etching and carbonnanotube coating of the metal sites.

P-739Protein-based integrated optical sensor deviceAnna Mathesz1, Sandor Valkai1, Istvan Ando2, Elmar K.Wolff3, Pal Ormos1 and Andras Der11Institute of Biophysics, Biological Research Centreof the Hungarian Academy of Sciences, P.O. Box 521, 6701Szeged, Hungary, 2Institute of Genetics, Biological ResearchCentre of the Hungarian Academy of Sciences, P.O. Box521, 6701 Szeged, Hungary, 3Institute for AppliedBiotechnology and System Analysis at the Universityof Witten/Herdecke, Herrhausenstrasse 44, 58455 Witten,Germany

The integrated optical Mach-Zehnder interferometer is ahighly sensitive device, considered a powerful lab-on-a-chiptool for specific detection of various chemical and biochemicalreactions. Despite its advantages, there is no commerciallyavailable biosensor based on this technique. The main rea-son is the inherent instability of the device due to slightchanges of environmental parameters. In this paper we offer asolution to this problem that enables the optimal adjustment ofthe working point of the sensor prior to the measurement. Thekey feature is a control unit made of a thin film of the light-sensitive chromoprotein bacteriorhodopsin deposited on thereference arm of the interferometer. After showing the transfercharacteristics of such a device, we demonstrate its appli-cability to sensing of specific protein–protein interactions. Weexpect our method to become a rapid and cost-efficient

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alternative of the commonly used measuring tools in proteinresearch and medical diagnostics.

P-740Nanobody-tagged polyplexes for transcriptionaltargeting of a lethal transgene and cancer cell killingD. Ahmadvand1, F. Rahbarizadeh2, E. Sadeqzadeh2,L. Parhamifar1, S.M. Moghimi11Center of Pharmaceutical Nanotechnologyand Nanotoxicology, University of Copenhagen, DK-2100Copenhagen Ø, Denmark, 2Department of MedicalBiotechnology, Faculty of Medical Sciences, Tarbiat ModaresUniversity, Tehran, Iran

MUC1 antigen is an aberrantly glycosylated glycoproteinover-expressed in tumours of epithelial origin. We have iso-lated an anti-DF3/Mucin1 (MUC1) single domain antibody(nanobody) and covalently linked this to the distal end ofpoly(ethylene glycol)3500 (PEG3500) in PEG3500-25 kDapolyethylenimine (PEI) conjugates. The resultant conjugates(average of 16 PEG chains and 8.7 nanobody per PEI)successfully condensed plasmids coding a transcriptionallytruncated-Bid (tBid) lethal transgene (under the control of thecancer-specific MUC1 promoter) into polyplexes of 130 nm insize and with zeta potential of close to neutrality. Polyplexesproved effective in dramatically elevating Bid/tBid expressionin both MUC1 over-expressing caspase 3-deficient andcaspase 3-positive tumour cell lines and induced consider-able cell death. Transgene expression and concomitant celldeath was exclusively promoter-specific. Our attempt notonly provide a powerful proof of concept in combiningnanobody-based targeting with transcriptional targeting as asafe way to deliver transgenes to specific cells, but alsoovercomes the known PEI-mediated cellular toxicity andminimize (or eliminate) non-targeted cell damage.

P-741Structure-function relationships underlying anticanceractivity of novel 4-thiazolidonesPanchuk R.R.1, Chumak V.V. 1,2, Fil’ M. R. 2,Havrylyuk D.Ya.3, Lesyk R.B.3, Stoika R.S.1,21Institute of Cell Biology, NAS of Ukraine, Drahomanov Str14/16, Lviv, Ukraine,2Ivan Franko Lviv National University,University Str 1, Lviv, Ukraine, 3Danylo Halytsky LvivNational Medical University, Pekarska Str. 69, Lviv, Ukraine

Thazolidone derivatives are novel synthetic compoundspossessing various biological activities. We selected threesuch compounds Les-3120, 3166, 3372 which passedNational Cancer Institute in vitro tests. Annexin V/PI andDAPI staining, DNA electrophoresis in agarose gel, andWestern-blot analysis using specific antibodies against 30cellular proteins involved in apoptosis were applied to studymolecular mechanisms of tumor cell death induced by thesecompounds. It was found that molecular targets of thiazoli-dones in target cells strongly depend on structure of theirside groups: Les-3120 containing isatine fragment, activatedcaspase-8 involved in receptor-mediated apoptosis, whileLes-3166 possessing benzthiazol residue, induced mito-chondrial apoptosis mediated by caspase-9, and Les-3372which has a unique chlorine atom in side chain, also led to

mitochondrial apoptosis mediated by AIF (apoptosis-induc-ing factor). To increase anticancer potential of thesemolecules, in silico study was performed and most activegroups of Les-3120 and Les-3372 were combined into onemolecule. In vitro studies showed that such hybrid moleculecalled Les-3661 possessed 10 times higher anticancerpotential (IC50=1 lM) comparing with initial compounds.

P-742Porous vaterite particles as drug delivery system:synthesis, encapsulation, and controlled releaseB. Parakhonskiy1,2, A. Haase1,F. Piccoli3, I. Caola3,F. Tessarolo1, T. Bukreeva2, R. Antolini11BIOtech - Interdipartimental Center for BiomedicalTechnologies, University of Trento,Trento, Italy, 2A.V.Shubnikov Institute of Crystallography Russian Academyof Science, Moscow, Russia, 3Section of electronmicroscopy, Department of Laboratory Medicine, APSS,Trento, Italy

We report on the synthesis and characterization of vateritemicrocontainers for controlled drug release. Moreover, wepresent experiments on possible release strategies ofencapsulated substances via recrystallization, pH controlled,or by desorption methods.Vaterite spherical particles were fabricated with controllableaverage sizes from 400±12nm till 10±1hm. We consideredtwo ways of functionalization of the containers: encapsulationof the substances during the vaterite synthesis or theiradsorption onto the prepared particles.As model experiments, vaterite containers, encapsulatingRhodamine 6G, were imaged by two-photon microscopy,showing dye release into the aqueous medium due torecrystallization to calcite within 3 days. Differently, in ethanolonly small amounts of the encapsulated markers were dif-fusion released after one week.The release mechanisms can be further controlled by cov-ering the microcontainers with additional polymer layers toincrease diffusion and recrystallization time. A change of thepH from neutral to acid conditions leads to the destruction ofthe vaterite matrix followed by a quick release of theencapsulated materials. These flexible control mechanismsmake this system an interesting candidate for pharmaceuti-cal applications.

P-743Label free biosensing using Grating CoupledInterferometryD. Patko1,2, A. Hamori2, K. Cottier3, S. Kurunczi2,R. Horvath2

Department of Nanotechnology, University of Pannonia,Egyetem u. 10., H-8200 Veszprem, Hungary, 2ResearchInstitute for Technical Physics and Material Science MFAHungarian Academy of Sciences, H-1120 Konkoly-Thege ut29-33, Budapest, Hungary, 3Creoptix GmbHEinsiedlerstrasse 25 CH-8820 Waedenswil, Switzerland

Grating Coupled Interferometry (GCI) is a label-free surfacesensitive sensor introduced by us recently. It is an ideal

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candidate for measuring extremely small concentrations ofanalytes in minute amount of samples.Thesystem isbasedonevanescentwavesensingandemploysmonomode thin film optical waveguides and a liquid crystalmodulator for phase interrogation. In thepresent contributionweshow some recent developments on the sensor in terms ofstability, temperature control and liquid handling.

P-744Analysis of magnetophoretic mobility of magneticnanoparticles in magnetic fieldMojca Pavlin1,2, Ana Dermota1, Andrej Znidarsic1, BostjanVladimir Bregar11Nanotesla Institute Ljubljana, Ljubljana, Slovenia,2University of Ljubljana, Faculty of Electrical Engineering,Ljubljana, Slovenia

Magnetic nanoparticles (NP) in combination with thera-peutic molecules represent one of most promising methodsfor targeted drug delivery. One of major current limitationsof magnetic drug targeting is to achieve efficient concen-tration of magnetic carrier-drug complexes at the targetedsites due to poor mobility of nanoparticles in tissue struc-tures. Interstitial delivery is hindered by microscopicextracellular matrix, which represents a major barrier fornanoparticles motilities. In order to achieve efficient mag-netic drug targeting it is crucial to know particle mobility ina given in vivo environment as well as to apply magneticfield having appropriate field gradient which drags mag-netic NPs.We used gel magnetophoresis in order to measure motilitiesof different magnetic NPs (Co-ferrite, c-Fe2O3) in agarose gel.Numerical modeling using FEMmethodwas used to determineappropriate settings of magnets, which generate sufficientmagnetic field gradient. Further, we used the numerical mod-eling to evaluate the magnetic force on the NPs for differentgeometries. We obtained that one of crucial factors whichdetermines final mobility in tissue is formation of larger aggre-gates of nanoparticles under physiological conditions andinteraction of nanoparticles with surrounding matrix.

O-745Defining the forces required to gate mechanosensitivechannels in mammalian sensory neuronsKate Poole and Gary LewinDepartment of Neuroscience, Max Delbrueck Centerfor Molecular Medicine, Robert-Roessle Str 10, 13125,Berlin-Buch, Germany

Our sense of touch and mechanical pain is based on mec-hano-electrical transduction (MET) at the terminal endings ofsubsets of dorsal root ganglion (DRG) neurons innervatingthe skin. To quantify the stimulus strengths required to gatemechanosensitive channels in these subsets of neurons, wedeveloped an approach using microstructured surfaces. TheDRG neurons are grown on laminin-coated PDMS pillararrays, mechanical stimuli are applied by deflecting individualpili and the deflection is monitored using light microscopy. As

the pili behave as light-guides, the center of each pilus canbe determined from a fit of the intensity values, allowingdetection of movements of a few nanometers. The responseto such stimuli is monitored using whole-cell patch-clamp. Pilideflections of 10nm can gate the rapidly adapting-current inmechanoreceptor cells, while deflections above 150nm arerequired for gating of slowly adapting-currents in nociceptors.Smaller stimuli are required to generate currents via pilideflection (10nm) vs neurite indentation (70-100nm), sug-gesting that gating occurs at the cell-substrate interface. Wehave also characterized the MET currents present in N2acells which we show are modulated by the substrate to whichthe cells are attached.

P-746Enhanced stimulation of Toll-like receptor 9via immunostimulatory nanoparticlesJan Rother, Anna Pietuch, Andreas JanshoffGeorg-August University Gottingen, Institute of PhysicalChemistry, Tammanstr. 6, D-37077 Gottingen, GermanyE-mail: jrother@gwdg.de

Among the Toll-like receptor family (TLRs), the TLR9 has beensubject of intensive research because of its predominantlocalization in the lysosomes of immune cells and its ligandrendering it a potential candidate for immunotherapy of auto-immune diseases and cancer. Additionally, a use as anadjuvant in vaccination is aimed using synthetic CpG-oligo-deoxynucleotides (CpG-ODN’s). Albeit, immunostimulatoryCpG-ODNs already showed promising results in animalexperiments and clinical trials, several groups found that TLR9is also expressed by tumor cells. First experiments show thatactivation of TLR9 displayed by cancerous cells leads to adecreased apoptosis rate and proliferation posing unpredict-able threat to tumor patients exposed to CpG-ODNs.Therefore, detailed knowledge about the impact of CpG-ODNson cancer cells is inevitable for a save use in pharmaceutics.Herein, we describe a sophisticated way to address TLR9 incancer cells using CpG-ODN functionalized ‘‘superpara-magnetic’’ MnO- and c-Fe2O3-nanoparticles (NPs) tostimulate TLR9 in A549 cells. Analysis of impedimetricmeasurements revealed a cytotoxic effect of the MnO-NPs.Cells treated with immunostimulatory Fe2O3-NPs showed anincreased micromotility as well as a higher long-term correl-eation of the impedance signal.

P-747Silver sol preparation history and effectiveness of SERSdiagnostics of intact red blood cellsAnna A. Semenova,*1 Venera V. Khabatova,3 Eugene A.Goodilin,1,2 Nadezda A. Brazhe,3 George V. Maksimov,3 YuriiD. Tretyakov1,21Faculty of Materials Science, Moscow State University,119991 Moscow, Russia, 2Chemistry Faculty, Moscow StateUniversity, 119991 Moscow, Russian Federation, 3BiologyFaculty, Moscow State University, 119991 Moscow, RussiaE-mail: semenova@fmlab.ru

Surface enhanced Raman spectroscopy (SERS) techniquehas a number of advantages related to promising

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biomedical diagnostics like high-sensitivity, single-moleculestudy, easy sample preparation. Furthermore, SERS allowsto conduct non-invasive studies of conformations of themolecules without destruction of living cells, i.e. in vivo [1].This work presents a SERS study of cytosolic hemoglobin(Hbc) using silver nanoparticles (AgNPs). The Hbc wasisolated from cytoplasm of red blood cells taken from raterythrocytes and diluted. AgNPs were prepared by devel-oping Leopold and Lendl method [2]. Three types of colloidswere prepared at various temperatures (25, 40 and 60 �C).The resulted AgNPs were characterized by UV-Vis-, FTIR-spectroscopy, DLS and TEM. Reduction of Ag ions leads tothe formation of predominantly spherical AgNPs but alsosilver nanorods, faceted and aggregated AgNPs in smallquantities with a surface plasmon resonance band in therange of 413 – 445 nm. For AgNPs synthesized at 25�C, forexample, a bimodal size distribution was observed (about 7and 45 nm medium sizes, respectively). SERS measure-ments were optimized for each type of AgNPs. It wasdemonstrated that AgNPs gave strong Raman enhance-ment from Hbc and types of SERS spectra differ from eachother.

[1] N. A. Brazhe, S. Abdali, A. R. Brazhe, O. G. Luneva, N.Y. Bryzgalova, E. Y. Parshina, O. V. Sosnovtseva, G.V. Maksimov, Biophysical Journal, 2009, 97,3206–3214.

[2] N. Leopold, B. Lendl, J. Phys. Chem. B., 2003, 107,5723–5727.

P-748Conjugation of zinc oxide nanoparticleswith cytochrome CMichaela Simsıkova1, Marian Antalık1,21Department of Biochemistry, Faculty of Science, P.J. SafarikUniversity, Kosice, Slovak Republic, 2Instituteof Experimental Physics, SAS, Kosice, Slovak Republic

Nano-ZnO is characterized by unique properties, low tox-icity and high biocompatibility instead of a lot of othersnanomaterials. For this fact nanoparticles of ZnO have greatpotential for applications in biosystems, for example biola-beling, biosensoring, delivery systems and others, whichcan be used in genetics, pathology, criminology, safety offood and many other industries. For these bioapplicationsare necessary surface modifications, which can made to thenanostructures to better suit their integration with biologicalsystems, leading to such interesting properties as enhancedaqueous solubility, bio-recognition or applicability for bio-logical systems.For synthesis of ZnO nanoparticles in aqueous solution weused 11-mercapto-undecanoic acid (MUA) as stabilizingagent. The coating of nanoparticles with MUA could allowtheir solubility in the water and the binding through carboxylgroups present in its structure. We defined the optimal pH forMUA modificated nano-ZnO solubility and their ability inter-action with positive charges.We studied the optical properties of pure and surface mod-ificated nanoparticles and their conjugates with cytochrome cand also the effect of pH on the interaction between nano-MUA and horse cytochrome c.

This work was financially supported by the research grantsVEGA No. 0038 and by Slovak Academy of Science in frameof CEX NANOFLUID.

P-749Permeation through nanochannels: revealing fastkineticsPratik Raj Singh, Mahendran Kozhinjampara, Niraj Modi,Ulrich Kleinekathoefer, Mathias WinterhalterJacobs University Bremen, Campus Ring 1, D-28759Bremen, Germany

The permeation of water soluble molecules across cellmembranes is controlled by channel forming proteins andparticularly the channel surface determines the selectivity. Anadequate method to study properties of these channels iselectrophysiology and in particular analyzing the ion currentfluctuation in the presence of permeating solutes providesinformation on possible interactions with the channel surface.As the binding of antibiotic molecules in the channels ofinterest is significantly weaker than that of preferentially dif-fusing nutrients in substrate-specific pores, the resolution ofconductance measurements has to be significantly increasedto be able to resolve the events in all cases. Due to thelimited time resolution, fast permeation events are not visible.Here we demonstrate that miniaturization of the lipid bilayer;varying the temperature or changing the solvent mayenhance the resolution. Although electrophysiology is con-sidered as a single molecule technique, it does not provideatomic resolution. Molecular details of solute permeation canbe revealed by combining electrophysiology and all atomcomputer modeling.

[1] Chimerel C et al., Eur. Biophys. J. 38, 121 (2008)[2] Mahendran KR et al., Eur. Biophys J. 38, 1141 (2009)[3] Hajjar E et al., Biophys J. 98, 569 (2010)[4] Mahendran KR et al., J. Biomol. Screen. 15, 302 (2010)[5] Mahendran KR et al, J. Phys: Condens. Matter 22,

(2010)

P-750Bio-applications of novel functionalized nanosizedcarriersR. Stoika1*, A. Zaichenko2*, N. Boyko1, N. Mitina2,Ye. Filyak1, R. Bilyy1, Yu. Kit1, R. Panchuk1, O. Shevchuk2,T. Skorokhoda2, N. Skorokhyd11Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine, 2LvivNational Polytechnic University, Lviv, Ukraine*E-mail: stoika@cellbiol.lviv.ua; zaichenk@polynet.lviv.ua

Novel functionalized nanocomposites (NC) were designedand synthesized on the basis of polymeric surface-active ol-igoelectrolytes. The developed technology permitscontrolling: 1) quality and quantity of structural blocks of NC,and size unimodality; 2) branching at specific sites in polymerchain of NC; 3) providing NC with reactive chemical groups;

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4) covalent conjugation of specific bio-targeting molecules.Provided bioactive elements were: a) specific anticancerdrugs, antibiotics, alkaloids; b) DNA and siRNA; c) immu-noglobulins and lectins; d) lipids and amino acids; e)polyethylene glycol. Fluorescent, luminescent, super-para-magnetic, or X-ray detectable compounds were alsoincorporated in NC to make them detectable and measur-able. Biocompatible NC possessing low toxicity towardsmammalian cells in vitro and in vivo (mice) were created.They were effective in delivery of: 1) drugs (doxorubicineand antibiotics) for chemotherapy in vitro and in vivo; 2)DNA for transfection of mammalian, yeast and bacterialcells; 3) protein antigens for animal immunization andspecific lectins for targeting apoptotic cells. These andother approaches in application of developed NC andnanobiotechnologies are considered.This work was supported by STCU grants #1930, #4140,#4953.

P-751Fluorescent nanodiamonds as cargo for siRNA deliveryto Ewing sarcoma cellsA. AlHaddad1, M.-P. Adam2, J. Botsoa2, G. Dantelle3, S.Perruchas3, Th.Gacoin3, C.Mansuy4, S. Lavielle4, C. Malvy1,F. Treussart2, J.-R. Bertrand11CNRS UMR8203, Universite Paris Sud 11, Institut GustaveRoussy, Villejuif, France, 2LPQM, CNRS UMR8537, ENSCachan, Cachan, France, 3Laboratoire PMC, CNRSUMR7643, Ecole Polytechnique, Palaiseau, France,4Laboratoire des Biomolecules, CNRS UMR7203, ENS,Universite Pierre et Marie Curie, Paris, France

In the anti-cancer drug delivery domain, nanotechnologiesare a promising tool, providing a good tissue distributionand a low toxicity. Drug delivery vehicles relying on solidnanoparticles have been proposed, among which diamondnanoparticle (size\20 nm) is a very promising candidate[1].We have investigated the delivery of siRNA by nanodia-monds (ND) into cells in culture, in the context of thetreatment of a rare child bone cancer (Ewing sarcoma), bysuch a gene therapy. siRNA was bound to NDs after NDscoating by cationic polymers, so that the interaction is strongenough to pass the cell membrane without loss of the drugand does not prevent its subsequent release.The cellular studies showed a specific inhibition of the geneexpression at the mRNA and protein level by the ND vec-torized siRNA. We also uses the fluorescence of color centercreated in the nanodiamonds [2] to monitor the release offluorescently-labeled siRNA in the intracellular medium. Thistechnique brings a quantitative insight in the efficiency ofsiRNA to stop cell proliferation.Considering the success of the cell model we recently startedthe drug delivery in tumor xenografted on nude mice.

References

[1] E.K. Chow et al., Sci Transl Med 3, 73ra21 (2011)[2] M. Baker, Nature Methods 7, 957–961(2010)

O-752Multiscale pattern fabrication for life-scienceapplicationsFrancesco Valle1, Beatrice Chelli1, Michele Bianchi1, EvaBystrenova1, Marianna Barbalinardo3, Arian Shehu1, TobiasCramer1, Mauro Murgia1, Giulia Foschi1, Pierpaolo Greco2,Massimiliano Cavallini1, Fabio Biscarini11ISMN-CNR, via Gobetti 101, I-40129 Bologna, Italy, 2ScribaNanotecnologie, Bologna, Italy, 3Nano4bio s.r.l.Viale G.Fanin 48, 40127 Bologna, ItalyE-mail: f.valle@bo.ismn.cnr.it

The combination of unconventional fabrication technologyand biomaterials allows both to realize state-of-the-art devi-ces with highly controlled lateral features and performancesand to study the main properties of the biomolecules them-selves by operating at a scale level comparable with the onecrucial for their activity. Soft lithography and microfluidicdevices offer a tool-box both to study biomolecules underhighly confined environments [1] and to fabricate in an easyway topographic features with locally controlled mechanicaland chemical surface properties, thus leading to a finercontrol of the interplay of mechanics and chemistry. I willpresent an application of this technology to the control of cellfate that is becoming a key issue in regenerative medicine inthe perspective of generating novel artificial tissues.Patterns of Extracellular Matrix (ECM) proteins have beenfabricated, by a modified Lithographically Controlled Wetting(LCW), on the highly antifouling surface of Teflon-AF to guidethe adhesion, growth and differentiation of neural cells(SHSY5Y, 1321N1, NE-4C) achieving an extremely accurateguidance [2]. Local surface topography is also known toinfluence the cell fate [3], thus, integrating this parameter inthe substrate fabrication could increase the complexity of thesignals supplied to the cells. In this perspective we havedeveloped a novel fabrication technique, named Litho-graphically controlled Etching (LCE), allowing, in one step, toengrave and to functionalize the substrate surface over dif-ferent lengthscales and with different functionalities.I will conclude showing how we have been developing ultra-thin film organic field effect transistors (OFETs) as label-freebiological transducers and sensors of biological systems.OFETs are low dimensional devices where ordered conju-gated molecules act as charge transport material.Unconventional patterning techniques and microfluidics havebeen adapted to proteins and nucleic acids to dose themolecules on the OFET channel with a high control of theconcentration. In another set of experiments, we have alsobeen addressing the signalling from neural cells and net-works grown on pentacene ultra-thin film transistosr [3,4].

References:

[1] E. Bystrenova, M. Facchini, M. Cavallini, M. G. Cacace,F. Biscarini, Angew Chem Int Ed Engl (2006), 45, 4779.

[2] F. Valle, B. Chelli, A. Bianchi, P. Greco, E. Bystrenova, I.Tonazzini, F. Biscarini, Advanced BioMaterials (2010),12: 6, B185-B191

[3] I. Tonazzini, E. Bystrenova, B. Chelli, P. Greco, P.Stoliar, A. Calo, A. N. Lazar, F. Borgatti, P. D’Angelo, C.

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Martini, F. Biscarini, Biophys. J. (2010), 2010 Jun16;98(12):2804-12.

[4] Bystrenova, B., Jelitai M., I. Tonazzini, A. N. Lazar, HuthM., Stoliar P., Dionigi C., Cacace MG., Nickel B., Madar-asz E., F. Biscarini, Adv. Func. Mat. (2008), 18(12):1751-56

P-753Alterations of immune response of lymphocytesexposed to PLGA and TiO2 nanoparticles in vitroMiroslava Kuricova1, Jana Tulinska1, Aurelia Liskova1,Eva Neubauerova1, Maria Dusinska2,1, Ladislava Wsolova,Dagmar Bilanicova3, Giulio Pojana3, Katarina Volkovova11Slovak Medical University, Bratislava, Slovakia, 2NorwegianInstitute for Air Research, Kjeller, Norway, 3University Ca’Foscari of Venice, Venice, ItalyEmail: miroslava.kuricova@szu.sk

Advances in nanotechnology are beginning to exert a sig-nificant impact in medicine. Increasing use of nanomaterialsin treatment of diseases has raised concerns about theirpotential risks to human health. In our study, the effect ofpoly(lactic-co-glycolic acid) (PLGA) and titanium dioxide(TiO2) nanoparticles (NPs) on function of B- and T- lympho-cytes was investigated in vitro.Human blood cultures were treated with PLGA and TiO2 NPsin concentrations: 0.12; 3 and 75 lg/cm2 for 72h. Lympho-cyte transformation assay was used to assess the effect ofNPs on lymphocyte function. Lymphocytes were stimulatedwith mitogens: concanavalin A, phytohaemmagglutinin(T-cell response) and pokeweed mitogen (B-cell response).Our findings indicate immunomodulatory effect of PLGANPs. Proliferative response of T- and B-lymphocytesexposed in vitro to the highest dose of PLGA for 72h wassuppressed significantly (p\0.01, p\0.05). On the otherhand, we observed stimulative effect of exposure to middledose of PLGA NPs on B-lymphocyte proliferation (p\0.05).No alteration was found in lymphocyte proliferation treated invitro with TiO2 NPs for 72h.In conclusion, proliferation of lymphocytes in vitro might beone of the relevant tests for evaluation of NPs immunotoxicity.AcknowledgementsSupported by EC FP7 [Health-2007-1.3-4], Contract: 201335and ITMS project: 26240120033. We thank Helena Nagyova,Edita Mrvikova, Marta Postrkova for their excellent technicalhelp.

P-754Using amino functionalized ORMOSIL nanoparticlesas a non-viral DNA vectorZsolt Wagner1, Gabor Racz2, Erzsebet Bori2, Tamas Tabi1,Eva Szok}o11Semmelweis University, Department of Pharmacodynamics,Budapest, 2Semmelweis University, Department of OralBiology, Budapest, Hungary

Silica nanoparticles are stable aqueous suspension ofcondensed siloxane nanocomposites, having an average

diameter between 10 and 100 nm. Particles containingorganic functional groups on their surface are calledOrganically Modified Silica Nanoparticles (ORMOSIL).Due to the various chemical and physical properties ofthe surface groups, ORMOSIL nanoparticles may have anenormous variety of biological applications, such as invivo bioimaging, non-viral gene delivery or targeted drugdelivery. Our aim was to synthesize both void and fluo-rescent dye doped amino functionalized ORMOSILnanoparticles through the microemulsion method and usethem for gene delivery. The obtained nanoparticles havebeen characterized by transmission electron microscopyand dynamic light scattering. Furthermore, the nanoparti-cles have been investigated to exploit their transfectionefficiency and the possible toxicity caused by surfactantsused in the synthesis. The transfection efficiency wastested on various cell cultures. Our further aim is the invivo transfection of salivary glands using ORMOSILnanoparticles. Our work has shown that the nanomedicineapproach, with nanoparticles acting as a DNA-deliverytool is a promising direction for targeted gene therapy.

Bioengineering & biotechnology

P-755In vivo amperoetric cells for detection of fast diffusing,physiologically important small moleculesLıvia Nagy, Bernadett Bareith, Tunde Angyal, Erika Pinter,Geza NagyUniversity of Pecs, Pecs, Hungary

H2S is a naturally occurring gas that is toxic in high con-centration. It exists also in different tissues of living animalssometimes in concentrations as high as 20 lM. It is generallyaccepted, that H2S has important roles in modulating differ-ent, physiologically important biochemical processessimilarly to other, fast diffusing molecules like NO, CO andH2O2.For investigation of the physiological effects of thesespecies their local concentration in the studied biologicalmedia is important to know. This means methods neededfor measuring the instantaneous concentration with highspatial resolution in living tissues without major invasion.Electrometric micro, and ultramicro sensors are often gainapplication in experimental life sciences for measurement oflocal ion concentration or following neurotransmitter speciesin vivo measurements.In our work efforts are being carried out to improve theapplicability of selective electrometric sensors in life scienceexperiments. As a result of these work an improved H2Smeasuring cell and improved electrode and method wasdeveloped for measurement of electroactive small moleculeslike NO or H2O2.In the poster to be presented the structure, the workingprinciples and the performances of the different sensorsmentioned will be described.

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O-756Engineered bacteriorhodopsin: a molecular scaleconductance photoswitchOlivia Berthoumieu1, Amol Patil2, Wang Xi2, LubicaAslimovska1, Jason Davis2, Anthony Watts1.1Biomembrane Structure Unit, Department of Biochemistry,University of Oxford, South Parks Road, Oxford OX1 3QU,UK, 2Department of Chemistry, Physical & TheoreticalChemistry Laboratory, University of Oxford, South ParksRoad, Oxford OX1 3QZ, UK

Bacteriorhodopsin (BR) is the only protein in the purplemembrane of the halophilic organism Halobacterium salina-rium. It is a light-driven proton pump converting light into atransmembrane proton gradient through isomerization of thecovently bound retinal chromophore. Its stability, as well asits photoactivity in dried films, has made BR an attractivematerial for biomolecular devices. Such studies, however,have used BR within the membrane, on relatively large sur-faces. Here, conducting-probe atomic force microscopy (C-AFM) analysis was performed after isolating the protein fromits native membrane environment while keeping its basictrimeric structure, and demonstrated that the molecularconductance of BR can be reversibly photoswitched withpredictable wavelength sensitivity. Intimate and robust cou-pling to gold electrodes was achieved by using a strategicallyengineered cysteine mutant located on the intracellular sideof the protein which, combined with a 75% delipidation,generated protein trimers homogenously orientated on thesurface. C-AFM proximal probe analysis showed a repro-ducible 3 fold drop of BR mean resistance over *5 cycles ofinterspersed illuminations at the same gold-bR-gold junctionwhen k[495 nm, while no shift was observed with otherwavelengths.

P-757Capture of circulating tumor cells with a highly efficientnanostructured silicon substrates with integratedchaotic micromixersShutao Wang1, Kan Liu1, Jian Liu2, Zeta T.-F. Yu1, XiaowenXu, Libo Zhao1, Tom Lee1, Eun Kyung Lee1, Jean Reiss1, Yi-Kuen Lee1, Leland W. K. Chung2, Jiaoti Huang1, MatthewRettig1, David Seligson1,Kumaran N. Duraiswamy1, CliftonK.-F. Shen1, Hsian-Rong Tseng11University of California at Los Angeles, 2Cedars-SinaiMedical Center, Los Angeles, California, USA

A new-generation technology for cancer diagnosis, led byH.R. Tseng at UCLA and L.W.K. Chung at Cedars-SinaiMedical Center, was developed for improved harvesting ofcirculating tumor cells from the blood of patients with prostatecancer. This technology has the capability to enrich circu-lating tumor cells (CTCs) with high efficiency by integratingan antibody-coated Silicon nanopillar (SiNP) substrate withan overlaid polydimethylsiloxane (PDMS) microfluidic chaoticmixer (Angew. Chem., doi:10.1002/ange.2010005853,2011). This core technology shows significantly improved

sensitivity in detecting rare CTCs from whole blood, thusprovides an alternative for monitoring cancer progression. Byassembling a capture-agent-coated nanostructured sub-strate with a microfluidic chaotic mixer, this integratedmicrochip can be applied to isolate CTCs from whole bloodwith superb efficiency. Ultimately, the application of thisapproach will open up opportunities for early detection ofcancer metastasis and for isolation of rare populations ofcells that cannot feasibly be done using existing technolo-gies. This technology helped to find a needle in a haystackand will open up the opportunity for single cell genomic andepigenetic sequencing and gene expression profiling.Results from further development of this technologywill assist the physicians in follow-up patients and testingvigorously the concept of personalized oncology with indi-vidualized therapy. This novel technology has recently beenreviewed and highlighted by Nature Medicine (17:266: March2011).

O-758Membrane biohybrid systems for tissue and organengineeringLoredana De BartoloInstitute on Membrane Technology, National ResearchCouncil of Italy, ITM-CNR, c/o University of Calabria,via P. Bucci cubo 17/C, I-87030 Rende (CS) Italy,E-mails: l.debartolo@itm.cnr.it; loredana.debartolo@cnr.it

The growing crisis in organ transplantation and the agingpopulation have driven a search for new and alternativetherapies by using advanced bioengineering methods. Theformation of organized and functional tissues is a verycomplex task: the cellular environment requires suitablephysiological conditions that, presently, can be achieved andmaintained by using properly-designed bioreactors repro-ducing all specific functions and bioactive factors that assureviability/regeneration of cells cultured in an appropriatescaffold. The creation of biomimetic environment requires theuse of biomaterials such as membranes with specific phys-ico-chemical, morphological and transport properties on thebasis of the targeted tissue or organ. Tailor-made mem-branes (organic, functionalized with specific biomolecules, inhollow-fiber configuration), designed and operated accordingto well-defined engineering criteria are able to sustain spe-cific biotransformations, to provide adequate transport ofoxygen, nutrients and catabolites throughout the cellularcompartment, and to supply appropriate biomechanicalstimuli of the developing tissue. In this talk the author willshow the development of membrane engineered constructsfocusing on liver and neuronal systems. The role of mem-brane surface and transport properties in providinginstructive signals to the cells for the guiding of proliferationand differentiation will be discussed. Membrane bioreactors,which through the fluid dynamics modulation may simulatethe in vivo complex physiological environment ensuring anadequate mass transfer of nutrients and metabolites and themolecular and mechanical regulatory signals, will bepresented.

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P-759An original microfabricated cell culture substratefor cell-based assaysFanny Evenou1, Benoıt Ladoux1,2, Jean-Marc Di Meglio1,Pascal Hersen1,21Laboratoire Matiere et Systemes Complexes (MSC), UMR7057 CNRS-Universite Paris-Diderot, Paris, France,2Mechanobiology Institute (MBI), National Universityof Singapore (NUS), Singapore

Here we present a novel but simple system for cell-basedassays enabling simultaneous testing of multiple sampleson a same tissue without cross-contamination betweenneighbouring assays, as well as sequenced or repeatedassays at the same tissue location. The principle of thismethod lies in the spatially-controlled diffusion of testcompounds through a porous matrix to the target cells. Asimple microfabrication technology was used to defineareas where diffusion processes are allowed or inhibited.We performed proof-of-principle experiments on Madin-Darby canine kidney (MDCK) epithelial cells using Hoe-chst nuclear staining and calcein-AM cell viability assay.Fluorescent staining superimposed properly on membranepattern with a dose-dependent response, indicating thatboth compounds specifically and selectively diffused tothe target cells. MDCK cells similarly treated with cyto-chalasin B showed their actin network rapidly altered, thusdemonstrating the suitability of this system for drugscreening applications. Such a well-less cell-basedscreening system enabling multiple compounds testing ona same tissue and requiring very small volumes of testsamples appears interesting for studying potential com-bined effects of different biochemicals applied separatelyor sequentially.

P-760Subpicosecond all-optical switching by the proteinbacteriorhodopsinLaszlo Fabian1, Zsuzsanna Heiner1, Mark Mero2, MiklosKiss3, Elmar K. Wolff4, Pal Ormos1, Karoly Osvay3

and Andras Der11Institute of Biophysics, Biological Research Centreof the Hungarian Academy of Sciences, Szeged, Hungary,2Hungarian Academy of Sciences, Research Group of LaserPhysics, Szeged, Hungary, 3Department of Opticsand Quantum Electronics, University of Szeged, Szeged,Hungary, 4Institute for Applied Biotechnology and SystemAnalysis at the University of Witten/Herdecke, Witten,Germany

It is generally believed that all-optical data processing is themost promising direction to achieve serious improvementsboth in capacity and speed of Internet data traffic. One of thebottlenecks of the state-of-the-art photonic integration tech-nology is to find the proper nonlinear optical (NLO) materialsthat are supposed to serve as cladding media in waveguide-based integrated optical circuits performing light-controlledactive functions. Recently, the unique chromoprotein bacte-riorhodopsin (bR) has been proposed to be used as anactive, programmable NLO material in all-optical integratedcircuits.

In integrated optical applications of bR, its light-inducedrefractive index change is utilized. In this paper we exploit therefractive index changes of a dried bR film accompanying theultrafast transitions to intermediates I and K, which allows evensub-ps switching, leading beyond Tbit/s communication rate. Inthe experiments direct pulses of a femtosecond laser system at800 nm were used along with synchronized ultrafast laserpulses at 530 nm. We believe that the results may be the basisfor the future realization of a protein-based integrated opticaldevice, and represent the first steps to a conceptual paradigmchange in optical communication technologies.

P-761SEREX-derived antigens for detection of breast cancerserum autoantibody profileR.G. Kiyamova1, O.I. Kostianets1, 2, M.A. Shyyan1,2,V.V. Filonenko11Institute of Molecular Biology and Genetics NAS Ukraine,Kyiv, Ukraine, 2Taras Shevchenko National Universityof Kyiv, Kyiv, Ukraine

Molecular alterations following malignant transformation ofcells stimulate systemic immune response in human body. Thisresponse can be avaluated by detection of circulated autoan-tibodies directed to corresponding tumor-associated antigens.Last years, such autoantibodies attract an increasing attentionof researchers as potential cancer biomarkers. Since the seraof cancer patients typically contain a unique set of antibodiesthat reflect the tumor-associated antigens expressed in aparticular malignant tissue, diagnosing and predicting theoutcome of disease such as breast cancer based on serumautoantibody profiling is an attractive concept.To create a representative panel of antigens for detecting ofbreast cancer autoantibody profile we selected 18 breastcancer associated antigens. These antigens were identifiedby screening of tumor cDNA libraries with autologous serausing SEREX (SErological investigation of RecombinantlyEXpressed clones) approach. All antigens were cloned,expressed, purified in bacteria and tested with sera of breastcancer patients and healthy donors in large-scale allogenicscreening using ELISA.The utility of selected tumor associated antigens for detectingof autoantibody profile in different types of breast cancer wasevaluated.

O-762Organ printing: the key to eternal life?Francoise Marga1, Cyrille Norotte1, Karoly Jakab1, ChiragKhatiwala2, Benjamin Shepard2, Bradley Hubbard1, StephenColbert1, Gabor Forgacs1,31University of Missouri, Columbia, MO, USA, 2Organovo, Inc.San Diego, CA, USA, 3The Shipley Center for Innovation,Clarkson University, Potsdam, NY, USA

We introduce a novel automated rapid prototyping method(organ printing) that allows engineering fully biological three-dimensional custom-shaped tissue and organ modules. In thistechnology bio-ink units (multicellular aggregates) composedof single or several cell types together with supporting materialare delivered by special printers. Printing of the bio-ink units

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(controlled by architectural software) is carried out according toa design template, consistent with the geometry and compo-sition of the desired organ module. Structure formation occursby the post-printing fusion of the discrete bio-ink units. Whenthe bio-ink units contain more than one cell type, fusion isaccompanied by sorting of the cells into the physiologicallyrelevant pattern. Thus structure formation takes place throughself-assembly processes akin to those utilized in earlyembryonicmorphogenesis. We demonstrate the technology bydetailing the construction of vascular and nerve grafts.Spherical and cylindrical bio-ink units have been employed tobuild fully biological linear and branching vascular tubularconduits and multiluminal nerve grafts. Upon perfusion in abioreactor the constructs achieved desirable biomechanicaland biochemical properties that allowed implantation into ani-mal models. Our results show that the printing of convenientlyprepared cellular units is feasible and may represent a prom-ising tissue and organ engineering technology.

P-765Femtosecond laser assisted microsurgeryof mammalian cells for medical and biotechnologyapplicationsM.M. Rakityanskiy1, I.V. Ilina1, D.S. Sitnikov1,A.V. Ovchinnikov1, M.B. Agranat1, Y.V. Khramova2,M.L. Semenova21Joint Institute for High Temperatures of the RussianAcademy of Sciences, Moscow, Russia, 2Moscow StateUniversity, Biological Faculty, Moscow, Russia

Femtosecond lasers have become important tools for non-contact microprocessing of biological specimens. Due to theshort pulse length and intensity-dependent nature of the mul-tiphoton ionization process, fs-laser pulses affect only a smallvolume of a treated cell, providing a high degree of spatiallocalization. We employed fs-laser to address topical bioengi-neering and biomedical problems such as cell fusion andembryo biopsy respectively. A tightly focused laser beam (Cr:Fseed oscillator and a regenerative amplifier, 620nm, 100 fs, 10Hz) was used for a fusion of blastomeres of two-cell mouseembryos and for a polar body (PB) biopsy. In order to fuseblastomeres the contact border of cells was perforated by asingle laser pulse. The fusion process usually completed within*60 min. In order to perform a noncontact laser based PBbiopsy we initially drilled an opening in the zona pellucida with aset of laser pulses, and then extracted the PB out of zygote bymeans of optical tweezer (cw laser, 1064 nm). The energy oflaser pulses was thoroughly optimized to prevent cell damageand increase the fusion and biopsy rates. The proposed tech-niques demonstrate high efficiency and selectivity and show agreat potential for using fs lasers as a microsurgical tool.

P-766New insights into mechanisms of electric field mediatedgene deliveryMasa Kanduser and Mojca PavlinUniversity of Ljubljana, Faculty of Electrical Engineering,SI-1000 Ljubljana, Slovenia

Gene electrotransfer is widely used for transfer of geneticmaterial in biological cells by local application of electric

pulses and is currently the most promising non-viral deliverymethod for gene therapy for a series of diseases as well asfor DNA vaccination. Current description of the processdefines several steps: electropermeabilization, DNA-mem-brane interaction, translocation, trafficking to nucleus andinto nucleus. But the mechanisms of electrotransfer are stillnot fully understood.We present results of the systematic in vitro analysisusing pEGFP of all steps involved in electrotransfectionfrom electropermeabilization, analysis of different pulsingprotocols, theoretical analysis of plasmid mobility tovisualization of the processes of DNA-membrane inter-action. We demonstrate that in order to translate in vitroresults to tissue level sub-optimal plasmid concentrationshave to be used. Furthermore, sofar the method of DNAentry into cytoplasm was only speculated. Our resultssuggest that it is crucial that first, membrane is electrop-ermeabilized, then sufficient electrophoretic force iscrucial for insertion of DNA into destabilized lipid bilayerfollowed by DNA translocation into cytoplasm via a slowprocess. Efficiency of electrotransfer depends also on thestage of of cell culture – cells in dividing phase are easerto electrotransfect.

P-767Gentamicin interaction with B16F10 cell membranestudied by dielectrophoresisI. M. Lazar1,2, L. Saplacan1, G. Lazar2, A. Sobetkii3,M. M. Iordache1, E Kovacs1, M. Radu1,4, M.G. Moisescu11Carol Davila Medical University, Biophysics and CellBiotechnology Dept., Bucharest, Romania, 2VasileAlecsandri University, Bacau, Romania, 3MGM STARCONSTRUCT SRL, Bucharest, Romania, 4Horia HulubeiNational Institute of Physics and Nuclear Engineering,Bucharest - Magurele, Romania

Dielectrophoresis (DEP) is the translational motion of polar-izable particles due to an electric field gradient. Positive-DEPand negative-DEP correspond to particle movement forwardor backward the region of high field intensity, respectively.Our study reveals some of the cell membrane modificationsinduced by Gentamicin (Gt), as they are reflected in thecrossover frequency fCO of B16F10 murine cells incubatedwith Gt for different concentrations and durations. fCO is theAC frequency when cells turn from positive-DEP to negative-DEP. Gentamicin is a positively charged aminoglycosidicantibiotic, with concentration-dependent killing action; it iswidely used because of its low cost and reliable bactericidalactivity. Gt drawbacks consist in high toxicity for renal andhearing cells; the molecular mechanisms of this toxicity arestill unclear. For low external medium conductivities (&0.0012 S/m), fCO of control and Gt-cells was found to rangefrom 3 to 10kHz. fCO shifts to higher frequencies with theincrease of Gt concentration and incubation time. Cells di-electrophoretic behavior is discussed using the cell single-shell based model.This work was supported by CNCSIS–UEFISCSU, project1193 PNII – Idei ID 1195/2008 and by CNCSIS–UEFISCSU,project 1174 PNII – Idei ID 1193/2008.

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P-768PEGylation increases mobility of chitosan-DNA deliverysystems in artificial extracellular matricesSylvie Lelu, Sabina Strand, Jan Steine, Catharina De LangeDaviesNorwegian University of Science and Technology,Trondheim, Norway

Extracellular matrix (ECM) is a major obstacle for succesfuldelivery of genes. Chitosan is a versatile and biocompatiblepolysaccharide derived from chitin and is a promising genecarrier. Chitosan-DNA interactions, and hence DNA polyplex-ation and release can be controlled through chitosande-acetylation degree, molecular weight and functionalizationof chitosan cationic groups. Grafting of poly(ethylene glycol)PEG to gene delivery vectors increases circulation time of genedelivery systems in blood vessels and reduces polyplexescharge. Diffusion and unpacking of PEGylated and non-PEGylated chitosan-DNA polyplexes through articial ECMsbased on collagen and collagen-hyaluronic acid (HA) gels werecompared using fluorescence correlation microscopy, confocalmicroscopy and colocalization analysis. Non-PEGylated poly-plexes were immobilized in the gels whereas PEGylatedpolyplexes were diffusing. The smaller charge of PEGylatedpolyplexes seems to decrease interactions betweenpolyplexesand ECM components. Furthermore, HA might also screencollagen fibers-PEGylated polyplexes interactions. PEGylatedpolyplexes also showed a higher degree of unpacking in gels,probably due to a looser compaction of DNA by PEGylatedchitosan compared to non-PEGylated chitosan.

O-769Continuous droplet interface crossing encapsulationfor high through-put monodisperse vesicle designEtienne Loiseau1,2, Manouk Abkarian1,2, Gladys Massiera1,21Universite Montpellier 2, Laboratoire Charles CoulombUMR5221 - 34095 Montpellier, France, 2CNRS, LaboratoireCharles Coulomb UMR5221 - 34095 Montpellier, France

Fabrication of vesicles, a close membrane made of an am-phiphile bilayer, has great potentiality for encapsulation andcontrolled release in chemical, food or biomedical industriesbut also from a more fundamental point of view for the designof biomimetic objects.Methods based on lipid film hydratation1, inverse emulsiontechniques2 and more recently microfluidic techniques suchas double emulsion3 or jetting4 method are limited either by alow yield, a low reproducibility, a poor control on the size, orby the presence of remaining solvent or defects.We propose a fast and robust method5 easy to implement:Continuous droplet interface crossing encapsulation(cDICE), that allows the production of defect-free vesicles athigh-yield with a control in size and content. The vesicleshave controlled bilayer composition with a polydispersity insize lower than 11%. we have shown that solutions asdiverse as actin, cells, micrometric colloids, protein and highionic strength solutions can easily be encapsulated using thisprocess. By adjusting the parameters of our set-up, we areable to produce vesicles in the range 4-100 lm in diameter,stable for weeks. We believe this method open new per-spectives for the design of biomimetic systems and evenartificial tissues.

[1] M. I. Angelova and D. S. Dimitrov, Faraday Discuss.,1986, 81, 303.

[2] S. Pautot, B. J. Frisken and D. A. Weitz, Langmuir,2003, 19, 28702879; S. Pautot, B. J. Frisken and D.A. Weitz, Proc. Natl. Acad. Sci. U. S. A., 2003, 100,10718.

[3] H. C. Shum, D. Lee, I. Yoon, T. Kodger and D. A. Weitz,Langmuir, 2008, 24, 7651.

[4] J. C. Stachowiak, D. L. Richmond, T. H. Li, A. P. Liu, S.H. Parekh and D. A. Fletcher, Proc. Natl. Acad. Sci.U. S. A., 2008, 105, 4697.

[5] M. Abkarian, E. Loiseau and G. Massiera, Soft Matter,2011, DOI: 10.1039/c1sm05239j.

O-770Acceleration neuronal precursors differentiationinduced by substrate nanotopographyElisa Migliorini1,3, Gianluca Grenci1, Jelena Ban2, ElisabettaRuaro4, Massimo Tormen1, Marco Lazzarino1,3 and VincentTorre21CNR-IOM Laboratorio TASC, Trieste, Italy, 2InternationalSchool for Advanced Studies, Trieste, Italy, 3CBM S.c.r.l.,Trieste, Italy, 4Glance Vision Technologie, Trieste, Italy

Embryonicstem (ES) cell differentiation inspecificcell lineage isstill a major challenge in regenerative medicine. Differentiationis usually achieved by using biochemical factors (BF) whichconcentration and sides effects are not completely understood.Therefore, we produced patterns in polydimethylsiloxane(PDMS) consisting of groove and pillar arrays of sub-micro-metric lateral resolution as substrates for cell cultures. Weanalyzed the effect of different nanostructures on differentiationofES-derivedneuronalprecursors intoneuronal lineagewithoutadding biochemical factors. Neuronal precursors adhere onPDMS more effectively than on glass coverslips but the elas-tomeric material itself doesn’t enhance neuronal differentiation.Nano-pillars increase both precursors differentiation and sur-vival with respect to grooves. We demonstrated that neuronalyield was enhanced by increasing pillars height from 35 to 400nm. On higher pillar neuronal differentiation reaches*80% 96hours after plating and the largest differentiation enhancementof pillars over flat PDMSwasobservedduring the first 6 hours ofculture. We conclude that PDMS nanopillars accelerate andincrease neuronal differentiation.

O-771A polymerizable GFP variantAdel Muskotal1, Agnes Klein1, Balazs Toth1,2, HajnalkaJankovics1, Ferenc Vonderviszt1,3,41Bio-Nanosystems Laboratory, Research Instituteof Chemical and Process Engineering, Faculty of InformationTechnology, University of Pannonia, Veszprem, Hungary,2Agricultural Research Institute, Martonvasar, Hungary,3Institute of Enzymology, Budapest, Hungary, 4ResearchInstitute for Technical Physics and Materials Science,Budapest, Hungary

Flagellin is the subunit protein of bacterial flagellar filaments.It has the ability to polymerize in vitro into long filaments

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under appropriate conditions. The extremely variable D3domain of flagellin subunits, comprising residues 190–283,protrudes at the outer surface of flagellar filaments. The D3domain has no significant role in the construction of thefilament structure. Thus, replacement of D3 may offer apromising approach for insertion of heterologous proteins ordomains without disturbing the self-assembly of flagellinsubunits. Our work aims at the construction of flagellin-based fusion proteins which preserve the polymerizationability of flagellin and maintain the functional properties ofthe fusion partner as well. In this work a fusion construct offlagellin and the superfolder mutant of green fluorescentprotein (GFP) was created. The obtained GFP variant washighly fluorescent and capable of forming filamentousassemblies. Our results imply that other proteins (enzymes,binding domains etc.) can also be endowed by polymeri-zation ability in a similar way. This approach opens up theway for construction of multifunctional filamentousnanostructures.The support of the Hungarian National Office for Researchand Technology and the Hungarian Scientific Research Fund(OTKA) (grants CK77819, NK77978, NANOFLAG) isacknowledged.

P-772Gene delivery targeted to the TAG72 overexpressingtumour cells using an nanobody conjugatedpolyethyleneglycol-modified polyamidoamine dendrimerFatemeh Safarian1, Fatemeh Rahbarizadeh1, ZahraSharifzadeh2, Davoud Ahmadvand3 and Seyed MoeinMoghimi31Department of Medical Biotechnology, Faculty of MedicalSciences, Tarbiat Modares University, Tehran, Iran, 2NationalCell Bank of Iran, Pasteur Institute of Iran,Tehran, Iran,3Centre for Pharmaceutical Nanotechnologyand Nanotoxicology, University of Copenhagen, DK-2100Copenhagen Ø, Denmark

Keywords: Polyamidoamine (PAMAM) Dendrimer, hetrobi-functional PEG, anti-TAG72 nanobody.

Generation 5 Polyamidoamine (PAMAM) Dendrimer hasbeen shown to be highly efficient nonviral carriers in genedelivery. However, their toxicity limits their applications. Inthis study, to improve their characteristics as gene deliverycarriers, G5 PAMAM Dendrimer was modified with anti-TAG72 nanobody through hetrobifunctional PEG, then com-plexed with t-Bid coding pDNA, yielding PAMAM-PEG-Anti-TAG72 nanobody/pDNA nanoparticles (NPs). Nuclear mag-netic resonance (NMR) spectroscopy, zeta sizing and gelretardation assay results provided evidence that the nano-vector was successfully constructed. The transfectionefficiency of vector/pDNA complexes were evaluated in vitro.Real time PCR results also demonstrated that Anti-TAG72nanobody modified NPs are more efficient in t-Bid killer geneexpressing in colon cancer cell line than the unmodified NPs.In conclusion, PAMAM–PEG–anti-TAG72 nanobody showedgreat potential to be applied in designing tumour-targetinggene delivery system.

P-773Macromolecular crowders and stem cell differentiationRafi Rashid1,2, Michael Raghunath4,5 & Thorsten Wohland2,31National University of Singapore (NUS) Graduate Schoolfor Integrative Sciences & Engineering (NGS), Singapore,2NUS Centre for BioImaging Sciences, National Universityof Singapore, Singapore, 3Dept of Chemistry, Facultyof Science, National University of Singapore, Singapore,4Dept of Biochemistry, Yong Loo Lin School of Medicine,National University of Singapore, Singapore, 5Divisionof Bioengineering, Faculty of Engineering, NationalUniversity of Singapore, Singapore

Macromolecular crowding (MMC) is a biophysical tool whichhas been used extensively to enhance chemical reactionsand biological processes by means of the excluded volumeeffect (EVE). The in vivo stem cell microenvironment con-tains macromolecules which are crucial for stem cell self-renewal and cell fate determination. In order to mimic thisphysiological microenvironment, crowders are included incell culture medium. We have observed that the ex vivodifferentiation of human mesenchymal stem cells (hMSCs)into the adipogenic lineage is significantly amplified when acrowder mixture comprising Ficoll 70 and Ficoll 400 is addedto the culture medium. Stem cell differentiation is modulatedby soluble chemical substances as well as interactionsbetween cells and the extracellular matrix (ECM), and boththese external influences may be affected by MMC. Mea-surements we have performed by fluorescence correlationspectroscopy (FCS) show that Ficoll additives causeanomalous subdiffusion within a crowder concentrationrange of 0 to 300 mg/ml. The diffusion of fluorophore-labelled molecules in artificial lipid bilayers and membranesof living cells is not changed by crowders, suggesting thatthese crowders do not directly alter membrane propertiesand cell surface signalling. However, we have data to sug-gest that crowders increase actin polymerization reactionrates in vitro. We have also observed that crowders aretaken up by stem cells and that they localize to specificcompartments. Based upon our observations, we hypothe-size that crowders can influence stem cell differentiation byinfluencing molecular kinetics.

P-775Inspecting laccase action through radical generationJ. Strancar1, A. Mehle1, M. Bozic2, V. Kokol21Jozef Stefan Institute, Ljubljana, Slovenia, 2Universityof Maribor, Maribor, Slovenia

Lignocellulose-based composites are becoming extremelyimportant and perspective sustainable and renewable naturalmaterials. Fibre modification enhancing their existing prop-erties can be obtained to broaden the application areas. Inresponse to shortcomings of traditional chemical and physi-cal methods, enzymes and chemo-enzymatic methods haveemerged as eco-friendly catalysts working under mild con-ditions and enable tailoring of the material surface propertiesby substrate specificity and regional selectivity. Recently,

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binding of different functional molecules to lignin-rich fibresby using an oxidative enzyme (e.g. laccase) has beenreported leading to their functionalisation through free radicalreactions. By the application of electron paramagnetic reso-nance spectroscopy (EPR) laccase action was inspected.Consumption of substrates was investigated and their poly-merization traced. Stable radical intermediates weredetected with EPR when substrate molecules were in contactwith active enzymes. Secondly, oxidation of mediators likenitroxides was determined via EPR spectroscopy of stablewater-soluble nitroxide radicals. Finally, the generation ofshort-lived radicals as well as their reduction was measuredvia EPR spin trapping using DMPO as sensitive water solu-ble spin trap.

P-776The effect of ovary hormone stimulation on mouseoocyte and early embryo electric conductivityO.A. Strikha1, E.I. Smolyaninova1, E.O. Gordienko1,V.A. Shigimaga2, A.A. Kolesnikova21The Institute for problems of cryobiology and cryomedicineof The National Academy of sciences of the Ukraine,Ukraine, 2The Institute of Animal Science of the NationalAgrarian Academy of the Ukraine, Kharkov, Ukraine

Mammalian ovary hormone stimulation (OHS) is known tobe an inalienable stage of reproductive biotechnology aswell as human infertility treatment. The basic aim of theOHS is to receive a stock of valuable oocytes and earlyembryos for subsequent utilization in the reproductivetechnology, experimental work et al. However, it is knownthat OHS itself affects the character of ovulation andoocyte quality, which in its turn affects the development ofembryos and even has distant consequences. The wide-ness of cell parameters and appropriate methods forinvestigation of gamete/embryo quality are very important.The aim of this study is determination of specific electricconductivity of mouse oocytes and early embryos whichhave been received after OHS in comparison with theones that have been received in natural animal sex cycle.Using techniques of electroporation the dependence ofspecific electric conductivity of mouse oocytes, zygotes,2-cell and 8-cell embryos on the external electric fieldintensity has been studied. It is shown that the whole poolof oocytes that were obtained in the result of OHS con-sists of two groups of oocytes that don’t differ from eachother morphologically, but differ by their electric parame-ters and resistance to electric breakdown. At the zygotestage, dividing of embryos into two groups is preserved,but is less expressed. At the stage of 2-cell and 8-celldividing of embryos into two groups on their electricconductivity disappeared but certain scattering of theparameters due to individual embryo peculiarities isobserved. The obtained data show that OHS may lead tolatent changes of oocyte state that in their turn affectembryo quality.

O-777FTIR spectroscopic study of biopolyester synthesistraits in the bacterium Azospirillum brasilenseA.V. Tugarova1, A.A. Kamnev1, P.A. Tarantilis2,M.G. Polissiou21Institute of Biochemistry and Physiology of Plantsand Microorganisms, Russian Academy of Sciences,Saratov, 410049, Russia, 2Agricultural University of Athens,Athens 11855, Greece

Many microbes synthesize and accumulate granules ofpolyhydroxyalkanoates (PHA, biodegradable storage mate-rials alternative to traditional plastics), which help themsurvive under stresses. In particular, the plant-growth-pro-moting rhizobacterium Azospirillum brasilense, that is underinvestigation worldwide owing to its agricultural and bio-technological significance, can produce poly-3-hydroxybutyrate (PHB) [1]. In our work, PHB synthesis in A.brasilense cells was studied under various stresses usingdiffuse reflectance FTIR spectroscopy. PHB in cells wasdetermined from the band intensity ratio of the polyesterm(C=O) at *1740 cm-1 to that of cell proteins (amide II bandat *1550 cm-1), showing A. brasilense to be able to pro-duce PHB up to over 60% of cells’ dry weight. Stressesinduced PHB accumulation, enhancing IR absorption in PHBspecific regions. Analysis of a few structure-sensitive PHBvibration bands revealed changes in the degree of intracel-lular PHB crystallinity (related to its enzymatic digestion rate)at different stages of bacterial growth, reflecting a novel traitof the bacterial adaptability to an enhancing stress, which isof great importance to agricultural biotechnology.

[1] Kamnev AA, Tugarova AV, Antonyuk LP et al. (2006)Anal Chim Acta 573-574: 445-452.

P-778Development of a xylanase A variant capable of formingfilamentous assembliesVeronika Szabo1, Adel Muskotal1, Balazs Toth1,2,Ferenc Vonderviszt1,31Bio-Nanosystems Laboratory, Research Instituteof Chemical and Process Engineering, Universityof Pannonia, Veszprem, Hungary, 2Agricultural ResearchInstitute, Martonvasar, Hungary, 3Institute of Enzymology,Budapest, Hungary

The aim of this work is to furnish enzymes with polymeriza-tion ability by creating fusion constructs with thepolymerizable protein, flagellin, the main component of bac-terial flagellar filaments. The D3 domain of flagellin, exposedon the surface of flagellar filaments, is formed by thehypervariable central portion of the polypeptide chain. D3 isnot essential for filament formation. The concept in this pro-ject is to replace the D3 domain with suitable monomericenzymes without adversely affecting polymerization ability,and to assemble these chimeric flagellins into tubular

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nanostructures. To test the feasibility of this approach, xy-lanase A (XynA) from B. subtilis was chosen as a modelenzyme for insertion. With the help of genetic engineering, afusion construct was created in which the D3 domain wasreplaced by XynA. The FliC(XynA) chimera exhibited cata-lytic activity as well as polymerization ability. These resultsdemonstrate that polymerization ability can be introduced

into various proteins, and building blocks for rationallydesigned assembly of filamentous nanostructures can becreated (Table 1).The support of the Hungarian National Office for Researchand Technology and the Hungarian Scientific Research Fund(OTKA) (grants CK77819, NK77978, NANOFLAG) isacknowledged.

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