Bio-NMR and EAST-NMRAnnual User Meeting

Brno, Czech RepublicJanuary 24 – 27, 2011

Projects Bio-NMR (No. 261863) and EAST-NMR (No. 228461)are funded by the European Commission’s Framework Programmme 7 (FP7)

Dear Colleagues:

Welcome to the 2nd Annual EAST-NMR and the 1st Annual Bio-NMR User Meetings.EAST-NMR and Bio-NMR are projects of the 7th Framework Program of EU aiming atthe advancement of nuclear magnetic resonance spectroscopy and its applications. Theconference will consist of lectures dedicated to the highlights in the field of NMR ordefining the state of the art in specific areas of joint research activities, and it will includeuser presentations, The General Assembly and User meetings of both projects as well asthe meeting of the local operators. Brigitte Sambain, a representative of the Unit ResearchInfrastructure, Directorate General for Research of European Commission, is present.

This year, the program also incorporates presentations of prominent representativesof other (non-NMR) essential techniques (cryo-EM, X-ray crystallography), illustratingtheir respective approaches to solving challenging problems of structural biology. Theexpected ”cross-fertilizing”effect of such interactions among experts from different fieldsshould help to integrate the research of the NMR-oriented groups into an multidisciplinaryeffort necessary to push further the current limits of structural biology.

The main aim of the meeting is to provide an effective forum for discussion, at bothformal and informal levels. The high number of registered participants (179) should guar-antee a stimulating environment and the conference program allows to maximize the timethe participants spend together. The previous, exceptionally successful meetings of thistype have proven to be at the forefront of biomolecular NMR and we hope that the currentmeeting will continue in this tradition.

Best wishes,Vladimır Sklenar

Organizing Committee:Ivano Bertini (coordinator of Bio-NMR)Rolf Boelens (coordinator of Bio-NMR JRAs)Harald Schwalbe (coordinator of EAST-NMR)Vladimır Sklenar (Chair)

Local Organization:Hana BrichackovaRadovan FialaVladimır Sklenar (Chair)Lukas Zıdek

Host Institutions:NCBR and CEITEC, Masaryk University, Brno, Czech Republic

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General Information

Internet ConnectionUnlimited Internet connection is available free of charge:

• In conference halls: WiFi, network hotel, no password• In rooms:

WiFi: network HotelContinental with the strongest signal, password alvaedisonCable: you need your own computer, fitted with a network card and dynamic IPaddress allocation set up (standard settings for all operating systems)

MealsDue to the limited capacity, identical meals are served in two rooms:

• in small Restaurant (see map on page 12) for

– external speakers (Blackledge, Driscoll, Peti, Plitzko, Stuart)– IEP and SAB speakers (Allain, Arseniev, Graslund, Konrat, Permi, Prestegard)– EC Project Officer (Sambain)– PI’s of the facilities providing TA (Bertini, Boelens, Emsley, Karlsson, Kover,

Kozminski, C. Luchinat, Meier, Oschkinat, Plavec, Redfield, Sklenar, Schwalbe)

• in large Dinning Hall (see map on page 12) for

– users (including user speakers)– staff of the consortium members and other participants

PostersPosters are presented in Hall 3. Please place your poster under the number indicated inthis book of abstracts, using the provided pins. Posters should stay in Hall 3 during thewhole meeting but should be removed by Thursday noon.

LecturesSpeakers should see the technical assistant at his desk in the lecture hall before theirsessions for technical arrangements. The speaker will receive a signal two minutes beforethe time limit and the chairpersons will be instructed to cut the presentation if it exceedsthe allotted time limit. Each lecture is followed by a five-minute slot for discussion andcomputer switching.

Tickets for Public Transportation (Wednesday Evening)You receive two 10-min tickets for public transportation to get to the location of theWednesday’s evening programme and back to the hotel. Note that you have to validatethe ticket in the tram. If you do not need the tickets, please return them to the ticketcollection box at the hotel reception).

Phone Numbers+420 731 102 557 AUM emergency number (Hana Brichackova)+420 541 519 305 Hotel reception desk+420 541 519 111 Hotel operator

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Programme

Monday Jan. 24

10:00 Registration open

13:30 Lunch

SESSION I, chair Harald Schwalbe14:30 Opening14:50 James Prestegard: Integrating NMR with Other Technologies: High Order

Assemblies of Chemokines and Glycosaminoglyans15:25 Jurgen Plitzko: Hybrid imaging – Novel approaches and recent advances in

correlative microscopy for structural biology

16:00 Tea & Coffee

SESSION II, chair Claudio Luchinat16:30 Astrid Graslund: NMR studies of the amyloid β peptide involved in Alzheimer’s

disease: molecular interactions, secondary structure conversions and aggregation17:05 Peter Tompa: Structural disorder and chaperone activity of plant dehydrins

in vitro and in vivo17:40 Torsten Herrmann: Protein structure determination directly from the NMR

spectrometer using UNIO

18:30 Dinner (EAST-NMR Management Committee meets in Restaurant)

Bio-NMR GENERAL ASSEMBLY Project Coordinator: Ivano Bertini20:00 Brigitte Sambain, Project Officer – DGRTD Research Infrastructure Unit

20:30 Harald Schwalbe – Transnational Access

20:40 Rolf Boelens – Joint Research Activity

21:00 Ivano Bertini – Networking

(contributions from Christina Redfield – WP 4.2 and 4.5, Chris Spronk – WP 3.3 and 5.6,

Adam Lange – WP 5.3, Rolf Boelens – WP 6.1)

21:30 Henriette Molinari – User Group

21:40 Astrid Graslund – notes from the Internal Evaluation Panel

21:45 James Prestegard – monitoring of the activities

21:50 Antonio Rosato – WeNMR

22:00 Bio-NMR Consorcium Members Meeting

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Tuesday Jan. 25

SESSION III, chair Ivano Bertini9:00 David Stuart:9:35 Wolfgang Peti: MAP Kinase Regulation

10:10 Rafal Augustyniak: NMR studies of homeoprotein Engrailed 2. Assignment,diffusion, dynamics

10:30 Tea & Coffee

SESSION IV, chair Jacob Anglister11:00 Hartmut Oschkinat: Structural investigations on the ABC transporter

ArtMP-J and on the interaction of αB-Crystallin with substrates by Solid-State NMR11:35 Miquel Pons: NMR Studies of bacterial nucleoid associated proteins and their

DNA complexes12:10 Irene Diaz Moreno: Tyr-modified cytochrome c and cell death in eukaryotic

systems

12:30 Lunch (EAST-NMR Management Committee meets in Restaurant)

SESSION V, chair Ago Samoson13:30 Robert Konrat: What’s behind Disordered – Unfolded – Unstructured?14:05 Beat H. Meier: Structure determination by Solid-state NMR14:40 Sam Asami: Proton detection of aliphatic resonances with ssNMR spectroscopy

15:00 Posters (odd numbers presented) and Refreshment

SESSION VI, chair Wiktor Kozminski16:30 Perttu Permi: Structural studies of EspFU, a novel SH3 ligand triggering the

pathogen-driven actin assembly17:05 Christina Redfield: Oxidation-state-dependent protein-protein interactions in

disulfide cascades17:40 Janez Plavec: From interactions of small molecule ligands and their self

assembly to DNA quadruplexes and prion proteins

18:30 Dinner (Bio-NMR Management Committee meets in Restaurant)

EAST-NMR GENERAL ASSEMBLY Project Coordinator: Harald Schwalbe20:00 Presentation of the EAST-NMR project (Harald Schwalbe)

20:10 Transnational Access (Vladimır Sklenar, Janez Plavec, Katalin Kover, Wiktor Kozminski)

20:35 Joint Research Activity (Rolf Boelens)

20:45 Networking Activities (Vladimır Sklenar)

20:55 Discussion

22:00 EAST NMR MC Meeting

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Wednesday Jan. 26

SESSION VII, chair Niels Chr. Nielsen9:00 Martin Blackledge: Exploring Multiple Timescale Motions in Folded and

Intrinsically Disordered Proteins using NMR9:35 Bernhard Brutscher: Recovering lost magnetization: polarization enhancement

in biomolecular NMR10:10 Mario Jug: Study of zaleplon/cyclodextrin complexation by 1H-NMR

spectroscopy

10:30 Tea & Coffee

SESSION VIII, chair Somer Bekiroglu11:00 Fred Allain: Insight into RNA splicing and editing mechanisms from the NMR

structures of protein-RNA complexes.11:35 Tobias Madl: Solvent PRE-assisted structural analysis of large protein

complexes12:10 Gabriele Giachin: NMR structure of the human prion protein with the

pathological Q212P mutation: insights into inherited human prion diseases

12:30 Lunch (Bio-NMR Management Committee meets in Restaurant)

SESSION IX, chair Katalin E. Kover13:30 Alexander Arseniev: NMR view on helix-helix interactions of membrane

proteins14:05 Norbert Muller: NMR Noise Spectroscopy14:40 Vasudevan Ramesh: NMR of RNA at 1GHz – A New Frontier in Structural

Biology

15:00 Posters (even numbers presented) and Refreshment

SESSION X, chair Andras Perczel16:30 Rolf Boelens: Structure and dynamics in gene regulation and DNA repair17:05 Goran Karlsson: NMR-optimized cell-free protein expression

EVENING PROGRAMME18:30 Mendel Museum and Banquet at The Queen Elizabeth wine cellar

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Thursday Jan. 27

SESSION XI, chair Edvards Liepinsh9:00 Paul Driscoll: Methyl-TROSY NMR of death domain assemblies: low symmetry

organisation suggested by non-degenerate chemical shifts9:35 Mateus Webba da Silva: DNA Quadruplex Topologies by Design

10:10 Richard Stefl: Transcription termination by the Nrd1 complex

10:30 Tea & Coffee

SESSION XII, chair Lyndon Emsley11:00 Adam Lange: Solid-State NMR as a Tool in Structural Biology: VDAC, Tau,

and the TTSS Needle11:35 Lukas Trantırek: In-cell NMR spectroscopy of nucleic acids12:10 Ildefonso Marin-Montesinos: Analysis of derivatized aminoacids in biological

samples by Dynamic Nuclear Polarization

12:30 Lunch

14:00 Departure

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Transport Routes for the Wednesday’s Evening

To get to the Mendel Square from the hotel, walk to the tram stop Ceska (red dottedroute on the map). Prepare the 10-min ticket and take trams No. 5 (direction Ustrednıhrbitov), No. 6 (direction Stary Lıskovec), or No. 7 (direction Bohunice, Svermova) tothe Mendlovo namestı stop (3 stops from Ceska, see the blue route on the map). Whenarriving at the Mendel square, you should see the historic building of The Abbey to yourright. The Queen Elizabeth wine cellar (“U kralovny Elisky”, address Mendlovo Namestı1b, GPS coordinates: 49◦11’32.614”N, 16◦35’38.183”E) is located behind The Abbey.

ContinentalHotel

Česká st.

Queen ElizabethWine cellar

("U Královny Elišky")

The AbbeyMendel Museum

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Lectures

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Lecture, Mon 14:50

Integrating NMR with Other Technologies: High Order Assemblies ofChemokines and Glycosaminoglyans

Prestegard JH, Wang X, Watson C, Sharp JS

Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, 30602,USA

Proteins seldom act in isolation. Instead they interact with themselves, with otherproteins, or with other types of molecules to function as higher order complexes. Under-standing the modes of these interactions is as important as understanding the structure ofthe individual components of the complexes. A particularly interesting example involvesthe organization of chemokines at the surfaces of cells. CCL5 or RANTES plays a role inthe activation and recruitment of T-cells to sites of injury or infection. While the forminvolved in some aspects of T-cell activation may be monomeric, binding to gycosamin-oclycans (GAGs) of the extra-cellular matrix and formation of oligomeric structures isnecessary for endothelial layer transmigration and actual function in vivo. We demon-strate key aspects of glycosaminoglycan interaction using NMR chemical shift mappingand develop a model for a CCL5 oligomeric structure by combining NMR with other bio-physical techniques. Development of the oligomeric model uses residual dipolar couplings(RDCs) to validate X-ray monomeric structures and provide orientational constraints forassembly of oligomers. Small angle X-ray scattering (SAXS) data are used to providetranslational constraints. NMR cross-saturation data and MS hydroxyl radical footprint-ing data are used to identify residues in the oligomeric interface. The resulting modelprovides a new picture of how GAG binding and receptor interactions may cooperate toinfluence recruitment and transmigration of T-cells.

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Lecture, Mon 15:25

Hybrid imaging – Novel approaches and recent advances in correlativemicroscopy for structural biology

Plitzko JM(1)

(1) Max Planck Institute of Biochemistry, Dept. of Molecular Structural BiologyD-82152 Martinsried (near Munich), Germany

Hybrid Imaging can be seen simply as the combined or fused imaging of two differ-ent datasets, where two different modalities contribute equally to image information. Togo beyond this elementary definition hybrid imaging could be viewed as any combina-tion of structural and functional information to facilitate a comprehensive and exhaustivedata interpretation. However, this combined approach can only be realized, if innova-tive hardware accessories, new methodologies and optimized preparation protocols are inplace. The well-coordinated interplay of optical and spectrometric techniques will facili-tate the functional and structural characterization at the molecular level, which will offerunique complementary insights. Thus, if combined successfully, the final outcome will begreater than the sum of its parts. Hybrid imaging, integrating the different methodologiesand options to quantitatively study cell architecture, protein organization and individualmacromolecular key players, will be indispensable to study biological systems on differentscales [1]. In this presentation on hybrid imaging, we overview novel approaches and recentadvancements in hybrid and correlative imaging with an emphasis on sample preparationand optimization, which are both decisive steps in combined biomolecular imaging andstructural analysis. We will discuss the current state of integrating optical (light) andelectron microscopy with a view to performing cryo-electron tomography in an efficientand targeted manner [2,3]. 1. Plitzko, J. and Baumeister, W. J Struct Biol 2010, 172:p1592. Plitzko, J.M., A. Rigort, and A. Leis. Curr Opin Biotechnol 2009, 20: 83-9. 3. Rigort,A, FJB. Baeuerlein, A. Leis, M. Gruska, C. Hoffmann, T. Laugks, U. Boehm, M. Eibauer,H. Gnaegi, W. Baumeister and J.M. Plitzko. J Struct Biol 2010, 172:169-179.

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Lecture, Mon 16:30

NMR studies of the amyloid β peptide involved in Alzheimer’s disease:molecular interactions, secondary structure conversions and aggregation

Graslund A(1)

(1) Department of Biochemistry and Biophysics, Stockholm University, S-106 91Stockholm, Sweden

The amyloid β peptide consists of 39-43 residues and is the major component ofneuritic plaques in the brain from patients suffering from Alzheimer’s disease. In aqueoussolution the Abeta peptide aggregates and forms β-sheet structures, finally forming solidfibrils. We study the structure conversions and aggregation properties of the Abeta(1-40)peptide using high resolution NMR spectroscopy. At low concentrations, low temperaturesand low ionic conditions in an aqueous solution, Abeta(1-40) is monomeric. Although thepeptide displays only weak propensities towards secondary structure, CD and NMR canbe used to characterize these propensities in different segments of the peptide, also afteradding metal ions like zinc or copper (1). The metal ions bind to ligands in the N-terminusof the peptide, and induce increased order in the N-terminus. Cyclodextrin or covalentlylinked cyclodextrin dimers interact with the aromatic sidechains of Abeta(1-40). Theinteraction probably changes the aggregation pathways and mediates the inhibition offibril formation effected by these compounds. Detergents like Congo red interfere with thepeptide structure conversions and also change the aggregation pathways of Abeta(1-40)(2).

By gradually adding the detergent lithium dodecyl sulphate (LiDS) or SDS to a diluteaqueous solution of Abeta(1-40), secondary structure conversions of Abeta(1-40) can beobserved (3). An initial transition after adding small amounts of LiDS involves conversionof the weakly structured peptide to beta-sheet structure, concomitant with formation oflarge aggregates. This structure transition may mimic the behavior of the Abeta peptide,which forms oligomeric structures at a crowded membrane surface. At concentrations closeto the detergent CMC or above, a second transition makes the peptide rearrange to forma partly α-helical structure, concomitant with disaggregation and formation of normalLiDS micelles which apparently partly dissolve the aggregates. This α-helical structureis similar to that previously observed by NMR at high SDS concentrations. It has twoα-helical segments, Abeta(16-24) and (29-35), separated by a flexible hinge, and flexibleunstructured N- and C-termini (4).

References:1. Danielsson, J., Pierattelli, R., Banci, L. and Graslund, A. FEBS J. 274 (2007) 46-59.2. Lendel, C., Bolognesi, B., Wahlstrom, A., Dobson, C. and Graslund, A. Biochemistry

49 (2010) 1358-13603. Wahlstrom, A., Hugonin, L., Peralvarez-Marin, A., Jarvet, J. and Graslund, A.

FEBS J. 275 (2008) 5117-5128.4. Jarvet, J., Danielsson, J., Damberg, P., Oleszczuk, M. and Graslund, A. J. Biomol.

NMR 39 (2007) 63-72.

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Lecture, Mon 17:05

Structural disorder and chaperone activity of plant dehydrins in vitro and invivo

Peter Tompa (1), Bianka Szalaine Agoston (1)(2), Denes Kovacs (1), Andras Perczel (2)

(1) Institute of Enzymology, Biological Research Center, Hungarian Academy ofSciences, Budapest, Hungary; (2) Laboratory of Structural Chemistry and Biology,Institute of Chemistry, ELTE University, Budapest, Hungary

Structurally disordered proteins (IDPs) can be classified into six functional categories[1, 2]. Based mainly on bioinformatic analysis, we have suggested a couple of years agothat disordered regions of traditional chaperones or even fully disordered proteins can havepotent chaperone activity, probably by an “entropy transfer” mechanism [3]. To carry outdetailed structure-function analysis of this phenomenon, we studied two dehydrins of A.thaliana, ERD10 and 14. Dehydrins are fully disordered stress proteins of plants, theexpression of which increases critically upon dehydration elicited by water stress, highsalinity or cold. In a range of chaperone assays with distinct substrates (e.g. alcohol de-hydrogenase, firefly luciferase, human citrate synthase), we have shown that ERD10 and14 are potent chaperones in vitro, with activities commensurable with that of Hsp90 [4].To address the structural background of this effect, we carried out full NMR resonanceassignment of the 185 amino acid-long ERD14. Secondary chemical shift and relaxationdata show that ERD14 is fully disordered, with five short regions of somewhat restrictedflexibility suggesting structural bias to local helicity. In-cell NMR of ERD14 overexpressedin E. coli shows that three of these regions (conserved K-segments) undergo further order-ing, probably due to binding partner molecules in the cell. Overexpressed ERD14 providessignificant protection to cells against stress conditions elicited by freezing, high salt (2MNaCl) or severe dehydration (6M gylcerol) [5]. These studies show that ERD14 is an IDPlargely disordered in vivo, with local elements undergoing function-related local folding;the implications of these findings are discussed in detail.

Reference:1. Tompa P (2002) Intrinsically unstructured proteins. TiBS 27, 527-533.2. Tompa P (2009) Structure and function of intrinsically disordered proteins CRC

Press, Taylor and Francis Group.3. Tompa P & Csermely P (2004) The role of structural disorder in the function of

RNA and protein chaperones. FASEB J. 18, 1169-1175.4. Kovacs D, Kalmar E, Torok Z & Tompa P (2008) Chaperone activity of ERD10

and ERD14, two disordered stress-related plant proteins. Plant Physiol. 147, 381-390.5. Szalaine Agoston B, Kovacs D, Perczel A & Tompa P (2010) Chaperone activity of

plant dehydrins in vivo. in preparation.

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Lecture, Mon 17:40

Protein structure determination directly from the NMR spectrometer usingUNIO

Paul Guerry, Torsten Herrmann

Centre Europeen de RMN a Tres Hauts Champs, Universite de Lyon, CRNS-FRE3008,ENS Lyon, UCB Lyon 1, CNRS, 5 rue de la Doua, 69100 Villeurbanne, France(torsten.herrmann@ens-lyon.fr)

The talk will present the first protein structure determination directly performed in afully unsupervised manner from the time-domain FID data acquired.

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Lecture, Tue 9:00

David Stuart

Abstract not available.

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Lecture, Tue 9:35

MAP Kinase Regulation

Wolfgang Peti

Dept. of Molecular Pharm., Physiology & Biotech. and Dept. of Chemistry, BrownUniversity, Providence, RI, USA 02903

Protein phosphorylation is a central mechanism for cellular regulation and communi-cation. Here we report on our efforts to elucidate the molecular basis of the regulation ofthe ser/thr Protein Phosphatase 1 (PP1), a phosphatase that catalyzes ∼30% of all de-phosphorylation reactions in humans with regulatory roles in cellular processes as diverseas cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism,transcription and neuronal signaling. PP1 is tightly regulated by its interaction with morethan 200 known regulatory proteins, which not only localize PP1 to distinct regions ofthe cell, but also modulate its activity. We will provide structural examples of numerousPP1:regulator complexes, as well as molecular insights into higher order heterotrimericPP1 complexes, which have enabled us to develop a comprehensive picture of PP1 regu-lation in the cell. We will also provide our novel structural insights into the regulation ofMAP kinases by both phosphatases and scaffolding proteins. While 100’s of structures ofser/thr kinases are available, there is a severe shortage of structural information on theirregulatory complexes. Our experimental approach combines multiple biochemical andstructural biology techniques to gather enough data to develop precise structural modelsof these multi-component complexes. Therefore, we are combining NMR spectroscopy,X-ray crystallography and small angle X-ray scattering to understand how these com-plexes mediate phosphorylation signaling in the cell. Taken together, our studies haveprovided essential new insights into the regulation of MAP kinases by their regulatoryand scaffolding proteins. Furthermore, using structural biology we were able to transformour molecular understanding of PP1 regulation, leading to a new model how PP1 regu-latory proteins direct PP1 activity in vivo. Funding for this work was provided by NIHNS056128, NS054493, RR016457, ACS RSG-08-067-01-LIB & NSF MCB0952550.

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Lecture, Tue 10:10

NMR studies of homeoprotein Engrailed 2. Assignment, diffusion, dynamics

Augustyniak R, Ferrage F, Lequin O, Pelupessy P, Bodenhausen G

Ecole Normale Superieure, Departement de Chimie, UMR 7203 CNRS-UPMC-ENS, 24rue Lhomond, 75005 Paris, France, rafal.augustyniak@ens.fr

Many proteins play a physiological role in living organisms although they lack well-defined secondary structure. Frequently they interact with other biological partners, whichrequire fast conformational changes and high flexibility. Therefore, such proteins possessparticular dynamic properties, which are not observed in well folded globular molecules.We are studying the construct of Engrailed 2 protein that comprises residues 146 to 259,corresponding to the homeodomain and its N-terminal extension. This extension containsseveral interaction sites for other regulatory proteins and seems to be intrinsically unstruc-tured according to protein sequence analysis. We expressed and purified the Engrailed 2construct using classical protocol designed for isotopic labeling of NMR samples. Theassignment of backbone resonances has been completed in spite of the weak dispersion inproton dimension that is often in the case of disordered proteins. The assignment of sidechain resonances was also performed thanks to multidimensional experiments recordedwith non-uniform sampling. Subsequently, pulse sequences based on the heteronuclearstimulated echo and optimized for disordered proteins were used to investigate Engrailedstability in solution by measurements of the translational diffusion coefficients. Then,intrinsic dynamics was probed by monitoring different auto- and cross-relaxation rateconstants.

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Lecture, Tue 11:00

Structural investigations on the ABC transporter ArtMP-J and on theinteraction of αB-Crystallin with substrates by Solid-State NMR

Oschkinat H

Leibniz-Institut fur Molekulare Pharmakologie, Robert-Rossle-Str. 10,13125 Berlin,Germany

Small heat shock proteins occur as polydisperse oligomers and are vital to maintainlong-living cells in a functional state. A main player in higher organisms is αB-crystallinwhich is accompanied by αA-crystallin in the eye lens. γ-S-crystallin, another eye lensprotein, is one of its ’tasks’ since it needs to be kept stably folded for the life time of theorganism. Previously, we determined a structure of the α-crystallin domain, and obtaindfirst insights into the structure of the oligomer. Now we were investigating further theactivation mechanism of αB-crystallin and its interaction with substrates such as γ-S-crystallin as well as the structure of its N-terminaus.

Investigations of membrane proteins may be facilitated by extensive deuteration, andsubsequent detection of protons under magic-angle spinning conditions. Various schemesfor the usage of proton chemical shifts for achieving sequence-specific assignments willbe discussed. Samples of Outer membrane protein G and an ABC transporter reconsti-tuted into native lipid bilayers are investigated in variously labelled forms, and spectra ofdifferent functional states of the will be presented together with initial assignments andfunctional considerations. An optimized pulse sequence, Triple-CP, is presented for theexcitation of carbon signals in deuterated spectra. The TCP experiment exploits abun-dant deuterium, rare remaining non-exchangeable and exchangeable protons at carbonsites. A quantitative comparison between 1H and 1H+2H (TCP) cross-polarization ex-periments is presented, to be able to identify the best strategy for carbon detected MASNMR experiments. This new type of CP experiment would be particularly important forsystems such as membrane proteins whose amide protons are difficult to back-exchange,and as a result suffer from lower sensitivity.

The application of dynamic nuclear polarisation (DNP) to the investigation of thesesystems requires further optimization of samples, experimental parameters and concepts.The application of DNP to various proteins systems will be discussed, and spectra of dif-ferent types of membrane protein samples presented. Spectra with reasonable line widthare obtained on membrane integrated complexes. Various technical aspects of the appli-cation of DNP to protein systems will be discussed. During the DNP process, the electronpolarization is transferred to the surrounding core nuclei and subsequently to the bulknuclei. The enhanced signal reaches its maximum intensity after a certain period of time.This process depends on several factors such as; relaxation behavior, proton concentra-tion, spin-diffusion and type of the nucleus. As a result, for each nucleus to be polarizedthere is a characteristic exponential polarization build-up behavior, with different timeconstant (τB) for each nucleus of interest. A systematic investigation of the polarizationbuild-up behavior for different nuclei (1H, 2H, 13C, 15N) is presented and for protonatedand deuterated SH3 proteins. This information will shed light on the choice of the nucleusto be polarized.

27

Lecture, Tue 11:35

NMR Studies of bacterial nucleoid associated proteins and their DNAcomplexes

Cordeiro T(1), Renault M(2), Schmidt H(3), Marimon O(1), Garcıa J(1), GriesingerC(3), Baldus M(2), Pons M(1,4).

1. Institute for Research in Biomedicine (IRB Barcelona). Baldiri Reixac, 10-12.08028-Barcelona. Spain 2. Bijvoet Center for Biomolecular Research. UtrechtUniversity, Padualaan 8, 3584 CH Utrecht, The Netherlands. 3. Max Planck Institutefor Biophysical Chemistry. Department of NMR-based Structural Biology, Am Fassberg11, D-37077 Gottingen, Germany 4. Departament de Quımica Organica. Universitat deBarcelona (UB). Martı i Franques, 1-11. 08028-Barcelona. Spain Presenting authore mail: mpons@ub.edu

Nucleoid associated proteins (NAP) are responsible for packaging the bacterial chro-mosome and actively regulating the expression of large number of genes to provide acoordinated global response to environmental changes, including the manifestation of apathogenic phenotype upon guest colonization. One of the most abundant proteins inGram negative bacteria is H-NS. Members of the H-NS protein family are involved inthe regulation of for horizontally acquired genes that include most genes involved inpathogenicity and antibiotic resistance. H-NS family members bind to DNA as oligomersand there is a clear distinction between the regions associated to oligomerization and theDNA binding domains. We shall present collaborative work carried out in the frameworkof EAST-NMR and BIO-NMR including a) the determination of the structure of a DNAcomplex of the DNA binding region of Ler, a nucleoid associated protein that competeswith H-NS for the activation of the LEE operon encoding for a type III secretion sys-tem in enterohaemorragic E. coli (EHEC), b) the solid-state NMR characterization of fulllength H-NS oligomers (in collaboration with the Utrecht NMR facility), and c) the ini-tial characterization of YmoB, a NAP of unknown structure involved in biofilm formationusing, among other, 13C-detected NMR experiments carried out in collaboration with theFlorence NMR facility.

28

Lecture, Tue 12:10

Tyr-modified cytochrome c and cell death in enkaryotic systems

Aroca A, Garcıa-Heredia JM, Dıaz-Quintana A, De la Rosa MA, Dıaz-Moreno I

Instituto de Bioquımica Vegetal y Fotosıntesis, Centro de Investigaciones CientıficasIsla de la Cartuja, Universidad de Sevilla-CSIC, Spain

T-cell-restricted intracellular antigen-1 (TIA-1) is a ubiquitous RNA binding proteinthat acts as an effector of programmed cell death (PCD) depending on post-transcriptionalregulation of cytochrome c (Cc)-coding mRNA. TIA-1 contains three RNA recognitionmotifs (RRMs) – along with a single glutamine-rich domain – which specifically bind tothe AU-rich elements (AREs) located at the 3’ UTR regions of the Cc mRNA. So TIA-1acts as a transcriptional repressor of Cc-coding mRNA by sequestering it into the cyto-plasmic ‘stress granules’ and by avoiding its translocation and further degradation into‘P-bodies’. As mRNA translation is transiently repressed by TIA-1, both the Cc expres-sion and the apoptotic programme can be restarted upon cell death signalling. PCD isalso regulated by post-translational modification of Cc, whose tyrosine residues can benitrated or phosphorylated inside the mitochondria. Both modifications take place underdifferent cellular conditions. Whereas Cc phosphorylation occurs in cells under physio-logical conditions, the haemprotein is nitrated in almost all its tyrosine residues understress or apoptotic conditions. So understanding the structural, dynamic and physiologi-cal features of Tyr-modified Cc may allow a deeper insight into the underlying molecularmechanisms of processes related to cell death in eukaryotic systems. This work has beenfunded by the Ministry of Science and Innovation (BFU2009-07190) and the AndalusianGovernment (BIO-198). We also acknowledge the EU-NMR and Bio-NMR Projects forsupport.

29

Lecture, Tue 13:30

What’s behind Disordered – Unfolded – Unstructured?

Gerald Platzer, Leonhard Geist, Gonul Kizilzavas, Nicolas Coudevylle andRobert Konrat

Department of Structural and Computational Biology, Max F. Perutz Laboratories,University of Vienna, Vienna Biocenter Campus 5, A-1030 Vienna, Austria;

The classical structure-function paradigm that protein functionality relies on the ex-istence of a stably folded protein scaffold has been put in question and it is now acknowl-edged that an increasing number of proteins are lacking stably folded tertiary structuresand that this intrinsic flexibility has significant impact on biological functionality. Thepopular dichotomic partitioning of proteins into ordered and disordered specimens thusdoes not seem to appropriately grasp the great variety of conformational ensembles offluctuating biological polypeptides. Here we demonstrate that the binary order-disorderconceptual framework can be overcome by the recently introduced meta-structure ap-proach. Adapting concepts from renormalization theory we provide evidence for multi-phase transition behaviour in intrinsically disordered proteins and a novel way to cal-culate comprehensive structural ensembles. Data obtained on Osteopontin, a cytokineimplicated in tumor growth and metastasis and BASP1, a tumor suppressor illustratethis novel approach.

30

Lecture, Tue 14:05

Structure determination by Solid-state NMR

Meier BH(1), Bockmann A(2), Gath J(1), Guntert P (3), Huber M(1), van MelkebekeH(1) Schanda P(B), Schutz A(1), Szekeley K(1), Verel R(1), Wasmer C (1)

(1) Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland (beme@nmr.ethz.ch) (2)Institut de Biologie et Chimie des Proteines, CNRS-UMR 5086, 69367 Lyon, France (3)Institut fur Biophysikalische Chemie, J.W. Goethe-Universitat, 60438 FrankfurtGermany

Structure determination from solid-state NMR spectra is still a challenge. While ahandful of atomic resolution proteins have indeed be solved already, structure determina-tion of completely unknown proteins and of larger proteins (¿150 residues) is still difficultand prone to error. The talk will describe some of the problems and pitfalls and sketch ordescribe some recipes that promise to advance the field in the future. These include betterand more efficient pulse sequences, enhanced spectral resolution, higher-dimensional spec-troscopy, and improved computational processes to arrive at the correct structure forma set of given solid-state spectra. The principles will be illustrated using microcrystallineproteins as well as fibrils.

31

Lecture, Tue 14:40

Proton detection of aliphatic resonances with ssNMR spectroscopy

Asami S(1), Schmieder P(2), Schanda P(3), Meier B(4), Reif B(5,6,7)

(1) Structural Biology – Solid-State NMR, Leibniz-Institut fuer MolekularePharmakologie (FMP), Berlin, Germany, asami@fmp-berlin.de (2) Structural Biology –Solution NMR, Leibniz-Institut fuer Molekulare Pharmakologie (FMP), Berlin,Germany, schmieder@fmp-berlin.de (3) Laboratory of Physical Chemistry – Solid-StateNMR, Eidgenoessische Technische Hochschule Zuerich, Zuerich, Switzerland,Paul.Schanda@nmr.phys.chem.ethz.ch (4) Laboratory of Physical Chemistry –Solid-State NMR, Eidgenoessische Technische Hochschule Zuerich, Zuerich, Switzerland,beat.meier@nmr.phys.chem.ethz.ch (5) Structural Biology – Solid-State NMR,Leibniz-Institut fuer Molekulare Pharmakologie (FMP), Berlin, Germany,reif@fmp-berlin.de (6) Department of Chemistry, Universitaet Muenchen, Muenchen,Germany, reif@tum.de (7) Helmholtz-Zentrum Muenchen, Muenchen, Germany,reif@tum.de

Biological magic angle spinning (MAS) solid-state nuclear magnetic resonance spec-troscopy has developed rapidly over the past two decades. For the structure determinationof a protein by solid-state NMR, dihedral, as well as 13C-13C and 15N-15N long rangedistance restraints, are employed. Long-range distance restraints define the tertiary struc-ture of a protein, but are difficult to detect in uniformly isotopically enriched samples.In protonated samples, long range distances are accessed by growing the bacterium on amedium which contains [1,3]-13C glycerol or [2]-13C glycerol to dilute the 13C spin sys-tem. Labeling schemes which rely on heteronuclei are insensitive both for detection andin terms of quantification of distances, since they are relying on low-γ nuclei. Due to thepresence of large 1H-1H dipolar interactions in uniformly protonated samples, resolutionin the proton dimension is typically compromised, even in case homonuclear decouplingsequences are employed. We report a labeling scheme, which enables 1H-detection ofaliphatic resonances with high-resolution in MAS solid-state NMR spectroscopy. High-resolution spectra allow easy assignment of side chain 1H resonances. We show furtherthat 13C resolved 3D-1H-1H correlation experiments yields access to long-range proton-proton distances in the protein.

32

Lecture, Tue 16:30

Structural studies of EspFU, a novel SH3 ligand triggering thepathogen-driven actin assembly

Aitio O(1), Hellman M(1), Mantylahti S(1), Kazlauskas A(2), Vingadassalom D(3),Leong JM(3), Saksela K(2), Permi P(1)

(1) Program in Structural Biology and Biophysics, Institute of Biotechnology, Universityof Helsinki, FI-00014, Helsinki, Finland (2) Department of Virology, HaartmanInstitute, University of Helsinki and HUSLAB, Helsinki University Central Hospital,FI-00014, Helsinki, Finland (3) Department of Molecular Genetics and Microbiology,University of Massachusetts Medical School, Worcester, MA 01655, USA

E. coli secreted protein F-like protein from prophage U (EspFU) is a translocatedbacterial effector, which together with translocated intimin receptor (Tir) and host’s in-sulin receptor tyrosine kinase substrate (IRTKS) activates actin polymerization leadingto actin pedestal formation that is required to firm attachment of enterohemorrhagic E.coli (EHEC) pathogen to the intestinal epithelia. While the first 20 N-terminal residues ofEspFU has been shown to establish a high-affinity complex with the GTPase binding do-main (GBD) of Wiskott-Aldrich syndrome protein (WASP), the proline-rich C-terminusof EspFU associates with the Src homology 3 (SH3) domain of IRTKS. Structural char-acterization of EspFU and structural basis of its novel SH3 interaction triggering thepathogen-driven actin assembly will be discussed.

33

Lecture, Tue 17:05

Oxidation-state-dependent protein-protein interactions in disulfide cascades

Mavridou DAI(1), Saridakis E(2), Kritsiligkou P(1), Stevens JM(1), Goddard AD(1),Ferguson SJ(1) and Redfield C(1)

(1) Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX13QU, United Kingdom (2) Institute of Physical Chemistry, NCSR Demokritos, AghiaParaskevi, PO Box 60228, Athens, Greece Email address of presenting author:christina.redfield@bioch.ox.ac.uk

Oxidative protein folding, involving the formation and isomerization of disulfide bonds,is essential for the stability and function of numerous proteins in all organisms. In vivo,thiol:disulfide exchange reactions are catalyzed by a range of thiol:disulfide oxidoreduc-tases which often contain the thioredoxin fold. The protein DsbD transfers electrons fromthe cytoplasm to the periplasm of Gram-negative bacteria. This reducing power is requiredin the otherwise oxidizing periplasm for the isomerization of incorrectly formed disulfidebonds (Dsb) and for cytochrome c maturation (Ccm). Reductant transfer occurs via aseries of sequential thiol:disulfide exchange reactions between pairs of conserved cysteinesin the three domains of DsbD, tmDsbD (the integral membrane domain), nDsbD andcDsbD (the N- and C-terminal periplasmic globular domains) and their partner proteinson both sides of the inner membrane. The flow of electrons starts from cytoplasmic thiore-doxin and proceeds to tmDsbD, then to cDsbD and nDsbD and ends with the periplasmicproteins DsbC, DsbG and CcmG. Here we have used NMR spectroscopy to analyze thefactors which contribute to the reduction of nDsbD by cDsbD. We address the questionsof what happens before and after electron transfer between the two globular domainsand why the reverse reaction is avoided in what has been described by others as the“thermodynamically neutral” step of this disulfide cascade. We show that interactionsbetween these domains are oxidation-state dependent such that the functionally-relevantcomplex will be markedly more populated than the product complex. Understanding thealtered affinities of the binary protein complexes in response to oxidation state is key tounderstanding thiol-disulfide cascades. The principles established have wider significancefor processes mediated by thioredoxin-like proteins which are critical in both prokaryotesand eukaryotes.

34

Lecture, Tue 17:40

From interactions of small molecule ligands and their self assembly to DNAquadruplexes and prion proteins

Plavec J(1,2,3)

(1) Slovenian NMR Centre, National Institute of Chemistry, Ljubljana, Slovenia (2)EN→ FIST Centre of Excellence, Ljubljana, Slovenia (3) University of Ljubljana,Slovenia

The steroelectronic properties of ligand-binding cavities of renin angiotensin aldos-terone system, which is stimulated by several signals including a drop in blood pressure,help in identification of new inhibitors such as aliskiren (T. Mavromoustakos, Universityof Athens).

The formation of supramolecular aggregates by self-assembly is a widely acceptedmethod to build complexes bearing cavities large enough to encapsulate guest molecules,and exhibit a number of exciting properties such as the possibilities to stabilize and studyreactive molecules or to act as nanoreaction vessels that permit formation of reactionproducts which are not obtainable in bulk solutions because of the unique confined envi-ronments offered by the cavities (A. Lutzen, University of Bonn).

In contrast to double-helical DNA the rules for self-assembly of G-quadruplexes arebeing established (M. Webba da Silva, University of Ulster). G-quadruplexes are built ofG-quartets and require cations for their formation, structural integrity and stabilization(1). In general, cations are localized along the central cavity of a G-quadruplex. Oligonu-cleotides containing a single run of guanines form tetramolecular G-quadruplex structures.We have shown recently that d(TG4T) is involved in the equilibrium of two monomericforms in the presence of K+, Na+ and 15NH4+ ions (2).

Prion propagation in transmissible spongiform encephalopathies involves the conver-sion of the cellular prion protein into the pathogenic conformer. Human familial forms ofthe disease are linked to the specific mutations such as in Gerstmann-Straussler-Scheinkersyndrome (GSS). Inherited point mutations may increase the likelihood of misfolding (G.Legname, SISSA, Italy). We determined high-resolution 3D structure of the truncatedrecombinant human PrP (residue from 90 to 231) containing the GSS-related Q212P mu-tation. The substitution of a glutamine by a proline at the position 212 reveals differencesin the C-terminal end of the protein and the β2–α2 loop region (3).

References:(1) M. Webba da Silva, M. Trajkovski, Y. Sannohe, N. Ma’ani Hessari, H. Sugyiama,

J. Plavec, Angew. Chem. Int. Ed. 2009, 48, 916.(2) P. Sket, J. Plavec, J. Amer. Chem. Soc. 2010, 132, 12724.(3) G. Ilc, G. Giachin, et al., PLoS One, 2010, 5: e11715.

35

Lecture, Wed 9:00

Exploring Multiple Timescale Motions in Folded and Intrinsically DisorderedProteins using NMR

Guillaume Communie, Frank Gabel, Jie-Rong Huang, Malene Ringkjøbing Jensen, LucaMollica, Valery Ozenne, Loic Salmon, Robert Schneider, Yai Mingxi, Martin Blackledge

Protein Dynamics and Flexibility, Institute de Biologie Structurale Jean-Pierre Ebel,CNRS; CEA; UJF; UMR 5075, 41 rue Jules Horowitz, F-38027 Grenoble, Cedex,France. martin.blackledge@ibs.fr

Proteins are inherently flexible, displaying a broad range of dynamics over a hierarchyof time-scales from pico-seconds to seconds. This molecular plasticity enables conforma-tional changes in protein backbone and sidechains that are essential for biomolecularfunction. NMR is sensitive to all conformational fluctuations occurring up to the millisec-ond and over the last five years we have developed robust approaches to quantitativelydescribe these molecular motions from NMR data. We combine analytical and numericalapproaches to develop a self-consistent representation of all motions occurring in proteinson timescales from the picosecond to the millisecond. The development of meaningful de-scriptions of the behaviour of intrinsically disordered proteins (IDPs), is a key challengefor contemporary structural biology. Explicit molecular ensembles representing a dynamicequilibrium of rapidly interconverting conformers are gradually becoming established asthe most appropriate descriptors to define protein conformational disorder. Due to theincrease in available degrees of conformational freedom compared to a static picture, theidentification of accurate protein conformational ensembles requires the development ofrobust approaches to determine the significance and uniqueness of any proposed equilib-rium. We have developed methods to determine local and long-range structural behaviourin IDPs from NMR and small angle scattering data and to elucidate the role of this flex-ibility. We have identified local conformational preferences in a number of importantunfolded proteins that were previously inaccessible to structural biology. We develop newtechniques to determine the level of intrinsic structure in IDPs and applied this to de-scribe the pre-recognition state of active sites of viral proteins. These same methods arebeing used to understand protein folding and stability and in particular the study of themolecular basis of chemical denaturation of proteins.

36

Lecture, Wed 9:35

Recovering lost magnetization: polarization enhancement in biomolecularNMR

Favier A, Brutscher B

IBS, Institut de Biologie Structurale Jean-Pierre Ebel, 41 rue Jules Horowitz, F-38027Grenoble; CEA; CNRS; Universite Joseph Fourier

Experimental sensitivity remains a major drawback for the application of NMR spec-troscopy to fragile and low concentrated biomolecular samples. Here we describe anefficient polarization enhancement mechanism in longitudinal-relaxation enhanced fast-pulsing triple-resonance experiments. By recovering undetectable 1H polarization origi-nating from longitudinal relaxation during the pulse sequence, the steady-state 15N po-larization becomes enhanced by up to a factor of ∼5 with respect to thermal equilibriumyielding significant sensitivity improvements compared to conventional schemes. The ben-efits of BEST-TROSY experiments with respect to BEST-HSQC or conventional pulseschemes at high magnetic field strength will be illustrated for various protein and RNAapplications.

37

Lecture, Wed 10:10

Study of zaleplon/cyclodextrin complexation by 1H-NMR spectroscopy

Jug M (1), Jablan J(2)

(1) Department of Pharmaceutics, Faculty of Pharmacy and Biochemistry, University ofZagreb, A. Kovacica 1, 10 000 Zagreb, Croatia, mjug@pharma.hr (2) Department ofAnalytical Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, A.Kovacica 1, 10 000 Zagreb, Croatia, jjablan@pharma.hr

Cyclodextrins (CDs) are a group of structurally related cyclic oligosaccharides derivedfrom bacterial starch degradation. The ground for their popularity from a pharmaceu-tical point of view is in their ability to favourably modify undesired biopharmaceuticalproperties of the drug, such low chemical stability and limited aqueous solubility, bythe inclusion complex formation. Numerous CD derivatives has been synthesized in or-der to extend complexing and solubilizing properties of naturally occurring CDs. Dueto their bioavailability and lack of oral toxicity, CDs are used as carriers in order toimprove bioavailability of drugs with limited aqueous solubility or to achieve controlleddrug release. Zaleplon (ZAL) is a hypnotic drug indicated for a short term therapy ofinsomnia. Due to low solubility in water, the oral bioavailability of the ZAL is only 30%of applied dose. In order to improve aqueous solubility and consequently increase oralbioavailability of the drug, the interaction of ZAL with natural β-CD and its randomlymethylated derivative (RAMEB) was studied. 1H-NMR spectroscopy was used to confirmthe actual inclusion complex formation and to put some insight into host-guest bindingmode. One dimensional 1H-NMR showed that in the presence of ZAL, H3 and H5 proton,which are lining the interior of the cavity of βCD and H6 proton, which is located at thenarrower end of the βCD torus, experienced a strong shielding effect, clearly confirmingthe actual inclusion complex formation. In the same time, the majority of ZAL protonsshowed a down-field shift in their resonances, due to deshielding effects caused by van derWaals interaction between the drug and carbohydrate ring upon complexation. In case ofRAMEB, deep penetration of ZAL into central cavity of CD was also evidenced. Furtherevidence for the inclusion complex formation between ZAL and CDs was obtained byDOSY spectroscopy, while ROESY spectra allowed determination of ZAL/CD bindingmodes

38

Lecture, Wed 11:00

Insight into RNA splicing and editing mechanisms from the NMR structuresof protein-RNA complexes.

Allain FHT

Institute of Molecular Biology and Biophysics, ETH Zurich, CH-8093 Zurich,Switzerland

RNA binding proteins (RBP) are often modular proteins being composed of one toseveral small RNA binding domains (80-100 aa) of different types, the most common onesbeing the RRM (RNA Recognition Motif), the dsRBM (double-stranded RNA BindingMotif) and the KH domain (hnRNP K homology domain). In order to understand thefunction and mechanism of action of RBP involved in RNA alternative-splicing and RNAediting, we recently solved the solution structures of five alternative-splicing factors con-taining RRMs (Tra2β1, SC35, ASF-SF2, hnRNP F and hnRNP L) and of one editingfactor containing two dsRBMs (ADAR2) bound to their respective RNA substrates. Thestructures of the RRM-containing proteins revealed very different mode of recognitiondespite embedding the same common RRM fold. While the β-sheet of the RRM remainsthe primary RNA binding surface for SC35, Tra2β1 and hnRNP L, several extension(large loops, N- and C-terminal extremities) are necessary to achieve high affinity andsequence-specificity. ASF-SF2 pseudo-RRM2 and hnRNP F quasi-RRMs use a very RNAdifferent binding surface to achieve equally high RNA binding affinity and specificity re-vealing the extraordinary versatility of this RNA binding domain. Implication of thesedifferent binding modes for splicing regulation have been experimentally investigated forTra2β1 and hnRNP F with SMN exon 7 and Bcl-x mRNA, respectively. The structureof the two dsRBMs of the Adenosine deaminase ADAR2 bound to RNA revealed thatRNA recognition by dsRBMs are more sequence-specific than previously thought andthat this sequence-specificity is achieved by a direct of readout of the minor-groove of theA-form RNA helix. Mutations in the RNA or in the protein confirmed the importanceof this sequence-specific readout for ADAR2 RNA binding and for editing. The structureof ADAR2 dsRBMs bound to RNA also suggests how ADAR2 edit selectively certainadenines over others.

39

Lecture, Wed 11:35

Solvent PRE-assisted structural analysis of large protein complexes

Madl T(1,2,3), Guttler T(4), Gorlich D(4), Sattler M(2,3)

(1) NMR Spectroscopy Research Group, Bijvoet Center for Biomolecular Research,Utrecht University, 3584 CH Utrecht, Netherlands, t.madl@uu.nl (2) Institute ofStructural Biology, Helmholtz Zentrum Munchen, 85764 Neuherberg, Germany (3)Department Chemie, TU Munchen, 85747 Garching, Germany (4) Max-Planck-Institutfur Biophysikalische Chemie, Gottingen, Germany

Determining structure and architecture of multi-component proteins and their com-plexes is essential for understanding cellular processes. Here we present an efficient ap-proach for structural analysis of large and transient protein complexes. Among the avail-able structure determination techniques, rigid-body assembly/docking approaches arewidely used. Nevertheless, when only a sparse restraint set is available, these approachesbecome highly ambiguous as they often converge to multiple distinct possible conforma-tions. We present an efficient, general and robust strategy for improving structure calcula-tions using paramagnetic relaxation enhancements (PREs) from the soluble paramagneticagent Gd(DTPA-BMA). Solvent PREs provide a rich source of structural/dynamic infor-mation and constitute an attractive because complementary alternative to covalent spinlabels[1,2]. The strength of this methodology is that it is readily implemented (no cova-lent modifications needed), and that (transient) interactions and local structure can beefficiently detected and refined. Motivated by understanding the general mechanisms be-hind nuclear export of cargos in the challenging ternary 150 kDa nuclear export complexwe show that accuracy and convergence of conventional approaches can be significantlyimproved by a very limited set of experimental solvent PRE data. First, solvent PREsare an excellent indicator of the quality of structural models and thus resolve ambiguities;second, direct refinement against solvent PRE data further improves structural accuracyand precision. Thus, our approach suggests that solvent PREs provide a new class ofrestraints that are easily accessible and applicable to large complexes. In particular forchallenging systems, our approach promises significant time-savings and significantly im-proved quality of structure calculations. [1] Angew Chemie, 48 (2009) 8259 [2] JACS, 124(2002) 372 [3,4] NSMB, 17, (2010) 1367; subm.

40

Lecture, Wed 12:10

NMR structure of the human prion protein with the pathological Q212Pmutation: insights into inherited human prion diseases

Giachin G* (1), Ilc G* (2,3), Biljan I (3,4), Jaremko M (5), Jaremko L (5,6), Benetti F(1,7), Plavec J (2,3,8) Igor Zhukov(3,5) and Giuseppe Legname(1,7,9)

(1) Laboratory of Prion Biology, Neurobiology Sector, Scuola Internazionale Superiore diStudi Avanzati (SISSA), via Bonomea 265, I-34136 Trieste, Italy (2) EN→FIST Centreof Excellence, Dunajska 156, SI-1001, Ljubljana, Slovenia (3) Slovenian NMR Centre,National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia (4)Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102A,HR-10000 Zagreb, Croatia (5) Laboratory of Biological NMR, Institute of Biochemistryand Biophysics, Polish Academy of Sciences, ul. Pawinskiego 5a, PL-02-106 Warsaw,Poland (6) Faculty of Chemistry, Warsaw University, Pasteura 1, PL-02-093, Warsaw,Poland (7) Italian Institute of Technology, SISSA Unit, via Bonomea 265, I-34136Trieste, Italy (8) Faculty of Chemistry and Chemical Technology University of Ljubljana,Askerceva cesta 5, SI-1000 Ljubljana, Slovenia (9) ELETTRA Laboratory, SincrotroneTrieste S.C.p.A., I-34149 Basovizza, Trieste, Italy * These authors contributed equally tothe work E-mail addresses of corresponding authors: janez.plavec@ki.si, legname@sissa.it

Prion diseases are a group of neuropathies characterized by a spongiform neurode-generation of the brain caused by prions. According to the “protein-only hypothesis”,during prion diseases the cellular prion protein, PrPC, is converted into an abnormalform, called PrPSc, by a not well-elucidated process of conversion from the α-helix intoβ-sheet secondary structures. One of the strongest arguments supporting the “protein-only hypothesis” is the link between prion diseases and inherited human mutations in thePRNP gene. Several point mutations leading to familial Creutzfeldt-Jakob (CJD) disease,Gerstmann-Straussler-Scheinker (GSS) disease, Fatal Familial Insomnia have been iden-tified in PRNP gene. Our understanding of the mechanisms by which mutations causedisease remains limited. We believe that high-resolution 3D structure of the mutated prionprotein, more susceptible for spontaneous conversion into the infectious form, will helpus to understand the molecular mechanism at early stages of the disease. In this studywe determined and examined a high-resolution 3D structure of the truncated recombi-nant human PrP (residue from 90 to 231) containing the GSS-related Q212P mutation.The substitution of a glutamine by a proline at the position 212 reveals novel structuraldifferences in comparison to the known PrP structures. The most remarkable differencesinvolve the C-terminal end of the protein and the β2–α2 loop region. This structuremight provide new insights into the early events of conformational transition of PrPCinto PrPSc. Indeed, the spontaneous formation of prions in familial cases might be dueto the disruptions of the hydrophobic core consisting of β2–α2 loop and α3 helix.

Reference:Ilc G, Giachin G, J et al., PLoS One, 5(7): e11715. doi:10.1371/journal.pone.001 1715,

(2010)

41

Lecture, Wed 13:30

NMR view on helix-helix interactions of membrane proteins

Arseniev AS, Bocharov EV, Shenkarev ZO, Mineev KS, Paramonov AS

Department of Structural Biology, Shemyakin& Ovchinnikov Institute of BioorganicChemistry, Russian Academy of Sciences, 117997, Moscow, Russian Federation,aars@nmr.ru

Helical membrane proteins are a major class of membrane proteins that are essentiallyinvolved in key processes including bioenergetics, signal transduction, ion transmission,catalysis, and so on. This class of proteins is characterized by the presence of highlyhydrophobic stretches of ≥ 20 amino acids, which span the membrane in a helical con-formation. Helical membrane proteins can exist as simple structures, with just one or afew helices spanning a membrane, as well as large oligomeric complexes with many trans-membrane helices. The lecture will present our experience with solution NMR study ofstructure and dynamics of several homo and hetero dimers of transmembrane domainsof EphA and ErbB families of bitopic receptor tyrosine kinases and four-helical voltage-sensing domain of the archaeal potassium channel KvAP. The following issues will bediscussed: a) effective peptide/protein expression, b) choice of solubilization media andprove of the native fold, c) search for NMR constraints, d)structure calculation and re-finement, e) specificity and details of helix-helix interactions and f) what can we learn atpresent about functioning of the membrane proteins based on NMR data.

42

Lecture, Wed 14:05

NMR Noise Spectroscopy

Muller N. (1), Nausner M. (1), Schlagnitweit J. (1), Smrecki V. (2)

(1) Institute of Organic Chemistry, Johannes Kepler University, Altenbergerstr. 69,A-4040 Linz, Austria, norbert.mueller@jku.at (2) NMR Center, Rudjer BoskovicInstitute, Bijenicka 54, HR-10002, Zagreb, Croatia

NMR noise spectra depend on the resonance circuit, spin density, relaxation, inho-mogeneous broadening, radiation damping and temperature. Although they are powerspectra, line shapes are generally mixed absorptive-dispersive. At the conventional tuningoptimum (CTO) noise signals should appear as dips in the thermal noise power base line[1]. However, this symmetrical dip line shape is usually found at large tuning offsets fromthe Larmor frequency, the spin noise tuning optimum, SNTO. At the CTO, often hun-dreds of kHz from the SNTO, dispersive noise line shapes prevail. Both pure spin noise,from incomplete cancellation of random fluctuations of magnetic moments, and absorbedcircuit noise ACN, due to radiation damping [2], contribute to noise spectra. The complextuning dependence [3] can be attributed largely to ACN. The absorptive pure spin noisecontribution can be observed essentially undisturbed in wide line solids spectra [4] andin presence of field gradients [5]. By adjusting probes for SNTO conditions up to 50%increase in sensitivity can be achieved for different types of solid and liquid state experi-ments [3,4,6]. [1]McCoy, M.A.; Ernst, R.R. Chem Phys Lett 1989, 139, 587 [2]Giraudeau,P.; Muller, N.; Jerschow, A.; Frydman, L. Chem Phys Lett 2010, 489, 107 [3]Nausner, M.;Schlagnitweit, J.; Smrecki, V.; Yang, X.; Jerschow, A.; Muller, N. J Magn Reson 2009,198, 73 [4]Schlagnitweit, J.; Dumez, J.N.; Nausner, M.; Jerschow, A.; Elena-Herrmann,B.; Muller, N. J Magn Reson 2010, 207, 168 [5]Muller, N.; Jerschow, A. Proc Natl AcadSci USA 2006, 103, 6790 [6]Nausner, M.; Goger, M.; Bendet-Taicher, E.; Schlagnitweit,J.; Jerschow, A.; Muller, N. J Biomol NMR 2010, 48, 157 This work was supported by:European Union (FP7 EAST-NMR, Contract # 228461; FP6 EU-NMR Contract# RII3-026145), Austrian Marshall Plan Foundation, Croatian Ministry of Science, Educationand Sports (098-0982929-2917); OAD (WTZ AT-HR 19/2008); Austrian Science FundsFWF (P19635-N17).

43

Lecture, Wed 14:40

NMR of RNA at 1GHz – A New Frontier in Structural Biology

Ramesh V

School of Chemistry and Manchester Interdisciplinary Biocentre, University ofManchester, 131, Princess Street, Manchester, M1 7DN, U.K,vasudevan.ramesh@manchester.ac.uk

The picornaviruses are a family of small icosahedral animal viruses that contain single-stranded RNA genomes (∼ 8K bases). A salient feature of these viral RNAs is that they areendowed with unusually long 5’-untranslated regions (5’-UTR) (600 – 1300 nucleotides)containing a high degree of secondary structure. The precise role and mode of recognitionof such secondary structures are unclear at this stage. As a consequence of this feature,initiation of protein synthesis has been shown to take place by a novel mechanism in whichtranslation of picornaviral mRNAs is directed by RNA elements (∼ 450 nucleotides)known as internal ribosomal entry sites (IRES) that occur within the 5’-UTR regions.Within the cardio-and aphthovirus IRES elements, at the end of one predicted domain,is a hammerhead motif that contains a GNRA tetraloop. Such tetraloops are highlyrepresented in complex RNA structures. The importance of this tetraloop is demonstratedby the complete loss of IRES function when the 3’ terminal A residue is modified. Wepropose to carry out a 1 GHz NMR structural investigation of this hammerhead motif (75-mer) from the encephalomyocarditis (EMCV) and foot and mouth disease virus (FMDV)IRES elements. Our preliminary studies provide support for a stable, well folded structurefor the predicted motif. We have also prepared segmentally labelled 75-mer RNA forstructure determination and are improving the RNA ligation method to generate NMRamounts of the labelled RNA. We have also initiated NMR studies of of RNA-RNAinteractions within the I-domains of EMCV and FMDV and RNA-protein interactionswithin the J-domain of EMCV to gain structural evidence for such interactions which arehighly relevant for elucidating the IRES mechanism. At present there are no commerciallyavailable drug therapies for any of the diseases caused by picornaviruses and thus theresults of the study will be significant.

44

Lecture, Wed 16:30

Structure and dynamics in gene regulation and DNA repair

Rolf Boelens

Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands

NMR spectroscopy has developed to an invaluable tool to investigate the structure anddynamics of biomolecules. NMR provides unique detailed information on the interactionsbetween biomolecules which can be weak and transient, that cannot be easily obtainedby other methods. This progress is due to technical advances in NMR spectroscopy (3Ddouble- and triple-resonance NMR, and high-field and sensitive NMR instruments), inprotein production (high-throughput protein production and uniformly 13C/15N/2H la-beling) and considerable advances in computing.

At Utrecht we use NMR spectroscopy to study structure and dynamics of protein-protein and protein-DNA complexes involved in DNA transcription and DNA repair.Studied examples are (i) E.coli Lac repressor, (ii) the Kid-Kis plasmid maintenance sys-tem, (iii) transcription factor TAF3 histone interactions and (iv) the Rad6/Rad18 ubiq-uitination complex. 13C/15N isotope labeling was required for all NMR studies, andin several cases complemented with 2H labeling. In many cases the NMR studies werecomplimented with biophysical data and results from complimentary structural biologytechniques.

The different examples demonstrate the strength and flexibility of NMR for studyingthe structure and dynamics of proteins and complexes involved in cellular regulation.

45

Lecture, Wed 17:05

NMR-optimized cell-free protein expression

Karlsson BG, Pedersen A, Isaksson L and Enberg J

Swedish NMR Centre, University of Gothenburg, SE405 30 Goteborg, Sweden,goran.karlsson@nmr.gu.se

Cell-free protein synthesis is a convenient method for producing preparative amountsof protein for structural biology. Highly specific isotope incorporation for NMR spec-troscopy is especially efficient as the absence of an active metabolism to a large extentprevents scrambling. However, only about 15% of the supplied amino acids are currentlyincorporated into the target protein even in the best reported systems, suggesting a sig-nificant potential for improvement by optimization of the existing system as well as byintroducing new additives. This is important as the amino acids account for more thanhalf of the costs associated with producing 13C,15N-labelled samples for NMR.

46

Lecture, Thu 9:00

Methyl-TROSY NMR of death domain assemblies: low symmetryorganisation suggested by non-degenerate chemical shifts.

Esposito D(1), Nematollahi L(1), Sankar A(1), Morgner N(2), Robinson CV(2),Rittinger K(1), Driscoll PC(1)

(1) Medical Research Council-National Institute for Medical Research, Mill Hill, LondonNW7 1AA, UK and (2) Department of Chemistry, University of Oxford, Oxford, UK;pdrisco@nimr.mrc.ac.uk

Death domains (DDs) exhibit a Greek-key α-helical fold and are known to take partin homotypic binary interactions between DD-containing proteins. We report heteronu-clear NMR investigations of the complexes formed between the DDs of (i) CD95/Fas andFADD, and (ii) PIDD and RAIDD. In the former example the NMR data unequivocallydemonstrate, contrary to recently published X-ray crystallographic evidence, that theglobular part of CD95-DD retains its native fold and the extreme C-terminal region ofCD95 remains in a flexible state. Instead, hydrodynamic and ESI-mass spectrometry datasuggest an overall size of the complex consistent with 5:5 stoichiometry, reminiscent of thecore of the complex formed between RAIDD and PIDD DDs. 13C-methyl-ILV labellingand methyl-TROSY spectra suggest that both complexes lack mirror image symmetry,consistent with the emerging concept that DDs can form multidomain assemblies withpseudo-helical DD packing arrangements.

47

Lecture, Thu 9:35

DNA Quadruplex Topologies by Design

Mateus Webba da Silva

School of Biomedical Sciences, University of Ulster, Cromore Road, BT51 1SA, UK

The duplex topology of DNA has a well-defined geometry, the sequential contextof its components is highly variable, and most importantly, its self-assembly is highlypredictable for forming programmable intra- and intermolecular interactions. Thus, rawmaterial for nanotechnological bottom-up approaches. (1) We have been working on thesystematization of the principles that form the basis of control for the self-assembly offour-stranded DNA architectures denominated G-quadruplexes. DNA quadruplex struc-tures are likely to be formed in vivo, having roles in key biological processes such as themaintenance of telomeres, regulation of gene transcription, DNA recombination, and pack-aging of the retroviral genome. However, current interest in these architectures includesthe programmed build up of DNA objects, devices, and materials. Thus a systematizationof the principles that form the basis of control for the assembly process is necessary. (2),(3) This new dimension will not only allow for structural versatility, but also result in theability to control motion within any structures built with these topologies.

We will briefly present results on our efforts to demonstrate control of self-assemblyof quadruplexes, and their application to the build-up of nanostructured materials.

References(1) Yan, H. Science 2004, 306, 2048.(2) Webba da Silva, M. Chemistry European Journal 2007, 13, 9738.(3) Webba da Silva, M.; Trajkovski, M.; Sannohe, Y.; Ma’ani Hessari, N.; Sugiyama,

H.; Plavec, J. Angew Chem Int Ed Engl 2009, 48, 9167.

48

Lecture, Thu 10:10

Transcription termination by the Nrd1 complex

Kubicek K, Hobor F, Cerna H, Pergoli R, Bacikova, V, Pasulka P, Stefl R

National Centre for Biomolecular Research, Central European Institute of Technology,Masaryk University, Brno, Czechia

Non-coding RNA polymerase II transcripts are processed by the poly(A) independenttermination pathway that requires the Nrd1 complex. Nrd1 complex is recruited to thetermination site by the C-terminal domain of RNA Polymerase II phosphorylated at serine5 (pSer5). It also binds to RNA termination signals and thus facilitates the terminationwith the assistance of TRAMP-exosome machinery. We will present the structural basisof how the Nrd1 complex recognizes the pSer5 C-terminal domain of RNA Polymerase IIand one of the RNA termination signal.

49

Lecture, Thu 11:00

Solid-State NMR as a Tool in Structural Biology: VDAC, Tau, and theTTSS Needle

Vijayan V(1), Daebel V(1), Loquet A(1), Schneider R(1), Gattin Z(1), Seidel K(1),Kolbe M(2), Zychlinski A(2), Griesinger C(1), Becker S(1), Mandelkow E(3), Lange A(1)

(1)Max Planck Institute for Biophysical Chemistry, Gottingen, Germany (2)Max PlanckInstitute for Infection Biology, Berlin, Germany (3)Max Planck Unit for StructuralMolecular Biology, Hamburg, Germany adla@nmr.mpibpc.mpg.de

Solid-state NMR has recently emerged as a powerful technique in structural biology.For instance, ssNMR opens new ways to study membrane proteins in their natural lipidenvironment, or insoluble disease-associated protein aggregates. Moreover, functional fil-amentous assemblies such as the needle of the Type Three Secretion System (TTSS) –composed of multiple copies of a single small protein – can be readily studied. In terms ofmembrane proteins our main interest lies on the voltage-dependent anion channel (VDAC)that is located in the mitochondrial outer membrane and constitutes the major pathwayfor the transport of ADP and ATP. Recently we have determined the native conformationof the N-terminal part of human VDAC1 in liposomes by ssNMR [Schneider et al. Ange-wandte Chemie 2010]. Here, we report the measurement of dipolar order parameters forresidues in the N-terminus as well as in other parts of the molecule. Tau paired helical fil-aments (PHFs) are the major constituent of neurofibrillary tangles (NFTs). A progressiveaccumulation of NFTs in neurons is one of the hallmarks of Alzheimer’s disease. Here, wepresent a detailed structural model of PHFs formed by a three-repeat isoform of tau. Thestructural model is based on the measurement of intra- as well as intermolecular distancerestraints. Site-directed mutagenesis experiments are in excellent agreement with the pro-posed model. Furthermore, we have started to structurally characterize the needle of theS. typhimurium TTSS [Poyraz et al. NSMB 2010]. Based on an optimized in vitro needlepreparation we can now obtain ssNMR spectra of very high quality. Progress in resonanceassignment of the needle protein PrgI and detection of distance restraints will be reported.In this regard, we are applying a novel approach for the study of intermolecular interfacesbased on equimolar mixtures of [1-13C]- and [2-13C]-glucose labeled proteins [Loquet etal. JACS 2010].

50

Lecture, Thu 11:35

In-cell NMR spectroscopy of nucleic acids

Hansel R(1,2), Foldynova-Trantirkova S (3), Lohr F (1,2), Buck J (2,4), Bongartz E(1,5), Bamberg E (1,5), Schwalbe H (2,4), Dotsch V (1,2),Trantirek L (3,6)

(1) Institute of Biophysical Chemistry, Goethe-UniVersity, Frankfurt am Main,Germany (2) Center for Biomolecular Magnetic Resonance and Cluster of ExcellenceMacromolecular Complexes, Goethe University, Frankfurt am Main, Germany (3)Biology Centre of the AS CR, VVi and Faculty of Sciences, University of SouthBohemia, Ceske Budejovice, Czech Republic (4) Institute for Organic Chemistry andChemical Biology, Goethe-UniVersity, Frankfurt am Main, Germany (5)Max-Planck-Institute of Biophysics, Department of Biophysical Chemistry, Frankfurt amMain, Germany (6) Department of Chemistry, Utrecht University, Utrecht, TheNetherlands, l.trantirek@uu.nl

In-cell NMR spectroscopy of proteins in different cellular environments is a well-established technique that, however, has not been applied to nucleic acids so far. Here, weshow that isotopically labeled DNA and RNA can be observed inside the eukaryotic envi-ronment of Xenopus laevis oocytes by in-cell NMR spectroscopy. One limiting factor forthe observation of nucleic acids in Xenopus oocytes is their reduced stability. We demon-strate that chemical modification of DNA and RNA can protect them from degradationand can significantly enhance their lifetime. Finally, we show that the imino region of theNMR spectrum is devoid of any oocyte background signals enabling the detection evenof isotopically nonlabeled molecules.

51

Lecture, Thu 12:10

Analysis of derivatized aminoacids in biological samples by Dynamic NuclearPolarization

Marin-Montesinos I.(1),(2), Ludwig C.(2), Saunders M.G.(2) and Gunther U.(2)

(1)Institute for Research in Biomedicine, Parc Cientıfic de Barcelona, C/ Baldiri Reixac10, Barcelona, Spain, ildefonso.marin@irbbarcelona.org (2)Henry Wellcome Building forBiomolecular NMR Spectroscopy, School of Cancer Sciences, University of Birmingham,Edgbaston, Birmingham, B15 2TT, United Kingdom

Dynamic Nuclear Polarisation (DNP) is used to transfer the high spin polarization ofunpaired electrons to coupled nuclear spins. Stable radicals are added to a solution of thesample and irradiatingwith microwaves is applied for 1-3h at the EPR lines of the radical.In such experiments enhancements of ¿10,000 were achieved after rapidly warming uppolarized samples to approx. 300K where spectra are recorded after transfer to a high fieldmagnet. This implementation of DNP requires efficient transfer of polarization from stableradicals to the samples which is facilitated by an optimal contact between the radicaland the sample in a glass state formed at low temperature. Although recent advancesin dynamic nuclear polarization techniques have boosted the otherwise low sensitivityof NMR spectroscopy, the efficiency of the hyperpolarization process depends on thecomposition of the polarization matrix, in particular on the contact between the radicaland the target molecule and the capability of the matrix to transfer polarization throughspin diffusion. A concept for optimal matrix design is presented and applied to obtain two-dimensional heterocorrelated spectra of small drug-like molecules in 1 – 2 min after 90min of hyperpolarization. We propose a new methodology for fast-HMQC using small flip-angle excitation pulse. Only a small amount of hyperpolarized magnetisation is used in theevolution period while the remain hyperpolarized magnetisation is stored along Zdirectionand ready to be used in the next evolution increment. These new tools have beencombinedto analyse pharmaceutical compounds and acetylated aminoacids in biological fluids likeserum, showing the applicability of these methods to complex samples.

52

Posters

53

Poster 1

NMR solution studies of the binding equilibria of an intracellular lipidcarrier protein in the presence of membrane mimetics and bile salt ligands

Pedo M(1), Lohr F(2), D’Onofrio M(1), Assfalg M(1), Dotsch V(2), Molinari H(1)

(1) Biotechnology Department, University of Verona, Italy (2) Institute of BiophysicalChemistry, Goethe University, Frankfurt, Germany

Bile acids are physiological detergents that generate bile flow and facilitate intestinalabsorption and transport of lipids, nutrients and vitamins. The enterohepatic circulationof bile acids, fundamentally composed of two major processes, namely secretion from theliver and absorption from the intestine, exerts important physiological functions not onlyin feedback inhibition of bile acid synthesis but also in control of whole body lipid home-ostasis. Detailed knowledge is available about the transporter proteins at the membranesof hepatocytes and enterocytes involved in vectorial bile acid movement both in normalphysiological condition and in disease. In contrast, the process of intracellular transfer ofbile acids between the basolateral and the apical membrane is largely unexplored. High-resolution NMR spectroscopy is here applied to the study of a bile acid-binding proteinfrom avian liver (L-BABP) and its interactions with membrane mimetics and physio-logical ligands. The here proposed description of chemical equilibria involving the threecomponents constitutes an important step forward in the understanding of the mechanismof bile acid binding and release by L-BABPs.

55

Poster 2

Directed Evolution of Bacillus subtilis Lipase A Towards Thermostability:Role of Aggregation

Augustyniak W (1), Brzezinska AA (1), Wienk H (2), Boelens R (2), Reetz MT (1)

(1) Max-Planck-Institut fur Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mulheiman der Ruhr, Germany, dreslaw@poczta.onet.pl (2) Bijvoet Center for BiomolecularResearch, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Lipase A from Bacillus subtilis is one of the best known lipases and a good target forexamination of concepts in biocatalysis. Recently, B-FIT driven iterative saturation mu-tagenesis yielded mutants that retain their activity upon heating and cooling down [1,2].The availability of these mutants opens up the opportunity of studying the evolutionaryprocess using biophysical tools. Three mutants and wild-type lipase were prepared andcharacterized using thermal inactivation profiles, circular dichroism, fluorescence, activityassays and NMR spectroscopy. The results suggest that the mutants retain their activ-ity upon heating and cooling due to prevention of aggregation. Wild-type Lipase A doesnot show this effect; it unfolds and precipitates upon heating and does not re-fold whilstcooling down. Interestingly, the mutants are thermodynamically slightly less stable thanthe wild-type enzyme. The wild-type and the best mutant were selected for comparativeNMR studies. Structure determination, dynamics, H/D exchange and temperature coef-ficients are reported for the mutant. Currently, similar studies of wild-type Lipase A areunderway.

References:[1] M. T. Reetz, J. D. Carballeira, A. Vogel: Iterative saturation mutagenesis on the

basis of B factors as a strategy for increasing protein thermostability, Angew. Chem. Int.Ed. Engl., 2006, 45, 7745-7751.

[2] M. T. Reetz, J. D. Carballeira: Iterative saturation mutagenesis (ISM) for rapiddirected evolution of functional enzymes, Nature Protocols, 2007, 2, 891-903.

56

Poster 3

Assessing the structural quality of 3D protein structures determined byMAS solid-state NMR spectroscopy

Bardiaux B(1), Oschkinat H(1)

(1) Leibniz-Institut fur Molekulare Pharmakologie, Campus Berlin-Buch, Berlin,Germany, bardiaux@fmp-berlin.de

During the last decade, solid-state NMR (ssNMR) spectroscopy has become a majorinstrument in structural biology. Solid-state NMR can now rely on a complete toolboxthat allows approaching not only high-resolution structural information, but also inter-nal dynamics of immobilized, insoluble proteins, such as fibrils and membrane proteins.Since 2002, when the first structure of a protein was determined by Magic-Angle Spinning(MAS) ssNMR (1), more than twenty other structures have been deposited in the ProteinData Bank (PDB). Still, interpretation of solid-state NMR data is often complicated bya lower spectral resolution (compared to solution NMR) and by the difficulty to mea-sure precise inter-atomic distances. To investigate the bona fide aptitude of solid-stateNMR for high-resolution structure determination, we performed a survey of the actualquality of 3D protein structures solved by MAS ssNMR. Using well-established measuresof structural quality, various quality scores were evaluated and compared, not only tocommon standards (often related to X-ray crystallography data), but also to the rangeof value observed in solution NMR structures. As judged by the constant improvementin quality over the different generations of structures, our results clearly established thatMAS solid-state NMR now produces true high-quality structures and competes well withsolution NMR in terms of overall structural quality. Moreover, through two examples, wewill show how a better application of structure calculation methods, initially developedfor solution NMR, could allow to drastically enhance the quality of protein structures de-termined from MAS solid-state NMR restraints. (1) Castellani F, van Rossum, B, DiehlA, Schubert M, Rehbein K and Oschkinat H, Nature, 420, 98-102 (2002)

57

Poster 4

Cold and hot unfolding of a higly stable antifungal protein

Batta G(1), Fizil A(1), Gaspari Z(3), Barna T(2) Kover KE(1), Nyitrai M(2), TomoriV(2), Leiter E(2), Pocsi I(2), Marx F(4)

(1) Institute of of Chemistry, University of Debrecen, Debrecen, Hungary,batta@tigris.unideb.hu. (2) Institute of Biology, University of Debrecen, Debrecen,Hungary (3) Institute of Chemistry, Eotvos Lorand University, Budapest, Hungary (4)Division of Molecular Biology,Innsbruck Medical University,Innsbruck, Austria

The functional aspects of the structure and dynamics of the small (55 aa) antifungalprotein PAF have recently been disclosed by NMR[1]. The hydrophobic core of the proteinis maintained by three disulfide bonds, that holds a greek-key supersecondary structureas long as possible. PAF is harmless for mammalian cells and has many potential appli-cations (e.g. against Aspergillosis). Reversible thermal unfolding/folding of PAF could bemonitored in the -10 ... +80o C temperature range using 15N-1H HSQC spectra. Ther-modynamic parameters of unfolding were determined residue by residue, either with twoor three-state models. It turned out that cold denaturation happens in a concerted wayin contrast to heat-shock.

Acknowledgement:This research was supported by the NKTH-OTKA CK-77515 and TAMOP-4.2.1./B-

09/KONV-2010-0007 Grants.

References:[1] G. Batta, T. Barna, Z. Gaspari, S. Sandor, K. E. Kover, U. Binder, B. Sarg, L.

Kaiserer, A. Kumar-Chhillar, A. Eigentler, E.Leiter, N.Hegedus, I. Pocsi, H.Lindner andF.Marx: FEBS Journal., 2009, 276, 2875-2890

58

Poster 5

Specific binding of herbal drug sinomenine with human serum albumin asdetermined by saturation transfer difference NMR study

Bazylak G (1), Ludwig C (2), Gunther UL (2), Pan TL (3)

(1) Department of Pharmaco-Bromatology, Faculty of Pharmacy,Collegium Medicum,Nicolaus Copernicus University,85-067 Bydgoszcz,POLAND,E-mail:gbazylak@cm.umk.pl (2) HWB NMR, CR UK Institute for Cancer Studies, University ofBirmingham, Edbagston,Birmingham,UK (3) School of Traditional Chinese Medicine,Chang Gung University, Tao-Yuan,Taiwan,ROC

The natural dextrorotatory morphinan analog as sinomenine (SN) is alkaloid isolatedfrom the Chinese medicinal plant Sinomenium acutum (Menispermaceae Juss). This com-pound is used in traditional Chinese anti-arthritic and anti-rheumatic therapies as pos-sessing broad immunomodulating and anti-inflammatory acitivities. However, until today,only limited data on the in vitro human serum albumin (HuSA) binding rate of SN havebeen determined with use of equilibrium dialysis assay [1]. In our study an interactionprocess occurring at the protein-solvent interface for SN-HSA system was investigatedby NMR using the saturation transfer difference (STD) technique. In addition, the STD-NMR study of interaction for the two various forms of HuSA (fatty-acid-loaded-HuSA andfatty-acid-free-HuSA) with the common anti-tussive alkaloid codeine, structurally similarto SN, and naproxen, an example of non-steroidal anti-inflammatory drug (NSAID), hasbeen also performed. We observed that SN binds exclusively to the second mentioned formof HuSA (i.e. fatty-acid-free-HuSA), what is contrary to naproxen, exhibiting high bind-ing affinity to the both form of studied HuSA, and contrary to codeine which indicatedno binding affinity to these both forms of HuSA. To explain these STD-NMR resultsthe location of specific SN binding site(s) of fatty-acid-free-HuSA have been proposed,thus enabling in vitro prediction of HuSA binding ability and evaluate pharmacokineticproperties of therapeutically used SN as the potential alternative agent for treatmentof rheumatoid arthritic (RA) subjects resistant to conventional NSAIDs drugs or RA-subjects failed in advanced, fusion protein based, biologic therapies [2].

References:[1] Z.-Q. Liu, K. Chan, H. Zhou, Z.-H. Jiang, Y.-F. Wong, H.-X. Xu, L. Liu: Life Sci.

77 (2005) 3197 – 3209.[2] Y.C. Chen, P.-W. Wang, T.-L. Pan, G. Bazylak, J.-J. Shen: Comb. Chem. High

Throughput Screen. 13 (2010) 469 – 481.

59

Poster 6

Solution structure of the STAS domain of Motor Protein Prestin

Gesiot L(1), Montecchio M(1), Pasqualetto E(1,2), Aiello R(1,2), Bonetto G(1,2),Mammi S(1), Battistutta R(1,2), Bellanda M(1)

(1) Department of Chemical Sciences, University of Padova, via Marzolo 1. 35131,Padova, Italy (2) Venetian Institute for Molecular Medicine (VIMM), via Orus 2, 35129Padova, Italy

Prestin is the motor protein responsible for the somatic electromotility of cochlearouter hair cells(1) and is essential for normal hearing sensitivity and frequency selectiv-ity of mammals. Prestin is a member of mammalian solute linked carrier 26 (SLC26)anion exchangers, a family of membrane proteins capable of transporting a wide varietyof monovalent and divalent anions. SLC26 transporters play important roles in normalhuman physiology in different tissues and many of them are involved in genetic diseases.SLC26 and the related SulP transporters carry a hydrophobic membrane core and a C-terminal cytosolic portion that is essential in plasma membrane targeting and proteinfunction. This C-terminal portion is mainly composed of a STAS domain, whose name(Sulfate Transporter and Anti-Sigma factor antagonist) is due to a remote but significantsequence similarity with bacterial ASA (Anti-Sigma factor Antagonist) proteins(2). Wehave recently solved the crystal structure at 1.57 A resolution of the cytosolic portionof prestin(3), the first structure of a SulP transporter STAS domain. Here we presentits characterization in solution by heteronuclear, multidimensional NMR spectroscopy.Prestin STAS significantly deviates from the related bacterial ASA proteins, especially inthe N-terminal region that, whereas previously considered merely a generic linker betweenthe domain and the last transmembrane helix, is actually fully part of the domain.

References:1. Zheng J., Shen W., He D. Z., Long K. B., Madison L. D. and Dallos P., Nature,

405, 149-155 (2000).2. Aravind L. and Koonin E. V., Curr Biol, 10, R53-55(2000).3. Pasqualetto E., Aiello R., Gesiot L., Bonetto G., Bellanda M. and Battistutta R.,

J. Mol. Biol., 400, 448-462 (2010)

60

Poster 7

NMR structure of the human prion protein Q212P mutant

Ilc,G.,(1),(2)* Giachin,G.,(3)* Biljan,I.(1),(4), Jaremko,M.(5),(6), Jaremko, L.(5),(6)Benetti,F.(3),(7), Zhukov,I.(1),(2),(5), Legname,G.(3),(7),(8), Plavec,J.(1),(2),(9)

(1)Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, SI-1000Ljubljana, Slovenia (2)EN→ FIST Centre of Excellence, Dunajska 156, SI-1001,Ljubljana, Slovenia (3) Laboratory of Prion Biology, Neurobiology Sector, ScuolaInternazionale Superiore di Studi Avanzati (SISSA), via Bonomea 265, Trieste, Italy (4)Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102A,HR-10000 Zagreb, Croatia (5) Laboratory of Biological NMR, Institute of Biochemistryand Biophysics, Polish Academy of Sciences, ul. Pawinskiego 5a, PL-02-106 Warsaw,Poland (6) Faculty of Chemistry, Warsaw University, Pasteura 1, PL-02-093, Warsaw,Poland (7) Italian Institute of Technology, SISSA Unit, via Bonomea 265, I-34136Trieste, Italy (8) ELETTRA Laboratory, Sincrotrone Trieste S.C.p.A., I-34149Basovizza, Trieste, Italy, (9) Faculty of Chemistry and Chemical Technology Universityof Ljubljana, Askerceva cesta 5, SI-1000 Ljubljana, Slovenia

Prion diseases are a group of neurodegenerative disorders that can arise spontaneously,through infection or through inheritance of mutations in the gene encoding the prion pro-tein (PrP). According to the “protein-only hypothesis”, the key event in the pathogenesisof prion disorders is the conversion of a cellular, highly α-helical PrP into a pathogenicβ-sheet enriched form, PrPSc. One of the strongest arguments supporting “protein-onlyhypothesis” is the linkage between inherited prion diseases and mutations in the genecoding for human PrP. However, the mechanisms by which mutations cause diseases arestill not resolved. Structural studies of PrP variants carrying familial mutations couldhelp us to understand the molecular mechanism at early stage of disease. In this study wehave determined a high-resolution 3D structure of the truncated recombinant HuPrP(90-231) containing Q212P mutation associated with Gerstmann-Straussler-Scheinker (GSS)syndrome. The obtained structure reveals unique structural features in comparison to theknown wild-type PrP structures with the most remarkable differences at the C-terminalend of the protein and in the β2-α2 loop. The C-terminal domain (residues 125-231) con-sists of a short anti-parallel β-sheet and four α helices. This is the first known example ofPrP structure where the α3 helix between Glu200 and Tyr226 is broken into two helices.The break results in dramatic changes in hydrophobic interactions between α3 helix andβ2-α2 loop. In comparison to WT a different mutal orientation of aromatic residues inβ2-α2 loop has been observed. In Q212P mutant Tyr169 is exposed to solvent and asa result the whole hydrophobic cluster is changed and shows increased exposure of hy-drophobic surface of the protein to solvent. Spontaneous generation of PrPSc in inheritedprion diseases might be due to the disruptions of the hydrophobic core consisting of β2-α2loop and α3 helix.

61

Poster 8

TPPP/p25: A NEW UNSTRUCTURED PROTEIN WITH GTPaseACTVITY

Bodor A (1), Perczel A(1), Zotter A(2), Ovadi J(2)

(1) Laboratory of Structural Chemistry and Biology, Institute of Chemistry, EotvosUniversity, 1117 Budapest, Pazmany Peter setany 1/A, Hungary. (2) Institute ofEnzymology, Biological Research Center, Hungarian Academy of Sciences, 1113Budapest, Karolina ut 29, Hungary. abodor@chem.elte.hu

Tubulin Polymerization Promoting Protein (TPPP) is a highly dynamic 25kDa proteinfor which the supreme target is the microtubule system. In normal human brain TPPPis expressed predominantly in oligodendrocyte; and in pathological inclusions TPPP co-accumulates with α-synuclein in both glial and neuronal cells leading to synucleinopathies.Multinuclear NMR investigations reveal two types of peaks: the intense peaks show lowsignal dispersion and are in accordance with a disordered protein structure; while the lowintensity peaks are dispersed and belong to the ordered part. SCS data obtained from theassignment of the 3D measurements (HNCA, (H)CC(CO)NH, TOCSY-HSQC, NOESY-HSQC) for the intense signals of TPPP prove that both the C- and N-terminal parts are‘disordered’. GTP binding of TPPP was monitored by HSQC measurements. Presumedbinding positions can be the Rossmann fold sequence and Switch II region, both situatedat the C-terminal part; however, GTP binding occurs in the ‘core’ region at the P loop.The specific hydrolytic activity of TPPP was followed by 31P NMR measurements. Realtime kinetic analysis showed that GTP hydrolysis leads to the formation of GMP and freephosphate, whilst GDP concentration is maintained at steady-state condition. GDP alonedoesn’t have hydrolytic activity in the presence of TPPP. This specific GTPase activityof TPPP is comparable with that of some non-activated small G proteins, suggestingits involvement in multiple physiological processes in addition to the regulation of GTP-mediated microtubule assembly. Study of Zn2+ effect on the structural and functionalfeatures showed partial folding with increased helical structure.

Acknowledgments:This work was supported by the Hungarian National Research Foundation, and the

Janos Bolyai Research Fellowship. 3D measurements were performed at the Oxford NMRFacility, in the EASTNMR Research Infrastructure framework.

62

Poster 9

NMR protein-protein interaction studies of the MRL proteins RIAM andlamellipodin

Bonet R (1), Reichenbach M (1), Campbell ID (1)

(1) Department of Biochemistry, University of Oxford, OX1 3QU, Oxford, UK,roman.bonetfigueredo@bioch.ox.ac.uk

The MRL (Mig-10/RIAM/lamellipodin) family of proteins have a role in the activationof integrins, via their interactions with RasGTPases and talin that lead to the recruitmentof the later to the plasma membrane. MRL proteins have an architecture consisting of aconserved central region containing a RA (Ras association) and a PH (pleckstrin homol-ogy) domain flanked by N-terminal coiled-coil regions and a C-terminal proline-rich region.Short sequences in the N-terminal region of MRL proteins are responsible for the bindingto talin. Using NMR spectroscopy we show that these N-terminal sequences of RIAMand lamellipodin bind specifically to the F3 domain of talin and, for the Lamellipodin-F3interaction, mapped the residues involved. In addition, for Lamellipodin, we detected anintra-molecular interaction between the N-terminal fragment and the PH domain, whichmaps in the same conserved region. We also observed that this interaction competes withthe talin F3 interaction. Thus, it is possible that the PH domain could play a regulatoryrole in MRL protein function

63

Poster 10

NMR investigation of human HAR1F RNA structure

Cevec M, Koschinat M, Richter C, Schwalbe H

Institute of Organic Chemistry and Chemical Biology, Goethe University,Max-von-Laue-Straße 7, D-60438, Frankfurt am Main, Germany

98.5% of the DNA sequences encoded in the human genome are non-coding, but at least70% of it is transcribed into RNA. Pollard et al. [1] identified 49 human accelerated regions(HARs) which displayed a significantly accelerated nucleotide substitution rate in humans.96% of the HARs are found in non-coding segments of the genome. 18 changes haveaccrued in the 118-nt long sequence of HAR1 instead of the expected 0.27 substitutionssince our last common ancestor with chimpanzees 7 million years ago. The forward RNAgene (HAR1F) is expressed together with reelin protein in the neocortex during embryonaldevelopment and later in Cajal-Retzius cells which are crucial in redirecting migratingneurons. Two different cloverleaf-like secondary structure models have been offered for thefull length hHAR1F RNA. We designed and prepared a full length and a 37-nt hairpinmodel RNA which mimics helix 1 of hHAR1F RNA. The non-labeled full length andthe 15N-labeled 37-nt RNA were transcribed in vitro with T7 RNA polymerase. Theresonance assignment was performed using 15N-HSQC, HNN-COSY and NOESY spectra.The NOE imino proton signal pattern of the 37-nt RNA was comparable to the patternof the full length hHAR1F RNA. We studied the dynamic properties of the 37-nt RNAconstruct with the help of 15N-relaxation NMR measurements. We estimated the fastinternal motions in the 37-nt RNA construct by the measurement of longitudinal andtransverse relaxation rates, along with heteronuclear NOEs. We were able to assign allaromatic, H1’ and H2’ and more than 50% of H3’ proton resonances, which allow us doa complete NOE sequential walk between aromatic and sugar proton resonances. Earlystructural calculations show that the 37-nt RNA forms a hairpin of two stems separatedby the internal loop.

Reference:1. Pollard K. S. et al. (2006), Nature, 443, 167-172.

64

Poster 11

Intrinsic Disorder in Measles Virus Nucleocapsids

Communie G(1,2),Ringkjøbing Jensen M(1), Ribeiro EA(1,2), Martinez N(2), DesfossesA(2), Salmon L(1), Mollica L(1), Narayanan T(3), Gabel F(1), Jamin M(2), LonghiS(4), Ruigrok RW(2), Martin Blackledge(1)

(1)Institut de Biologie Structurale Jean-Pierre Ebel, CEA-CNRS-UJF, UMR 5075, 41Rue Jules Horowitz, 38027 Grenoble, France. (2)Unit for Virus Host Cell Interactions,UJF-EMBL-CNRS, UMI 3265, 6 Rue Jules Horowitz, BP 181, 38042 Grenoble, France.(3)European Synchrotron Radiation Facility, F-38043 Grenoble Cedex, France.(4)Architecture et Fonction des Macromolecules Biologiques (AFMB), CNRS, UMR6098, France and Aix-Marseille Universite, 163 avenue de Luminy, Case 932, 13288Marseille Cedex 09, France.

The genome of measles virus is encapsidated by multiple copies of the nucleoprotein(N), forming helical nucleocapsids of molecular mass approaching the gigadalton. Theintrinsically disordered C-terminal domain of N (NTAIL) is essential for transcription andreplication of the virus via interaction with the phosphoprotein P of the viral polymerasecomplex. The molecular recognition element (MoRE) of NTAIL that binds P is situated90 amino acids from the folded RNA-binding domain (NCORE) of N, raising questionsabout the functional role of this disordered chain. Here we report the first in situ structuralcharacterization of NTAIL in the context of the entire N-RNA capsid. Using nuclearmagnetic resonance spectroscopy and small angle scattering, we demonstrate that the C-terminal domain of NTAIL is highly flexible in intact nucleocapsids and that the MoRE isin transient interaction with NCORE. We present a model whereby the first 50 disorderedamino acids of NTAIL are conformationally restricted as the chain escapes to the outside ofthe nucleocapsid via the interstitial space between successive NCORE helical turns. Thisprovides a structural framework for understanding the role of NTAIL in the initiation ofviral transcription and replication, placing the flexible MoRE close to the viral RNA and,thus, positioning the polymerase complex in its functional environment.

65

Poster 12

The anti-apoptotic Bcl-xL protein, a new piece in the puzzle of cytochrome cinteractome.

Bertini I.(1), Chevance S.(1), Del Conte R.*(1), Lalli D.(1), Turano P.(1)

(1) Magnetic Resonance Center (CERM), University of Florence, Via L. Sacconi 6,50019 Sesto Fiorentino, Italy

A model structure of the adduct between human cytochrome c and the human anti-apoptotic protein Bcl-xL was obtained from solution NMR data, providing insights atthe atomic level on the mechanism by which cytochrome c translocated to cytosol can beintercepted, such that the apoptosome is not assembled. This model allows the definitionof the protein-protein interaction surface and reveals key intermolecular contacts thatidentify new potentially druggable spots on cytochrome c and Bcl-xL.

66

Poster 13

SAS-6 oligomerization lies at the onset of centriole formation

Erat M. C. (1), Kitagawa D. (2), Keller D. (2), Steinmetz M. (3), Gonczy P. (2),Vakonakis I. (1)

(1) Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UnitedKingdom, michele.erat@bioch.ox.ac.uk (2) Swiss Institute for Experimental CancerResearch (ISREC), Swiss Federal Institute of Technology, Lausanne, Switzerland (3)Biomedical Research, Paul Scherrer Institute, Villigen, Switzerland

Centrioles are macromolecular structural assemblies that lie at the heart of the cen-trosome, the major microtubule organizing center of animal cells. Furthermore, they arefundamental for the assembly of cilia and flagella across eukarya. Despite their criticalrole in genome stability, the way in which the centrioles assemble is still poorly under-stood. Five essential proteins have initially been identified in Caenorhabditis elegans,and homologs of most components have been found across eukaryotic evolution. In mostspecies, the centriole assembly is initiated by a large nine fold symmetrical structuretermed the “cartwheel”. We employ a wide range of biophysical, biochemical and cellbiological techniques to elucidate the protein structures and interactions at the onset ofcentriole biogenesis. A major structural component of the early centriole is SAS-6 in C.elegans and its homologs in other species. SAS-6 proteins contain an evolutionarily con-served N-terminal domain, followed by a long coiled coil and a less ordered C-terminus.Electron microscopy with recombinant ceSAS6 revealed a globular headgroup attached toa 35nm long rodlike structure, in good agreement with the predicted length of the coiledcoil. Here, we present the crystal structure of the N-terminal domain of ceSAS6 and showevidence that the dimeric form found in the crystal is relevant for in vivo centriole for-mation. A further crystal structure containing part of the coiled coil region in addition tothe N-terminus suggested a ceSAS-6 multimer as possible nucleus for centriole formation.Intriguingly, this multimeric model establishes nine fold symmetry through a mechanismdifferent than that recently proposed for Chlamydomonas. AUC and NMR experimentsare currently performed to probe the existence of this model in solution.

67

Poster 14

Felli I. C.

Abstract not available.

68

Poster 15

Interactions of Cellular Retinol-Binding Proteins with Membrane MimeticSystems

Franzoni L(1), Gunther UL(2), Cavazzini D(3), Rossi GL(3) and Lucke C(4)

(1) Department of Experimental Medicine, Sect. of Chemistry and StructuralBiochemistry, University of Parma, Italy, lorella.franzoni@unipr.it; (2) CR UK Institutefor Cancer Studies, University of Birmingham, United Kingdom; (3) Department ofBiochemistry and Molecular Biology, University of Parma, Italy; (4) Max PlanckResearch Unit for Enzymology of Protein Folding, Halle (Saale), Germany.

Retinoids regulate a wide range of fundamental biological processes. The studies of theplasma and cytoplasmic carriers of retinoids gave insights into their metabolism; moreover,understanding the functions of these proteins may assist in developing compounds thathave specific therapeutic effects. For some time we have been investigating the interactionsof retinol with its two primary cellular binding proteins (1-3). CRBP-I shows wide tissuedistribution, while CRBP-II is expressed only in the enterocytes; they play central roles inthe maintenance of vitamin A homeostasis by directing it to the proper enzymes, either forstorage as retinyl esters or for oxidation to retinoic acid. The study of proteins dynamicson microsecond to millisecond time scales combined with 2D 1H-15N line shape analysisof spectra recorded during ligand titrations revealed distinct modes of retinol uptake bythe two carriers. Considering that both proteins deliver retinol to several membrane-associated enzymes, we have recently started to address the mechanism of ligand releaseby performing NMR and CD experiments in the presence of membrane mimetic systems.The results again suggest a different behaviour of CRBP-I with respect to CRBP-II.Transfer of retinol from CRBP-I seems to be mediated by interactions of the proteinportal region with the phospholipid vesicles, whereas a diffusional process may apply forCRBP-II. These peculiar differences between the two homologs might account for theirdifferent tissue-specific expression patterns and distinct functional roles. The EU-NMRinfrastructure HWB•NMR @ Birmingham (UK) is acknowledged for providing access toinstrumentation.

Reference:(1) Mittag T., Franzoni L., Cavazzini D., Schaffhausen B., Rossi G.L., Gunther U.L.

(2006) J. Am. Chem. Soc. 128, 9844-9848.(2) Franzoni L., Cavazzini D., Rossi G.L., Lucke C. (2010) J. Lipid Res. 51, 1332-1343.

(3) Reed M., Gunther U.L., Lucke C., Cavazzini D., Rossi G.L, Franzoni L. In preparation.

69

Poster 16

Gesiot L.

Abstract not available.

70

Poster 17

Antithrombin binding octasaccharides: effects on molecular conformation andanticoagulant activities of extensions of the active pentasaccharide sequence.

Guerrini M (1), Elli S. (1),Gaudesi D(1), Torri G(1), Casu B(1), Mourier P(2),HermanF(2),Boudier C(3),Lorenz M(2),Viskov C(2)

(1) G. Ronzoni Institute for Chemical and Biochemical Research, via G. Colombo 81,20133 Milan, Italy, (2)Sanofi-Aventis, 13 Quai Jules Guesde, 94403 Vitry sur Seine,France, and (3)CNRS UMR 7175, Departement Physicochimie et Pharmacochimie desInteractions Moleculaires et Cellulaires, Faculte de Pharmacie, Universite LouisPasteur, Strasbourg I, F 67401, France. guerrini@ronzoni.it

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) play their role in manyphysiological and pathological pathways by interacting with different proteins. Sequencespecificity for protein binding and activity has emerged, showing that it is the combinationof overall conformation, charge distribution, and flexibility exhibited by heparin/heparansulfate saccharides that determines the extent of protein binding and related activity.The accelerating effects of heparin and heparan sulfate on antithrombin (AT)-proteasereactions are dependent on the binding of a sequence-specific pentasaccharide AGA*IA(GlcNNAc/NS,6S-GlcA-GlcNNS,3,6S-IdoUA2S-GlcNNS,6S) to the serpin. This study wasaimed at investigating the role of the structure and molecular conformation of different dis-accharide extension of both sides of the pentasaccharide in modulating the affinity for AT.In the present work, the binding of different HSGAG oligosaccharides with antithrombin(AT) have been investigated by docking studies, 1D saturation transfer difference (STD)and 2D transferred NOE (tr-NOE) methods. The highly specific interaction of HSGAGswith AT mediated by a well defined pentasaccharide sequence as well as the active roleof both reducing and non-reducing extensions have been demonstrated.

71

Poster 18

Bacillus sp. 3B6, a bacterium isolated from cloud water, producing unusualvariety of fructans

Husarova S(1,2), Matulova M(1), Capek P(1), Sancelme M(2), Delort AM (2)

(1) Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences,Dubravska cesta 9, SK - 845 38 Bratislava, Slovakia, (slavomira.husarova@savba.sk) (2)Laboratoire de Synthese et Etude de Systemes a Interet Biologique, UMR 6504Universite Blaise Pascal – CNRS, 63 177 Aubiere, France

Besides an importance of microbial exopolysaccharides (EPS) for their different ap-plications in biotechnologies, theses molecules are well known to play a role in ecologicalniches. Their precise role in EPS-producing bacteria depends on their natural environ-ment. Most of functions ascribed to EPS are of a protective nature. For instance fructanswere shown to protect microbial cells against abiotic and biotic stress, such as desiccation,freezing, antibiotics or toxic compounds. Recent studies showed that microbial communi-ties are present in clouds and provoke new investigations of their structure and functioning.Studied Bacillus sp. 3b6 was isolated from cloud water collected in free troposphere of Puyde Dome in France. Incubation of Bacillus sp. 3B6 on sucrose afforded complex mixtureof metabolites. NMR structural analysis showed that this bacterium is able to transformsucrose into fructan EPS called levan, composed of 2,6-linked β-fructofuranoses. This re-sult might have some implications considering the environment: First, production of levancould be an efficient way for this strain to survive in clouds which represent an extremeenvironment for living cells, characterized by low temperatures, acidic pH and UV radi-ation. Second, it was found that EPS could be an important factor for the formation ofcloud droplets by changing the “wettability” of biological particles. Indeed the presenceof lipopolysaccharide, polysaccharidic or proteic structures was shown to enhance the hy-drophilicity of the cell surface and thus its hydroscopy. It should have a great importanceconcerning the cloud condensation nuclei (CCN) properties of strains in the atmosphere.Our results highly suggest that Bacillus sp. 3B6, which was isolated directly from cloudwater, could present the same properties in the atmosphere.

72

Poster 19

NMR studies on the pB state of full-length photo-active yellow protein(PYP)

Ippel JH(1),Hospes M(2),Boelens R(1),Hellingwerf K(2)

(1) NMR spectroscopy group, Dept. of Chemistry, University of Utrecht, Padualaan 8,3584 CH Utrecht, The Netherlands, j.h.ippel@uu.nl (2) Laboratory for Microbiology,Swammerdam Institute for Life Sciences, Nieuwe Achtergracht 166, 1018 WVAmsterdam, The Netherlands.

PYP is a 14-kDa water-soluble protein that act as a photosensor for the negativephototactic response toward the intense blue light of Ectothiorhodospira halophila [1], amotile bacterium which survives in Egyptian salt lakes. Light excitation induces a series ofstructural and dynamic changes yielding a blue-shifted, long-lived intermediate pB witha lifetime on the order of 500 milliseconds. We studied the pB kinetic intermediate byNMR spectroscopy using light irradiation from an argon laser, directed into the NMRprobe via an optical fiber. Previous NMR studies mainly focused on mutant PYP (E46Qand truncated delta25-PYP) exhibiting significantly slower photokinetics [2,3], whereaspB of wild type full-length PYP show extreme line broadening of NMR resonances, thuspreventing a detailed NMR analysis. Here, we show recent NMR data for the pB statein full-length PYP. New measurements at elevated temperatures allowed us to assignmost resonances, including that of the the N-terminus, and to determine the structureof PYP pB in solution. The results show that the pB state is structurally heterogeneousat low temperature and exists as a family of conformers that exchange on a millisecondtimescale. Increasing the temperature drives the the conformational ensemble towards asingle state that is in structural terms still quite different compared to the dark state,pG. The great challenge is to eventually obtain agreement between dynamic alterationsin the pB structure of PYP and its biological function. Literature: [1] Sprenger WW,Hoff WD, Armitage JP, Hellingwerf KJ, J. Bacteriol. (1993) 175, 3096-3104. [2] DerixNM, Wechselberger RW, van der Horst MA Hellingwerf KJ, Boelens R, Kaptein R, vanNuland NAJ, Biochemistry (2003) 42, 14501-14506. [3] Bernard C, Houben K, Derix NM,Marks D, van der Horst MA, Hellingwerf KJ, Boelens R, Kaptein R, van Nuland NAJ,Structure (2005) 13, 953-962.

73

Poster 20

Jablan J.

Abstract not available.

74

Poster 21

Structural and thermodynamic characterization of nucleotide binding to theR3H domain from human Sµbp-2

Jaudzems K(1), Zhulyenkov D(2), Otting G(3), Liepinsh E(1)

(1) Laboratory of Physical Organic Chemistry, Latvian Institute of Organic Synthesis,LV-1006, Riga, Latvia, kristaps.jaudzems@osi.lv (2) Latvian Biomedical Research andStudy Centre, LV-1067, Riga, Latvia (3) Research School of Chemistry, AustralianNational University, ACT 0200, Canberra, Australia

The R3H domain presents a small α/β motif from putative nucleic-acid-binding pro-teins [1]. In this work the binding affinities of mononucleotides to the R3H domainfrom human Sµbp-2 were determined by NMR titration experiments. Notable bindingoccurs only with 5’-monophosphorylated nucleotides and exhibits a preference for purine-containing deoxyribonucleotides supporting earlier findings that the Sµbp-2 proteins bindsingle-stranded DNA with 5’-phosphorylated G-rich sequences [2]. The NMR structureof the R3H domain in complex with 2’-deoxyguanosine 5’-monophosphate is presentedand reveals that two highly conserved arginines interact with the monophosphate andthe invariant histidine stacks with the nucleotide base. Additional interactions are iden-tified that could account for nucleotide selectivity. The conformation of bound dGMPsuggests that only the 5’-end-nucleotide of ssDNA interacts with R3H. On the basis ofthese observations the possible modes of DNA/RNA recognition of the R3H domain areevaluated.

References:[1] E. Liepinsh, A. Leonchiks, A. Sharipo, L. Guignard, G. Otting. (2003). Solution

structure of the R3H domain from human Sµbp-2. J. Mol. Biol. 326, 217-223.[2] Y. Fukita, T.R. Mizuta, M. Shirozu, K. Ozawa, A. Shimizu, T. Honjo. (1993).

The human Sµbp-2, a DNA-binding protein specific to the single-stranded guanine-richsequence related to the immunoglobulin mu chain switch region. J. Biol. Chem. 268,17463-17470.

75

Poster 22

Defining Conformational Ensembles of Intrinsically Disordered and PartiallyFolded Proteins Directly From Chemical Shifts

Jensen MR, Salmon L, Nodet G, Blackledge M

Protein Dynamics and Flexibility, Institut de Biologie Structurale, CEA-CNRS-UJFUMR 5075, Grenoble, France

The development of meaningful descriptions of the conformational behavior of intrin-sically disordered proteins represents a key challenge for contemporary structural biology.Here, we have developed an approach, based on the combination of ensemble descrip-tions of disordered proteins and state-of-the-art chemical shift prediction algorithms, todescribe backbone dihedral angle conformational behavior on the basis of 13C and 15NNMR chemical shifts alone. This allows the identification and characterization of entiresecondary structural elements and their associated populations, as well as providing in-dications of the subtle detail of local conformational sampling in unfolded proteins. Thistechnique raises the prospect of probing the conformational behavior of unfolded pro-teins under conditions where additional parameters cannot be easily measured but wherechemical shifts are still accessible, for example in crowded or cellular environments.

76

Poster 23

A Solution Model of the Complex Formed by Adrenodoxin and AdrenodoxinReductase Determined by Paramagnetic NMR Spectroscopy

Keizers PHJ (1), Mersinli B (2), Reinle W (2), Donauer J (2), Hiruma Y (1),Hannemann F (2), Overhand M (1), Bernhardt R (2), Ubbink M (1)

1 Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box9502, 2300 RA Leiden, the Netherlands 2 Universitat des Saarlandes, FR 8.8Biochemie, Campus B2.2, 66041 Saarbrucken, Germany p.keizers@chem.leidenuniv.nl

Lanthanide tags offer the opportunity to retrieve long-range distance information fromNMR experiments that can be used to guide protein docking. To determine whethersufficient restraints can be retrieved for proteins with low solubility and availability, Ln-tags were applied in the study of the 65 kDa membrane associated protein complex formedby the electron carrier adrenodoxin and its electron donor, adrenodoxin reductase. Thereductase is only monomeric at low concentration and the paramagnetic iron sulfur clusterof adrenodoxin broadens many of the resonances of nuclei in the interface. Guided bythe paramagnetic restraints obtained using two Ln-tag attachment sites, protein dockingyields a cluster of solutions with an RMSD of 3.2 A. The mean structure is close to thecrystal structure of the cross-linked complex, with an RMSD of 4.0 A. It is concluded thatwith the application of Ln-tags paramagnetic NMR restraints for structure determinationcan be retrieved even for difficult, low-concentration protein complexes.

77

Poster 24

Proton-detected solid-state NMR of the 153-residue protein SOD

Knight MJ(1), Webber A(1), Pell AJ(1), Bertini I(2), Felli I(2), Pieratelli R(2), EmsleyL(1), Lesage A(1), Pintacuda G(1)

(1) CRMN, 5 Rue de la Doua, ENS de Lyon, Lyon 69100, France (2) CERM, ViaSacconi 6, Sesto Fiorentino 50019, Italy

Proton-detected solid-state NMR of proteins has traditionally been difficult, since pro-ton homonuclear dipolar couplings cause severe line-broadening. However, the sensitivityof proton detection and the additional spectral resolution afforded by the inclusion of aproton dimension are very much desirable. Perdeuteration partially solves this problem bydiluting the protons in the sample, and proton detection has been successfully applied tosmall proteins already using MAS speeds of 24-40 KHz. We have used ultra-fast MAS (at60 KHz) and a very high magnetic field (1 GHz) together with perdeuteration to studythe enzyme superoxide dismutase (SOD) in microcrystalline form. SOD is dimeric with153 residues per monomer and is the largest protein to which proton detection has beenapplied in solid-state NMR to date. We show that amide proton assignments can be easilymade using a 3D CONH experiment, acquired in less than 2 days using only 2.5 mg ofprotein. We also show, for the first time, that RFDR mixing can be used in a 3D 15N-edited proton-proton correlation spectrum superficially similar to an HSQC-NOESY, andgather useful structural restraints. This demonstrates the feasibility of proton-detectionto study moderately sized proteins in the solid state using NMR.

78

Poster 25

Use of lanthanide chelating probes to study the allostery of the Lac repressor

Kovacic L(a), Folkers G(a), Wienk H(a), Keizers P(b), Ubbink M(b) and Boelens R(a)

From (a)Bijvoet Center for Biomolecular Research, University of Utrecht, Utrecht and(b)Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, TheNetherlands

Lac repressor (LacR) is a textbook example for understanding genetic control andallostery, tracking back to the pioneering work of Jacob, Monod and Changeux and repre-sents one of the first described heterotropic allosteric regulators. The allosteric regulationof LacR has been extended to a variety of cellular signalling pathways in all organismsand used as a model system for experimental and theoretical understanding the allostericmechanisms. However, knowledge of structures at a detailed atomic level of all LacR dy-namic allosteric states that occur as a consequence of interaction with its ligands, arestill poorly described. Crystal structures exist only for a few allosteric states of LacR, in-sufficient for understanding all regulatory events. High-resolution NMR can visualize allstates in solution and probe both structural and dynamical parameters. Using lanthanidechelating probes we can study the changes in structure and dynamics of all allosteric statesof the LacR by high-resolution NMR, by following pseudo contact shifts and relaxationbroadening. For this fully functional repressors were designed and prepared to specificallyreact with Cys-reacting lanthanide probes. Several different positions on the LacR werechosen to introduce surface exposed single or double Cys to test two types of tags. To beable to precisely monitor the LacR conformational changes in different allosteric states,the probe should be firmly attached. For that reason, the double-Cys reacting lanthanidechelating probe CLaNP-5 designed by Keizers and Ubbink was used. Sites were chosenon positions, where no conformational changes have been observed in the crystal struc-tures and for which it has been demonstrated that mutagenesis will not interfere withLacR activity. By comparing spectra of LacR in all allosteric states we can further ourunderstanding of allostery taking place in this protein.

79

Poster 26

Interaction between separated CCP modules from human C1r

Andras Lang1*, Katalin Szilagyi2, Balazs Major2, Peter Gal2, Peter Zavodszky2,Andras Perczel1

1 Eotvos Lorand University of Sciences, Institute of Chemistry, Pazmany Peter setany1/a, Budapest, Hungary 2 MTA, SzBK, Enzimologiai Intezet, Karolina ut 29, Budapest,Hungary

NMR spectroscopy has been applied to study the interaction between separated tan-dem CCP modules from C1r, the first serine protease. We show previously undetectedweak protein-protein interaction between the two modules. The interacting surface is dif-ferent from that of the linked tandem modules. Based on these experimental data andsolvent accessibility calculation, protein-protein docking was used to highlight the orien-tation of this novel interaction between the two modules.

80

Poster 27

Liepinsh E.

Abstract not available.

81

Poster 28

Insights on SOD1 maturation steps in the cellular cytoplasm via NMR

Banci L(1,2), Barbieri L(1,2), Bertini I(1,2), Cantini F(1,2), Luchinat E(1,2)

(1) Magnetic Resonance Center, University of Florence, Via Luigi Sacconi 6, 50019Sesto Fiorentino, Florence, Italy; (2) Department of Chemistry, University of Florence,Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy

Cu,Zn-superoxide dismutase (SOD1) is a 32 kDa homodimeric protein involved in thecellular defence systems against oxidative stress. It exerts its function in the cytoplasm andin the mitochondrial IMS (1). In order to reach its mature form, SOD1 has to incorporateone Zn2+ ion and one catalytic Cu+ ion per subunit. Additionally, two conserved cysteineresidues form an intramolecular disulfide bridge. These maturation steps occur in vivothrough a complex mechanism which involves the CCS metallochaperone. It is not yetclear in which way the zinc binding is involved in this mechanism.

The metal free form of SOD1 has been recently linked to the familial form of amy-otrophic lateral sclerosis (fALS), a fatal motor neurodegenerative disease. (2) It gives riseto insoluble protein aggregates in vivo under a number of conditions (3) which have notyet been fully elucidated.

In order to investigate the first maturation steps of SOD1 in a cellular environment,we have performed in-cell NMR experiments on wild type human SOD1 (WT hSOD1) inbacterial cells. In-cell NMR has the unique ability to acquire structural and conformationalinformation of biomolecules in their native cellular environment at atomic level (4).

We characterized the folding state of WT hSOD1, analyzing samples of E. coli cellsover-expressing the protein either in defect or in the presence of zinc(II) ion. The lattercondition allowed us to characterize the first step of WT hSOD1 maturation inside thecytoplasm: the incorporation of the zinc ion, and its effects on the protein structure. Thisstudy can help to shed light on the reasons why the alteration of these processes can leadto immature, non stable forms of SOD1 which have been found to be prone to aggregation.

References:(1) Okado-Matsumoto et al. J.Biol.Chem. 2001, 276, 38388-38393.(2) Banci et al. Proc.Natl.Acad.Sci.USA 2007, 104, 11263-11267.(3) Furukawa et al. Proc.Natl.Acad.Sci.USA 2006, 103, 7148-7153.(4) Reckel et al. Chembiochem. 2005, 6, 1601-1606.

82

Poster 29

Quantum Rotor Induced Hyperpolarization

C. Ludwig, I. Marin-Montesinos, M.G. Saunders, U.L. Gunther

School of Cancer Sciences, University of Birmingham, Birmingham, UK

Dynamic nuclear polarization (DNP) is used to transfer the high spin polarisationof unpaired electrons to coupled nuclear spins by doping samples with a stable radicaland irradiating with microwaves at the EPR lines of the radical. Signal enhancements of∼200 were achieved in the solid state whereas enhancements ∼10,000 were reported afterpolarizing the sample at low temperature (1.4K), followed by dissolution with hot solventand transfer of the sample to an NMR spectrometer (ex situ DNP). In the context ofthis experiment we recently observed an additional polarization mechanism arising fromhindered quantum rotors (QRs) in methyl groups, which generates polarizations at tem-peratures ¡1.5K. This polarization mechanism interferes with DNP at such temperatures.QR polarization generated by this mechanism is efficiently transferred via carbon boundprotons. Although QR polarizations have been studied for a small range of moleculesin great detail, we have observed such effects for a much broader range of substanceswith very different polarization rates at temperatures ¡1.5K. Moreover, we report transferof quantum rotor polarization across a chain of protons. The observed effect not onlyinfluences the polarization in low temperature DNP experiments but also opens a newindependent avenue to generate enhanced sensitivity for NMR.

83

Poster 30

Improving the maximum occurrence analysis of calmodulin conformations insolution by placing paramagnetic ions in both protein domains

Ivano Bertini, Soumyasri Das Gupta, Xiaoyu Hu, Peter H. J. Keizers, Claudio Luchinat,Malini Nagulapalli, Giacomo Parigi, Luca Sgheri, Marcellus Ubbink

CERM, University of Florence, Italy; Gorlaeus laboratories, Leiden University,Netherlands; Istituto per le Applicazioni del Calcolo, CNR, Sesto Fiorentino, Italy

Paramagnetism-based restraints have been shown to monitor the presence of confor-mational rearrangements among protein domains and to provide information on the max-imum occurrence (MO) of any conformation that is sterically allowed. The MO strategy isfocused on determining the maximum weight that any given conformation can have in anyconformational ensemble in agreement with all available experimental data obtained, e.g.,through solution NMR or small angle scattering measurements. Therefore, it can providethe maximum percent of time that a system can spend in any conformation. MO valuesfor the possible conformations of calmodulin (CaM), a protein composed of two domainswhich are free to reorient with respect to one another, were previously obtained by sub-stituting a calcium ion with paramagnetic lanthanides in the N-terminal domain of theprotein. We have now placed paramagnetic lanthanides also in the C-terminal domain ofCaM to analyze how the MO values are affected by the availability of these new restraints.In order to place a metal ion in the C-terminal domain of CaM, the Caged LanthanideNMR Probe 5 (CLaNP-5) was attached to the H107C/N111C CaM mutant. This tagbinds rigidly to the protein backbone through two cysteine residues. We have found thatthe simultaneous use of paramagnetism-based restraints arising from paramagnetic metalions located alternatively in both protein domains is quite effective in discriminating thedifferent possible protein conformations from their MO values.

References:1. Bertini, I.; Giachetti, A.; Luchinat, C.; Parigi, G.; Petoukhov, M. V.; Pierattelli, R.;

Ravera, E.; Svergun, D. I. J.Am.Chem.Soc. 2010, 132, 13553-13558.2. Bertini, I.; Gupta, Y. K.; Luchinat, C.; Parigi, G.; Peana, M.; Sgheri, L.; Yuan, J.

J.Am.Chem.Soc. 2007, 129, 12786-12794.3. Keizers, P. H. J.; Saragliadis, A.; Hiruma, Y.; Overhand, M.; Ubbink, M. J.Am.Chem.Soc.

2008, 130, 14802-14812.

84

Poster 31

NMR-based Structural Characterization of ApoSOD1 AmyloidogenesisPathways

Banci (1,2), Bertini I(1,2), Blazevits O(1), Cantini F(1,2), Lelli M(1,3), Luchinat C(1,2),Mao J(1), and Vieru M(1)

(1) Magnetic Resonance Center (CERM), UniVersity of Florence, Via L. Sacconi 6,50019, Sesto Fiorentino, Italy, mao@cerm.unifi.it (2) Department of Chemistry “UgoShiff”, UniVersity of Florence, Via della Lastruccia 3, 50019, Sesto Fiorentino, Italy (3)Current Address: Centre de RMN a Tres Hauts Champs, FRE 3008 CNRS/EcoleNormale Superieure de Lyon, 5 Rue de la Doua, 69100, Villeurbanne, France

Human superoxide dismutase 1 (hSOD1), a dimeric Cu,Zn metalloprotein, has beengenetically linked to a fatal motor neurodegenerative disease called familial amyotrophiclateral sclerosis (fALS). A large body of evidences has shown that loss of the post-translational modifications (e.g. metal ions) and/or acquisition of pathological mutationsare able to destabilize the hSOD1 protein and lead to the formation of amyloidal aggre-gates in vitro and in vivo. Amyloidal aggregation of demetallated SOD1 are very complexprocesses during which a variety of folding/unfolding events and interactions take placeand could be even coupled. In order to elucidate the complex pathways of apo wild-typeSOD1 amyloidogensis, we have performed the structural characterizations on four dis-tinct forms of apoSOD1 (solution, microcrystals, oligomers, fibrils) through solution andsolid-state NMR (SSNMR).First, a comparative analysis based on the backbone chemicalshifts of apoSOD1 in solution and in microcrystals reveals the unique high β-propensity ofseveral small stretches located on the long loops in apoSOD1 molecule in solution ratherthan in microcrystals. This suggests that extended/extra β-strands could form transientlyin these regions and may even drive the oligomerization/fibrillation. Second, the 13C-13CTOCSY or DARR spectra patterns of apoSOD1 solution, oligomers and fibrils indicatethat this protein experiences more significant conformational changes during fibrillationthan within oligomerization. A preliminary analysis on some well resolved signals on theSSNMR spectra of apoSOD1 fibrils indicates that some part of the protein undergoesmarked structural alterations toward β-conformation while forming fibrils. Moreover, wealso observed highly mobile parts in the apoSOD1 fibrils. Taking together, all these re-sults permit a structural glimpse into the complex apoSOD1 amyloidogenesis pathwaysand produce valuable mechanistic information on the fALS pathology.

85

Poster 32

Solid-state NMR investigation of the hormone binding receptor PYR1

Marchetti A(1,2), Lewandowski J(1), Dupeux F(3), Blackledge M(4), Pintacuda G (1),Marquez J(3), Emsley L(1)

(1) Universite de Lyon, CNRS/ENS Lyon/UCB Lyon 1, Centre de RMN a Tres HautsChamps, 5, rue de la Doua, F-69100, Villeurbanne, France.alessandro.marchetti@ens-lyon.fr (2) Scuola Normale Superiore, Piazza dei Cavalieri 7,I-56126, Pisa, Italia (3) EMBL Grenoble, BP 181, 6 rue Jules Horowitz, F-38042Grenoble Cedex 9, France (4) IBS/FDP, 41 rue Jules Horowitz, F-38027 GrenobleCedex 1, France

The plant stress-response pathway controlled by the hormone S-2-cis-4-trans abscisicacid (ABA), has a pivotal role in coordinating the adaptive response in situations ofdecreased water availability as well as the regulation of plant growth: the ABA sens-ing mechanism is for example crucial in seed development, inhibition of germinationand dormancy, as well as in stimulating root growth to improve water uptake. It hasbeen recently proved that interaction of abscisic acid with a 14-member proteins family,named PYR/PYL/RCAR, inhibit in an ABA-dependent manner the activity of a familyof key negative regulators of the ABA signaling pathway, the group-A protein of type-2C phosphatases (PP2Cs). 3D structures of some key complexes of these proteins withtheir coreceptor have hence been characterized through X-ray single crystal diffractionbut,nonetheless, many questions concerning the mechanism of plant drought resistanceinvolving this molecule remain. We have therefore undertaken the study by solid-stateNMR of one of these challenging systems, the PYR1 dimer (193*2 residues, 44 kDa)bound with its hormonal substrate ABA (PYR1:ABA). Using the expertise of the X-raydiffraction oriented HTX facility at the EMBL Grenoble, crystallization conditions withlower salt and buffer concentrations were found by optimization of the recipe used for X-ray crystallogenesis, yielding nanocrystals with optimal spectral properties. We will showhow the application of high field ultra fast MAS allows the acquisition of well resolved15N-13C correlations providing the basis for the assignment of numerous resonances ofthe protein backbone and at the same time constituting a probe of dynamics associatedwith conformational changes of the binding process.

86

Poster 33

Solid-state NMR on full-length oligomeric α-B crystallin and its mutantR120G

Markovic S(1), Brubaker W(2), Mainz A(3), Rehbein K(1), Reif B(3), van RossumBJ(1), Martin R(2), Oschkinat H(1)

(1) Structural Biology, Leibniz-Institut fur molekulare Pharmakologie (FMP), Berlin,Germany (2) Department of Chemistry, University of California, Irvine, USA (3)Department Chemie, Technische Universitat Munchen, Munich, Germany

α-B crystallin (αB) belongs to the group of small heat shock proteins and serves as achaperone to prevent heat-induced cellular stress and aggregation. Prominent substratesare the β/γ-crystallins in the eye lens, amyloid-β and α-synuclein, which cause neurode-generative diseases, and several cytoskeletal proteins such as desmin and microtubules.αB forms polydisperse oligomers up to 900 kDa, where oligomer size and chaperone ac-tivity are regulated by intermolecular binding. We apply solid-state NMR techniques toperform structural studies on oligomer activation, malfunction and substrate interactions.

87

Poster 34

NMR Studies of the Interaction between T-20 and gp120

Moseri A(1), Sagi1 Y(1) Biron Z(1), Naider F(2) and Anglister J(1).

(1)Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100,Israel, adi.moseri@weizmann.ac.il and (2) Department of Chemistry, College of StatenIsland of the City University of New York, Staten Island, New York 10314

Background: T-20 is the first FDA approved entry inhibitor, comprised of a 36 aminoacid linear segment from gp41 C-terminal heptad repeat (HR2). It was proposed to actby binding to the gp41 HR1 domain preventing the formation of the six-helix bundle.Interestingly T-20 mediated inhibition is modulated by co-receptor usage determined bythe sequence of the V3 loop. Recent studies demonstrated a direct binding between T-20 and gp120 preferably from CXCR4 utilizing strains. This binding was enhanced bysCD4 and proposed to be localized in the gp120 co-receptor binding site. Method: Theinteraction between NN-T-20-NITN and a gp120 construct based on the JR-FL R5 strainwas studied by SPR and NMR. The gp120 used was homogenously glycosylated withMan5GlcNAc2 and missing the V1/V2 loops in order to facilitate exposure of the V3loop. Results: NN-T20-NITN binding to gp120 was detected by SPR and was enhancedin the presence of the CD4M33 peptide which induces the CD4 bound conformation.NN-T20-NITN was 15N labeled for NMR studies. Using 1H-15N HSQC titrations thegp120 binding site on NN-T20-NITN was mapped to the tryptophan rich region at the C-terminus of T-20 667A S L W N W672. Conclusions: This study provided residue specificcharacterization of T-20 interaction with gp120. Further studies will be conducted toclarify T-20 co-receptor specificity and the role of the above interaction in T-20 mediatedinhibition.

88

Poster 35

NMR Characterization of Long-Range Order in Intrinsically DisorderedProteins

V. Ozenne (1), L Salmon (1), N. Gabrielle (1), G. Yin (2), M. Ringkjøbing Jensen (1),M.Zweckstetter (2), and M. Blackledge (1)

(1)Protein Dynamics and Flexibility, Institut de Biologie Structurale Jean-Pierre Ebel,Grenoble, France (2)NMR-Based Structural Biology, Max Planck Institute forBiophysical Chemistry, Gottingen, Germany

Intrinsically disordered proteins (IDPs) are predicted to represent a significant fractionof the human genome, and the development of meaningful molecular descriptions of theseproteins remains a key challenge for contemporary structural biology. In order to describethe conformational behavior of IDPs, a molecular representation of the disordered statebased on diverse sources of structural data that often exhibit complex and very differ-ent averaging behavior is required. We propose novel approaches using a combination ofparamagnetic relaxation enhancements (PREs) and residual dipolar couplings (RDCs)to determine simultaneously long-range and local structure in highly flexible proteins.Indeed, IDPs exhibit transient or persistent long-range tertiary structure that may berelated to biological activity or simply confer protection from proteolysis or amyloidosis.We investigate the effects of long-range contacts on the expected values of RDCs andshow that they can induce a severe distortion of the RDC baseline that compromises thedescription of local conformational sampling. By analytically describing these baselinedistortions and their dependence on the position of the contacting regions, we proposea way to simultaneously determine long-range and local structure in highly flexible pro-teins. This combined analysis is shown to be essential for the accurate interpretation ofexperimental data from R-synuclein, an important IDP involved in human neurodegener-ative disease, confirming the presence of long-range order between distant regions in theprotein.

89

Poster 36

Metal binding properties of the N-terminal domain of ZntA P1-type ATPase

Lucia Banci (1,2), Ivano Bertini (1,2), Simone Ciofi-Baffoni (1,2), Bharati Mitra (3) andRiccardo Peruzzini (1,2)

(1) Department of Chemistry “Ugo Schiff”, University of Florence, Italy; (2) MagneticResonance Center (CERM) – University of Florence, Via L. Sacconi 6, 50019 SestoFiorentino, Italy (peruzzini@cerm.unifi.it); (3) Department of Biochemistry andMolecular Biology, School of Medicine, Wayne State University, Detroit, Michigan48201

Zinc is an essential element required for the growth and metabolism of cells. Bacteriaand eukaryote use it as cofactor in a large number of proteins. However, excess of zinc canbe quite toxic. So, while the actual concentration of zinc ions in cells can reach up to 0.5mM, actually the free ion concentration in cell never goes over 10−5 M. Therefore, cellshave developed a mechanism for homeostasis of zinc that regulates the uptake and thedistribution of the ion among different target. Large part of this mechanism still remainunknown.

In the Escherichia coli the detoxification of excess of Zn(II) is achieved through severalmechanisms. ZntA, a P1-type ATPase, is one of the proteins involved in these processes.Proteins of this family are widely used by several organisms to transport across the mem-brane mono and bivalent metal ions and all share the same structure with eight trans-membrane domains and two or more N-terminal cytoplasmic domains. ZntA has beenpreviously characterized as a transporter of Zn(II), Cd(II) with a characteristic DCXXCbinding motif in the N-terminal domain. [1] ZntA can also transport Pb(II) for detoxifica-tion and it has been proposed that the mechanism of Pb(II) transport could be differentfrom the one of Zn(II), involving further cysteine residues at the N-terminus, which indeedcontains an additional cystein-rich motif (CCCDGAC). [2]

Our goal in this study is the characterization by NMR of the solution structure of theN-terminal domain of ZntA and its interaction with Pb(II) and Zn(II) ions, in order todefine coordination-related metal selectivity of this domain toward the two metal ions.

Our results shows that:The first 46 residues at the N-terminus are largely unfolded, while the rest of the

residues of the domain are folded forming a βαββαβ ferrodoxin-like fold.A domain with a deletion of the first 46 residues (named ∆46) has the same structure

and backbone chemical shifts of the folded part in the full length N-terminal domain.Binding of Zn(II) in the DCXXC site does not occur with the involvement of the

CCCXXXC motif. Zn(II) binds indeed both full length and ∆46 N-terminal domain inthe same way.

Pb(II) binds only the full length N-terminal domain of ZntA with a required involve-ment of both DCXXC and CCCXXXC motifs.

References:[1] Banci L, Bertini I, Ciofi-Baffoni S, Finney LA, Outten CE, O’Halloran TV, J Mol

Biol. 2002; 323(5):883-97.[2] Liu J, Stemmler AJ, Fatima J, Mitra B, Biochemistry. 2005; 44(13):5159-67.

90

Poster 37

Investigation of steady-state kinetics and dynamics of the multistep bindingof bile acid molecules to a lipid carrier protein

Ragona L(1), Cogliati C(1,2), Assfalg M(2), Zanzoni S(2), Tomaselli S(1), WhittakerS(3), Ludwig C(3), Gunther U(3), Molinari H(2)

(1) Laboratorio NMR, ISMAC, CNR, via Bassini15, 20133 Milano, Italy; (2)Dipartimento Scientifico e Tecnologico, Universita di Verona, Strada Le Grazie 15,37134 Verona, Italy; (3) School of Cancer Sciences, University of Birmingham, VincentDrive, Birmingham, B15 2TT, United Kingdom.

The investigation of multi-site ligand protein binding and multistep mechanisms ishighly demanding. Advanced NMR approaches, such as 2D 1H-15N line shape analysis,allowing a reliable investigation of ligand binding occurring on microsecond to millisec-ond time scales, have been here extended to model a two-step binding mechanism. Themolecular recognition and the complex uptake mechanism of two bile salt molecules bylipid carriers is an interesting example showing that protein dynamics has the potentialto modulate the macromolecule-ligand encounter. Kinetic analysis supports a conforma-tional selection model as the initial recognition process, where the dynamics observedin the apo-form is essential for ligand uptake, leading to conformations with improvedaccess to the binding cavity. Subsequent multistep events could be modelled, for severalresidues, with a two-step binding mechanism. The protein, in the ligand-bound state, stillexhibits a conformational rearrangement occurring on a very slow time-scale, as observedfor other proteins of the family. A global mechanism suggesting how bile acids access themacromolecular cavity is thus proposed.

91

Poster 38

Maximum Occurrence of conformations and regions: defining theconformational space sampled by Calmodulin

Bertini I(1,2), Giachetti A(1), Luchinat C(1,2), Parigi G(1,2), Petoukhov MV(3),Pierattelli R(1,2), Ravera E(1,2) and Svergun DI(3)

(1) Magnetic Resonance Center (CERM),University of Florence, 50019 SestoFiorentino, Italy, (2) Department of Chemistry “Ugo Schiff” – University of Florence,50019 Sesto Fiorentino, Italy, (3) EMBL, Hamburg Outstation, D-22603 Hamburg,Germany, and Institute of Crystallography, Russian Academy of Sciences, 117333Moscow, Russia

Structure and dynamics of proteins are strictly intertwined in defining the functionof the proteins themselves: probing protein plasticity is required to uncover key aspectsof the function. This kind of study represents a difficult task for X-ray crystallography,which may at best yield the structure of a single conformation trapped in the crystal. Solu-tion techniques, such as small angle scattering (SAS) techniques and paramagnetic NMRspectroscopy can provide experimental observables that are averages over a manifold ofconformations with different weights. The problem of recovering the protein conforma-tional ensemble from average data is an ill-defined inverse problem that admits an infinitenumber of solutions. Maximum Occurrence (MO) is defined to score each and every con-formation according to its largest weight possible in any ensemble in agreement with theaveraged experimental data. The two-domain protein calmodulin was used as a test case,using SAXS data and three sets of pseudocontact shifts and residual dipolar couplingsobtained after substitution of a lanthanide ion (Tb3+, Tm3+ or Dy3+) to the secondbinding site in the N-terminal domain. The method is universally applicable as it onlyrequires NMR data on paramagnetic derivatives of the protein (using native metal sitesor lanthanide tagging) and possibly SAS measurements and, thanks to distributed com-puting accessed through the e-nmr virtual organization, a big number of structures canbe routinely scored. Another important piece of information can be obtained by consider-ing the collective Maximum Occurrence for all the conformations belonging to a definedregion. At the same time, the complementary region is scored by a minimum Occurrencethat can be calculated as mO=(1-MO).

References:1. Bertini I., et al. Proc. Natl. Acad. Sci. USA, 101, 6841 – 6846 (2004)2. Bertini I., et al, J. Am, Chem. Soc., 129, 12786 – 12794 (2007)3. Bertini I., et al. J. Am, Chem. Soc., 132, 13553 – 13558 (2010)

92

Poster 39

Rosato A.

Abstract not available.

93

Poster 40

Temperature Dependent Intramolecular Dynamics of Exendin-4

Rovo P(1), Nyitray L(2), Perczel A(1)

(1) Eotvos Lorand University, Institute of Chemistry, Laboratory of StructuralChemistry and Biology (2) Eotvos Lorand University, Institute of Biology, MotorProteins, Structural Biology

The temperature dependence of the backbone dynamics of Exendin-4 (Ex-4) was stud-ied by 15N nuclear magnetic relaxation measurements. Longitudinal relaxation rates,transverse relaxation rates and heteronuclear Overhauser effects were collected for back-bone amide nitrogen at 277 K, 300 K and 312 K in water and in 30 vol% trifluoroethanol(TFE). The measured relaxation parameters were analyzed using both the Reduced Spec-tral Density Mapping approach to determine values of spectral density function at threefrequencies and the Lipari-Szabo model free formalism to characterize overall and in-tramolecular motions. We found that in water Ex-4 tumbles like a peptide while in thereceptor-bound mimicking state (30% TFE) it behaves more like a protein and has uni-form globular dynamics. At almost all conditions the N-terminal 9-residue segment rotatesindependently from the central α-helix. This N-terminal segment has extremely high in-ternal motions while the core helix is relatively rigid even at high temperatures.

94

Poster 41

Structure, Dynamics and Kinetics of Weak Protein-Protein Interactionsfrom Titration of NMR Relaxation

Salmon L(1), Roldan JL(2), Lescop E(3), Van Nuland N(4), Jensen MR(1), BlackledgeM(1)

(1) Protein Dynamics and Flexibility, Institut de Biologie Structurale,CNRS/CEA/UJF, Grenoble, France (2) Departamento de Quımica Fısica, Universidadde Granada, Granada, Spain (3) LCBS, ICSN CNRS, Gif sur Yvette, France (4)Structural Biology Brussels, Vrije Universiteit, Brussels, Belgium

The study of weak protein-protein interactions is a biologically important problem asthey are involved in a vast range of cellular events, such as transcription and replication orsignal transduction.They are notoriously difficult to characterize experimentally, defyingco-crystallization and because of the difficulties encountered when differentiating informa-tion deriving from bound and free forms of the proteins. Here an approach is developed tomeasure transverse and longitudinal 15N relaxation rates for a weak interaction betweenCD2AP SH3-C and Ubiquitin (Kd=0,2 mM). This study allows the quantitative deriva-tion of directly unmeasurable relaxation rates in the complex and thereby the extractionof accurate rotational diffusion tensors that provide essential conformational constraints.An estimation of the kinetics of complex formation was obtained using the exchange con-tribution to the transverse relaxation rate, present for each residue exhibiting differentchemical environment in the free and the bound form. Finally the fast dynamics in thecomplex can be determined and compared with the dynamics in the free forms of theproteins.

95

Poster 42

Elucidation of lignin structure by quantitative 2D-NMR

Sette M(1),Wechselberger R(2), Crestini C(1)

(1) Dipartimento di Scienze e Tecnologie Chimiche, Tor Vergata University, Via dellaRicerca Scientifica, 00133, Rome, Italy; (2) Janssen Pharmaceutica, Belgium

Quick Quantitative HSQC (QQ-HSQC) was applied to the quantitative evaluation ofdifferent interunit bondings in an array of milled softwood and hardwood and technicallignins using the guaiacyl C2 and syringyl C2-C6 signals as internal standard. The re-sults were found to be highly reproducible and comparable with earlier literature reports.The advantage of QQ-HSQC NMR analysis of lignin is the contemporary detection andquantification of lignin interunit bonding with a direct, non-destructive method requiringshort acquisition times.

96

Poster 43

The conformation and interactions of a 27-residue N-terminal peptide ofCCR5 in complex with gp120

Schnur E(1), Scherf T(1), Naider F(2), Anglister J(1)

(1) Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100,Israel. (2) Department of Chemistry and Macromolecular Assembly Institute, College ofStaten Island of the City University of New York, Staten Island, New York 10314, USA.

Human immunodeficiency virus type 1 (HIV-1) is the retrovirus that causes the ac-quired immunodeficiency syndrome (AIDS). HIV infection in humans is a pandemic af-fecting millions of people around the world. HIV-1entry into target cells is mediatedby the successive interaction of the viral envelope glycoprotein gp120 with the cellularreceptor CD4 and with a G protein-coupled chemokine co-receptor, mainly CCR5 orCXCR4. Interaction of CCR5 with the HIV-1 gp120-CD4 complex involves the amino-terminal domain of the CCR5 receptor (Nt-CCR5) and requires posttranslational sulfa-tion of its tyrosine residues. We studied a 27-residue peptide corresponding in sequence toNt-CCR5 (residues 1-27) and containing two sulfated tyrosine residues at positions Y10,Y14, in complex with a truncated gp120 (JR-FL, residues 88-492, -V1, -V2, homogenouslyGlcNAc-glycosylated) and a CD4-mimic miniprotein. NMR studies allowed calculation ofthe structure of Nt-CCR5 in its gp120- bound conformation. T1-rho-filtered NOE ex-periments revealed evidence for a helical conformation in the center of Nt-CCR5(1-27),induced upon gp120 binding, as well as a helical propensity of the peptide in its free form,especially at low temperatures (274 K). Saturation Transfer Difference (STD) experimentsallowed identification of the Nt-CCR5 residues that participate in gp120 binding.

97

Poster 44

Tetramolecular DNA Quadruplexes in Solution

Sket P(1,2), Plavec J(1,2,3)

(1) Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, SI-1001,Ljubljana, Slovenia (2) EN-FIST Centre of Excellence, Dunajska 156, SI-1001,Ljubljana, Slovenia (3) Faculty of Chemistry and Chemical Technology, University ofLjubljana, Askerceva cesta 5, SI-1000 Ljubljana, Slovenia, primoz.sket@ki.si

Guanine-rich DNA sequences can fold into four-stranded G-quadruplex structurescomposed of G-quartets, planar arrays of four guanines held together by eight Hoog-steen hydrogen bonds. G-rich sequences are overrepresented in the promoter regions ofa number of genes, including oncogenes, ribosomal DNAs as well as in telomeric DNAregions and immunoglobulin heavy chain switch regions of higher vertebrates. Forma-tion of G-quadruplexes has been implicated in association with human diseases. Additionof cations such as K+ or Na+ induces formation of G-quadruplexes. Sequence detailsand the nature of metal ions play a major role in formation and stabilization as well asstructural diversity of G-quadruplexes. Oligonucleotides containing only a single run ofguanines form tetramolecular G-quadruplex structures consisting of four strands. By theuse of solution-state NMR spectroscopy we have deepen our understanding of the struc-tural diversity of a simple oligonucleotide d(TG4T) as well as its U analogues. To date, asingle form of d(TG4T)4 in solution has been described, whereas the current NMR studyhas demonstrated the coexistence of two forms. One of the forms exhibits a T-quartet inaddition to four G-quartets. Substitution of U for T of the parent sequence provides fur-ther insights into dimerization and macromolecular association of G-quadruplex units. Inaddition, we have unequivocally proved that 15NH4+ ion movement inside the central ioncavities of tetramolecular G-quadruplexes is ca. 10 times faster than in bimolecular andmonomolecular G-quadruplexes. However, a T-quartet at the 5’ end decelerates cationmovements.

Reference:1. Sket P. and Plavec J., J. Am. Chem. Soc., 132, 12724 – 12732 (2010)

98

Poster 45

A tale of two halves: a NMR based study of stability and dynamics ofdifferent forms of BS-RNase

Picone D(2), Ercole C(2), Tancredi T(3), Boelens R(4), Batta G(5) and Spadaccini R(1)

(1)Dip. Scienze Biologiche ed Ambientali, Universita del Sannio,, 82100 Benevento,Italy, rspadacc@unisannio.it (2)Dip. Chimica, Universita degli Studi di Napoli “FedericoII”, 80126 Napoli, Italy, carmine.ercole@unina.it , picone@unina.it, (3)ICB del CNR,Pozzuoli, 80078 Italy, ttancredi@icmib.na.cnr.it (4) Bijvoet Center for BiomolecularResearch, Utrecht University, 3584 Utrecht, The Netherlands, r.boelens@uu.nl (5)Department of Organic Chemistry, University of Debrecen, H-4010 DebrecenHungary,batta@tigris.unideb.hu

One of the main targets of cancer therapy is the development of more selective, bio-logical mechanism-based agents that can overcome tumor resistance and minimize toxiceffects to normal cells. Several ribonucleases seem to be a good candidate for this ap-proach because of their strong cytotoxic activity towards malignant tumour cells. Amongribonucleases, bovine seminal ribonuclease (BS-RNase ) is the one that shows some ofthe most interesting features. This is the only mammalian dimeric ribonuclease and ithas been proposed that the interchange (swapping) of N-terminal helices is often asso-ciated with new biological functions including cytotoxic activity.The current hypothesislinking the swapping to the antitumor activity is that the entanglement of N-terminaltails hinders the neutralizing effect of the cytosolic ribonuclease inhibitor (RI), a proteinextremely abundant in mammalian cells that protects endogenous RNA by binding withvery high affinity endogenous ribonucleases. Thanks to two disulfide bonds bridging thetwo identical subunits of BS-RNase, the native protein exists as an equilibrium mixture oftwo isoforms, MxM and M=M, with and without exchange respectively, which show onlyminor structural differences in their X-ray structures, located essentially at level of the16-22 hinge regions, i.e. the loop connecting the dislocating arm to the main body of theprotein. High resolution NMR experiments allow a fine characterization of the structuraland dynamical properties of the different forms that the BS-RNase adopts in solution, i.e.monomer, swapped and un-swapped dimers and can be helpful to define the mechanismof interconversion and, perhaps, to design mutants with improved biological activity. Herewe present recent results based on relaxation, H/D exchange and chemical stability NMRexperiments acquired on both monomeric and dimeric forms of BS-RNase.

99

Poster 46

STRUCTURE-ACTIVITY RELATIONSHIP OF ARKADIA RINGFINGER E3 UBIQUITIN LIGASE THROUGH NMR SPECTROSCOPY

A. Loutsidou1, C.T Chasapis1, N.G Kandias1, D. Bentrop2, V. Episkopou3,G.A. Spyroulias1

1 Department of Pharmacy, University of Patras, GR-26504, Patras, Greece. 2 Instituteof Physiology II, University of Freiburg, D-79104 Freiburg, Germany. 3 MammalianNeurogenesis, MRC Clinical Sciences Centre, Imperial School of Medicine,Hammersmith Hospital, London W12 0NN, United Kingdom

E3 ubiquitin ligases play a key role in the recognition of target proteins by catalyzingthe covalent attachment of the ubiquitin and degradation by 26S proteasome[1]. Arkadiais the first example of an E3 ligase that positively regulates TGF–β family signaling.Arkadia has been suggested to induce ubiquitin-dependent degradation of negative regu-lators of TGF–β signaling, through its C-terminal RING finger domain[2]. The 68 a.a. ofthe Arkadia C-terminal, including the RING finger, was cloned and expressed in its zinc-loaded form, as suggested by atomic absorption and studied through multi-nuclear andmulti-dimensional NMR Spectroscopy[3]. The vast majority of the backbone 1H-15N res-onances have been assigned and deposited in the BioMagResBank (Accession No. 15948).The 3D NMR solution structure of Arkadia RING finger was determined and its atomiccoordinates have been deposited in Protein Data Bank (i.d. 2KIZ). Additionally, titra-tion studies monitored by NMR were also performed to probe the interaction interface ofArkadia RING and the partner E2 (UbcH5B) enzyme. The RING-E2 complex structurewas also constructed through an NMR-driven docking protocol (using HADDOCK). Fi-nally, mutations identified in the RING domain were analyzed in the light of the NMRmodel and new non-native RING finger forms (uniformly labeled in 15N/13C) were pro-duced with the aim to study the ZN-dependent structural integrity of the RING fingerand its ability to interact with E2 partner enzyme. NMR structures of these mutants willcontribute significantly to the Structure-Activity study of Arkadia and its role in TGF-bsignaling pathway.

References1. Hershko A & Ciechanover A, Annu Rev Biochem 1998, 67, 425–479.2. Mavrakis KJ, Andrew RL, Lee KL, Petropoulou C, Dixon JE, Navaratnam N, Norris

DP, Episkopou V. PLoS Biol 2007, 5, e673. Kandias NG, Chasapis CT, Bentrop D, Episkopou V, Spyroulias GA. BBRC 2009,

378, 498-502

100

Poster 47

Stanek J.

Abstract not available.

101

Poster 48

Light induced dynamics of proteorhodopsin investigated by NMR

Stehle J(1), Reckel S(2), Dotsch V(2), Schwalbe H(1)

(1)Institute for Organic Chemistry and Chemical Biology, Goethe University, Frankfurtam Main, Germany, stehle@nmr.uni-frankfurt.de (2)Institute of Biophysical Chemistry,Goethe University, Frankfurt am Main, Germany

Abstract Green-absorbing proteorhodopsin (PR), a light-driven proton pump, showsa strong dependence of its function on the pH. The primary proton acceptor D97 has anunusually high pKa value around 7.5 and its protonation state affects the absorption char-acteristics of the retinal cofactor. Furthermore, the direction of proton pumping switchesin response to pH between an outward directed transport at alkaline pH and an inwarddirected transport at acidic pH. The potential function of this pH-dependency includ-ing the changing vectoriality is, however, still debated and a possible regulatory activitycannot be excluded. For further insights into the dynamic properties of PR, illuminationexperiments using in situ NMR were conducted. Distinct light-induced structural rear-rangements could be observed at alkaline and acidic pH values supporting the idea of apH-dependent activation mode where only defined pH values allow a light response of PR.Mapping the chemical shift perturbations induced by illumination, revealed completelydiverse shift patterns for alkaline and acidic pH values, suggesting different conformationalchanges consistent with the isomerization-switch-transfer model.

102

Poster 49

Lanthanide binding CLaNP-5.2 for NMR of proteins: exploiting PCS andRDCs in MMP-1 internal dynamics

Bertini I(1), Cerofolini L(1), Fragai M(1), Geraldes CFGC(2), Luchinat C(1),Teixeira JMC(1,2)

(1)CERM, University of Florence, Italy (2)Department of Life Sciences and Center ofNeurosciences and Cell Biology, University of Coimbra, P.O. Box 3046, 3001-401Coimbra, Portugal, joaot@ci.uc.pt

The structure and dynamics of proteins functionalized with paramagnetic Lanthanidescan be studied using NMR tools provided by the paramagnetic center, such as pseudocon-tact shifts (PCS) and residual dipolar couplings (RDC). Lanthanide functionalization ofproteins can either be achieved by direct chelation of the ion by the protein(1) or throughthe attachment of artificial paramagnetic chelating tags(2,3). It was shown before thatMatrix Metalloproteinase-1 (MMP-1) has a relevant degree of internal freedom regardingits two domains, catalytic (CAT) and hemopexin-like (HPX) domains(4). In this work,(Ln)CLaNP-5.2(5) was attached to double Cys mutated CAT and the paramagnetic sus-ceptibility tensor was calculated and validated through PCS and RDCs for three differentLanthanides – Yb3+, Tm3+, and Tb3+. Recent work performed at CERM in collabo-ration with M. Ubbink shows that (Ln)CLaNP-5.2 can be attached to the C-terminaldomain of calmodulin and used to increase the information content generated by Lan-thanides bound to site II in the N-terminal domain on the conformational space sampledby this flexible two-domain protein(6). With this in mind, we functionalized the FullLength MMP-1 with the (Ln)CLaNP-5.2 and, considering the constancy of the tensors,we expect to be able to calculate the internal dynamics of the HPX domain with respect tothe CAT domain of MMP-1. 1. Bertini, I. et al. ChembioChem. 6, 1536-49(2005) 2. Nitz,M. et al. Angewandte Chemie Int.Ed. Engl. 43, 3682-5(2004) 3. Rodriguez-Castaneda,F. et al. Magn.Reson. Chem. 44 , S10-6(2006) 4. Bertini, I. et al. J. Biol. Chem. 284,12821-8(2009) 5. Keizers, P.H.J. et al. J. Am. Chem. Soc. 129, 9292-3(2007) 6 IvanoBertini, Soumyasri Das Gupta, Xiaoyu Hu, Peter H. J. Keizers, Claudio Luchinat, MaliniNagulapalli, Giacomo Parigi, Luca Sgheri, Marcellus Ubbink, Improving the maximumoccurrence analysis of calmodulin conformations in solution by placing paramagnetic ionsin both protein domains, in preparation.

103

Poster 50

NMR study of arabinan from sugar beet fiber

Uhliarikova I(1), Simkovic I(1)

(1) Institute of Chemistry, Slovak Academy of Sciences, 845 38 Bratislava, SlovakRepublic

Sugar beet pulp was extracted and chemically modified under neutral [1], acidic [2]and alkaline/peroxide conditions. The goal was to find out how the used conditions couldaffect the yield of soluble polysaccharide, its molar mass, monosaccharide compositionand structure. Under neutral conditions when extracted in water the yield is the smallest(1 %). In the presence of acids the yields were from 2 to 4 % and the molar mass closeto extracts with water (120 to 175 kg/mol) when treated with trifluoroacetic acid. In thepresence of hydrochloric or phosphoric acids the molecular mass increased from 1297 to1760 kg/mol. In the presence of NaOH or NaOH/H2O2 the yield were around 20 % andthe molar mass smaller than 681 kg/mol. According to NMR analysis (COSY, TOCSY,HSQC, and HMBC) the branching of the arabinan is demonstrated by many arabinoseunits with different chemical shifts. The branching of the extracted arabinan was moreevident when the extraction took place under neutral or acidic conditions than in thepresence of NaOH or NaOH/H2O2.

References1. I. Simkovic, A. Nunez, G. D. Strahan, M. P. Yadav, R. Mendichi, K. B. Hicks: Frac-

tionation of sugar beet pulp by introducing ion-exchange groups. Carbohydrate Polymers,76, 806-812 (2009).

2. I. Simkovic, I. Uhliarikova, M. P. Yadav, R. Mendichi: Branched arabinan obtainedfrom sugar beet pulp by quaternization under acidic conditions. Carbohydrate Polymers,82, 815-821 (2010).

104

Poster 51

Malaria cytoadherence: understanding the structural changes in the infectederythrocyte.

Christina Mayer, Leanne M. Slater, Michele C. Erat and Ioannis Vakonakis

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX13QU, United Kingdom

Upon infection of host erythrocytes, Plasmodium falciparum expresses proteins whichalter the shape and mechanical properties of red blood cells (RBC). The modified RBCfeature membrane protrusions (“knobs”) where parasite proteins accumulate. Knob for-mation is a critical step in the life cycle of P. falciparum as it allows infected erythrocytesto adhere to blood vessel walls and avoid clearance by the spleen. Knobs are equallyimportant in malaria pathology since RBC cytoadherence results in obstruction of themicrovasculature and tissue damage. However, despite the medical interest relatively lit-tle is known about the molecular details of this system. The P. falciparum ErythrocyteMembrane Protein 1 (PfEMP1) family of proteins localize to knobs and mediate erythro-cyte adherence through direct interactions with epithelial cell receptors. PfEMP1 featurevariable extracellular segments and a conserved ∼400 residue intracellular domain that ispresumed important for localization. Here, we show using NMR that PfEMP1 intracellulardomains are only partly structured; our data extend to multiple members of this proteinfamily. We present the structure of the core PfEMP1 fragment and discuss the variousintermolecular interactions present in the literature. In particular, we examine the effectsof the parasite Knob Associated Histidine Rich Protein (KAHRP). KAHRP is postulatedto interact directly with PfEMP1 and spectrin, thereby anchoring knobs to the cytoskele-ton and altering the mechanical properties of the spectrin-actin mesh. We discuss ouron-going efforts at isolating and defining these interactions. NMR and other biophysicalmethods are crucial to obtain detailed structural information on malarial cytoadherence.Our long-term goal is to understand the interactions between parasite and host proteinsthat help to form and maintain RBC knobs. We believe that better understanding of thesemechanisms would be of great value for later drug design studies.

105

Poster 52

De novo structure of the Yad A membrane protein by solid-state MAS NMR

Shahid SA(1), Bardiaux B(1), Markovic S(1), Franks T(1), Habeck M(2), Linke D(2),van Rossum BJ(1)

(1)Leibniz-Institute of Molecular Pharmacology (FMP), 13125 Berlin, Germany,brossum@fmp-berlin.de (2)Max Planck Institute for Developmental Biology, 72076Tubingen, Germany

Yad A is the membrane protein from Yersinia Enterocolitica and is a distinctive mem-ber of non-fimbrial, non-pillus adhesins, called trimeric autotransporter adhesins. Its trans-membrane anchor domain (YadA-M, 315 residues) forms a highly stable trimeric β-barrel.It plays a role as autotransporter, where it transports its own C-terminal domain throughthe membrane. Yad A adheres to the host cell surface and plays an important role inenteric food borne diseases. We used solid-state MAS NMR to study the structure of themembrane protein. We managed to fully assign the NMR data and to calculate a de novostructure of the YadA-M anchor domain.

106

Poster 53

NMR Studies of the extracellular domain of a prokaryotic Ligand-Gated IonChannel(LGIC)

Vlachou P.M. (1), Chasapis C.T. (1), Argyriou A.(1), Bentrop D.(2), Bocquet N.(3),Corringer P-J(3), Spyroulias G.A.(1)

(1)Department of Pharmacy, University of Patras, GR-26504, Patras, Greece(2)Institute of Physiology II, University of Freiburg, D-79108 Freiburg, Germany(detlef.bentrop@physiologie.uni-freiburg.de) (3)Pasteur Institute,G5 Group ofChannel-Receptor, CNRS URA 2182

Pentameric ligand-gated ion channels of the Cys-loop family are of special importancefor the rapid chemo-electrical signal transduction at synapses [1-3], but the mechanisms ofion permeation and gating of these membrane proteins remain elusive. Recently the X-raystructures of two prokaryotic homologues of the nicotinic acetylcholine receptor (nAChR),the best studied member of the LGIC family, have been determined: 1) the bacterialGloeobacter violaceus pentameric ligand-gated ion channel homologue 4 (GLIC; 2.9 A) inan open conformation [2] and 2) a homologue from the bacterium Erwinia chrysanthemi(ELIC; 3.3 A) in a closed conformation [3]. The 200-residue extracellular domain of GLIC,which is found to be a monomer in solution, was cloned and expressed in high yields in E.coli. The 1H-15N HSQC exhibits signal dispersion typical for polypeptides with mainlyβ structure. 13C/15N labeled GLIC is now studied using heteronuclear multidimensionalNMR spectroscopy and more than 40% of the backbone 13C/15N nuclei have alreadybeen assigned. Additionally, protein deuteration from 60 to 100% is expected to increasethe spectral sensitivity and resolution and allow the complete resonance assignment of theprotein and the extraction of NOE constraints (Chasapis C.T. et al. work in progress).

References:1. Corringer, PJ & Changeux, JP Scholarpedia, 3, 3468 (2008).2. Bocquet N, Nury H, Baaden M, Le Poupon C, Changeux JP, Delarue M, Corringer

PJ. Nature, 457, 111-4 (2009).3. Hilf RJ, Dutzler R. Nature 457, 115-8 (2009).

107

Poster 54

13C band-selective experiments for the determination of heteronuclearlong-range couplings: bsHSQC-TOCSY-IPAP, bsHR-HMBC3

Zalibera M(1),Hricovıni M(1)

(1) Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, SK-845 38Bratislava, Slovak Republic, zalibera.m@gmail.com

The determination of long-range heteronuclear coupling constants (nJXH; n ¿ 1) isan important parameter in the structural and conformational analysis of biomolecules.Two different strategies have been mostly used for the nJXH determination in pulse se-quences: (i) HSQ-TOCSY-type experiments for the measurement of nJXH on protonatedcenters and (ii) HMBC or long-range HSQC (HSQMBC) based sequences that providethe coupling constants also for nonprotonated heteronuclei. Here we present semi-selectiveversions of recently reported 2D HSQC-TOCSY-IPAP[1] (bsHSQC-TOCSY-IPAP) andHR-HMBC3[2] (bsHR-HMBC3) sequences. The series of these experiments can providean improved F1 resolution when compared to a single nonselective experiment withinthe same spectrometer time. This can be advantageously used in molecules sufferingwith severe overlap of resonances, such as saccharides with crowded ring carbon region.The presented sequences have been tested on disaccharide sample, α-Me-maltopyranoside.Measured proton-carbon coupling constants were compared with those obtained by DFTmethod using double-zeta basis set. The advantages of the presented methods, as well astheir limitations, are also discussed. [1] K. Kobzar, B. Luy, J. Magn. Reson. 2007, 186,131. [2] K. Furihata, M. Tashiro, H. Seto, Magn. Reson. Chem. 2010, 48, 179

108

List of Participants

Frederic HT AllainETH ZurichSwitzerlandallain@mol.biol.ethz.ch

Jacob AnglisterWeizmann Institute of ScienceIsraelJacob.Anglister@weizmann.ac.il

Alexander S. ArsenievRussian Academy of SciencesRussiaaars@nmr.ru

Sam AsamiFMP-Berlin, TU MuenchenGermanyasami@fmp-berlin.de

Michael AssfalgUniversity of VeronaItalymichael.assfalg@univr.it

Rafal AugustyniakEcole Normale Superieure, ParisFrancerafal.augustyniak@ens.fr

Wojciech AugustyniakMPI for Coal ResearchGermanydreslaw@poczta.onet.pl

Martin BabinskyNCBR, Masaryk UniversityCzech Republicmartbab@chemi.muni.cz

Veronika BacıkovaNCBR, Masaryk UniversityCzech Republicbacikova@chemi.muni.cz

Benjamin BardiauxFMP-BerlinGermanybardiaux@fmp-berlin.de

Gyula BattaUniversity of DebrecenHungarybatta@tigris.unideb.hu

Grzegorz BazylakNicolaus Copernicus UniversityPolandgbazylak@cm.umk.pl

Johanna Becker-BaldusGoethe University FrankfurtGermanyj.baldus@em.uni-frankfurt.de

Somer BekirogluTUBITAK MRCTurkeysomer.bekiroglu@mam.gov.tr

Massimo BellandaUniversity of PadovaItalymassimo.bellanda@unipd.it

Ivano BertiniCERM, University of FlorenceItalybertini@cerm.unifi.it

109

Ivana BiljanNational Institute of ChemistrySloveniaivana.biljan@ki.si

Martin BlackledgeIBS GrenobleFrancemartin.blackledge@ibs.fr

Andrea BodorEotvos Lorand UniversityHungaryabodor@chem.elte.hu

Rolf BoelensUtrecht UniversityNetherlandsboelens@nmr.chem.uu.nl

Roman BonetUniversity of OxfordUnited Kingdomroman.bonetfigueredo@bioch.ox.ac.uk

Bernhard BrutscherInstitute of Structural BiologyFrancebernhard.brutscher@ibs.fr

Hana BrichackovaNCBR, Masaryk UniversityCzech Republichanab@chemi.muni.cz

Peter BystrickyNCBR, Masaryk UniversityCzech Republicpeterbys@chemi.muni.cz

Mirko CevecGoethe University FrankfurtGermanycevec@nmr.uni-frankfurt.de

Jolyon K ClaridgeUniversity of OxfordUnited Kingdomjolyon.claridge@bioch.ox.ac.uk

Guillaume CommunieIBS GrenobleFranceguillaume.communie@ibs.fr

Alessandra CorazzaUniversity of UdineItalyalessandra.corazza@uniud.it

Hana CernaNCBR, Masaryk UniversityCzech Republichannka83@gmail.com

Rebecca Del ConteCERM, University of FlorenceItalydelconte@cerm.unifi.it

Irene Diaz MorenoUniversity of Seville - CSICSpainidiazmoreno@us.es

Michal DolezalIOCB, AS CRCzech Republicdolezal@uochb.cas.cz

Paul DriscollMRC-NIMRUnited Kingdompdrisco@nimr.mrc.ac.uk

Matus DurecNCBR, Masaryk UniversityCzech Republic211166@mail.muni.cz

Benedicte Elena-HerrmannUniversity of Lyon, RALF-NMRFrancebenedicte.elena@ens-lyon.fr

Lyndon EmsleyENS LyonFrancelyndon.emsley@ens-lyon.fr

110

Frank EngelkeBruker BioSpin GmbHGermanyfrank.engelke@bruker.de

Michele C. EratUniversity of OxfordUnited Kingdommichele.erat@bioch.ox.ac.uk

Gennaro EspositoUniversity of UdineItalyrino.esposito@uniud.it

Adrien FavierCNRSFranceadrien.favier@ibs.fr

Isabella C. FelliCERM, University of FlorenceItalyfelli@cerm.unifi.it

Radovan FialaNCBR, Masaryk UniversityCzech Republicfiala@chemi.muni.cz

W Trent FranksFMP-BerlinGermanyfranks@fmp-berlin.de

Lorella FranzoniUniversity of ParmaItalylorella.franzoni@unipr.it

Anna K FuzeryUniversity of OxfordUnited Kingdomanna.fuezery@bioch.ox.ac.uk

Jesus GarcıaIRB BarcelonaSpainjesus.garcia@irbbarcelona.org

Lorenzo GesiotUniversity of PadovaItalylorenzo.gesiot@unipd.it

Gabriele GiachinSISSAItalygiachin@sissa.it

Astrid O. GraslundStockholm UniversitySwedenastrid@dbb.su.se

Marco GuerriniG. Ronzoni InstituteItalyguerrini@ronzoni.it

Torsten HerrmannCRMN, ENS LyonFrancetorsten.herrmann@ens-lyon.fr

Chandralal HewageUniversity College DublinIrelandchandralal.hewage@ucd.ie

Fruzsina HoborNCBR, Masaryk UniversityCzech Republicfruzsi.hobor@gmail.com

Milos HricovıniSlovak Academy of SciencesSlovakiahricovini@savba.sk

Otakar HumpaNCBR, Masaryk UniversityCzech Republichumpa@chemi.muni.cz

Slavomira HusarovaSlovak Academy of ScienceSlovakiaslavomira.husarova@savba.sk

111

Josef ChmelıkInstitute of Microbiology, AS CRCzech Republicchmelik@biomed.cas.cz

Gregor IlcNational Institute of ChemistrySloveniagregor.ilc@ki.si

Hans IppelUtrecht UniversityNetherlandsj.h.ippel@uu.nl

Nadia Izadi-PruneyreInstitut PasteurFrancenadia.izadi@pasteur.fr

Jasna JablanUniversity of ZagrebCroatiajjablan@pharma.hr

Imre JakliEotvos Lorand UniversityHungaryjimre@chem.elte.hu

Olga JasnovidovaNCBR, Masaryk UniversityCzech Republicolga.jasnovidova@gmail.com

Kristaps JaudzemsLatvian Inst. Organic SynthesisLatviakristaps.jaudzems@osi.lv

Malene R. JensenIBS GrenobleFrancemalene.ringkjobing-jensen@ibs.fr

Mario JugUniversity of ZagrebCroatiamjug@pharma.hr

Simonas JurksaSpronk NMR ConsultancyLithuaniasimonas@spronknmr.eu

Pavel KaderavekNCBR, Masaryk UniversityCzech Republickada@ncbr.chemi.muni.cz

Goran KarlssonSwedish NMR CentreSwedengoran.karlsson@nmr.gu.se

Jitka KasalovaNCBR, Masaryk UniversityCzech Republicjitkak@chemi.muni.cz

Peter HJ KeizersLeiden Institute of ChemistryNetherlandsp.keizers@chem.leidenuniv.nl

Michael J KnightENS LyonFrancemichael.knight@ens-lyon.fr

Robert KonratUniversity of ViennaAustriarobert.konrat@univie.ac.at

Lidija KovacicUtrecht UniversityNetherlandslidija@nmr.chem.uu.nl

Katalin E. KoverUniversity of DebrecenHungarykover@tigris.unideb.hu

Wiktor KozminskiUniversity of WarsawPolandkozmin@chem.uw.edu.pl

112

Vojtech KubanNCBR, Masaryk UniversityCzech Republic236585@mail.muni.cz

Karel KubıcekNCBR, Masaryk UniversityCzech Republickarelk@chemi.muni.cz

Petr KulhanekNCBR, Masaryk UniversityCzech Republickulhanek@chemi.muni.cz

Andras LangELTE UniversityHungarylangax@chem.elte.hu

Adam LangeMPI for Biophysical ChemistryGermanyadla@nmr.mpibpc.mpg.de

Moreno LelliCRMN, ENS LyonFranceMoreno.Lelli@ens-lyon.fr

Ainars LeonciksASLA BiotechLatviaainleo@asla-biotech.com

Karolis LesickasSpronk NMR ConsultancyLithuaniakarolis@spronknmr.eu

Jozef R. LewandowskiCRMN, ENS LyonFrancejozef.lewandowski@ens-lyon.fr

Edvards LiepinshLatvian Inst. Organic SynthesisLatviaedv@osi.lv

Frank LohrGoethe University FrankfurtGermanymurph@bpc.uni-frankfurt.de

Christian LudwigUniversity of BirminghamUnited KingdomC.Ludwig@bham.ac.uk

Claudio LuchinatCERM, University of FlorenceItalyluchinat@cerm.unifi.it

Enrico LuchinatCERM, University of FlorenceItalyeluchinat@cerm.unifi.it

Antonın LyckaRes. Inst. of Organic SynthesesCzech Republicantonin.lycka@vuos.com

Vasantha Kumar M VenkateshaiahCERM, University of FlorenceItalykumar@cerm.unifi.it

Tobias MadlUtrecht UniversityNetherlandst.madl@uu.nl

Lena MalerStockholm UniversitySwedenlena@dbb.su.se

Katerina MalinakovaNCBR, Masaryk UniversityCzech Republickamala@ncbr.chemi.muni.cz

Despoina MallioriU of Patras, Dept of PharmacyGreeceG.A.Spyroulias@upatras.gr

113

Jiafei MaoCERM, University of FlorenceItalymao@cerm.unifi.it

Radek MarekNCBR, Masaryk UniversityCzech Republicrmarek@chemi.muni.cz

Alessandro MarchettiCRMN, ENS LyonFrancealessandro.marchetti@ens-lyon.fr

Ildefonso Marin-MontesinosIRB BarcelonaSpainildefonso.marin@irbbarcelona.org

Stefan MarkovicFMP-BerlinGermanymarkovic@fmp-berlin.de

Thorsten MarquardsenBruker BioSpin GmbHGermanythorsten.marquardsen@bruker.de

Kathleen S. McGreevyCERM, University of FlorenceItalymcgreevy@cerm.unifi.it

Beat H. MeierETH ZurichSwitzerlandbeme@ethz.ch

Henriette MolinariUniversity of VeronaItalyhenriette.molinari@univr.it

Adi MoseriWeizmann Institute of ScienceIsraeladi.moseri@weizmann.ac.il

Norbert MullerJohannes Kepler University LinzAustrianorbert.mueller@jku.at

Niels Chr. NielsenAarhus UniversityDenmarkncn@inano.dk

Jirı NovacekNCBR, Masaryk UniversityCzech Republicnovej@chemi.muni.cz

Jan NovotnyNCBR, Masaryk UniversityCzech Republicgiovannin@seznam.cz

Hartmut OschkinatFMP-BerlinGermanyoschkinat@fmp-berlin.de

Valery OzenneIBS GrenobleFrancevalery.ozenne@ibs.fr

Petr PadrtaNCBR, Masaryk UniversityCzech Republicpadrta@chemi.muni.cz

Veronika PapouskovaNCBR, Masaryk UniversityCzech Republicveverunka@chemi.muni.cz

Josef PasulkaNCBR, Masaryk UniversityCzech Republicpepap@chemi.muni.cz

Andras PerczelELTE UniversityHungaryperczel@chem.elte.hu

114

Perttu PermiUniversity of HelsinkiFinlandPerttu.Permi@helsinki.fi

Thelma PertinhezUniversity of ParmaItalythelma@unipr.it

Riccardo PeruzziniCERM, University of FlorenceItalyperuzzini@cerm.unifi.it

Wolfgang PetiBrown UniversityUnited States of Americawolfgang peti@brown.edu

Roberta PierattelliCERM, University of FlorenceItalypierattelli@cerm.unifi.it

Guido PintacudaCNRS, University of LyonFranceguido.pintacuda@ens-lyon.fr

Janez PlavecNational Institute of ChemistrySloveniajanez.plavec@ki.si

Juergen M PlitzkoMPI of BiochemistryGermanyplitzko@biochem.mpg.de

Miquel PonsUniversity of BarcelonaSpainmpons@ub.edu

James H. PrestegardUniversity of GeorgiaUnited States of Americajpresteg@ccrc.uga.edu

Jan PrchalInst. of Chem. Technol., PragueCzech Republicprchalj@vscht.cz

Jana PrecechtelovaNCBR, Masaryk UniversityCzech Republicjanap@ncbr.chemi.muni.cz

Laura RagonaISMAC-National Research CouncilItalylaura.ragona@ismac.cnr.it

Vasudevan RameshUniversity of ManchesterUnited Kingdomvasudevan.ramesh@manchester.ac.uk

Enrico RaveraCERM, University of FlorenceItalyravera@cerm.unifi.it

Christina RedfieldUniversity of OxfordUnited Kingdomchristina.redfield@bioch.ox.ac.uk

Christian RichterGoethe University FrankfurtGermanyric@uni-frankfurt.de

Janos RohonczyEotvos Lorand UniversityHungaryrohonczy@chem.elte.hu

Antonio RosatoCERM, University of FlorenceItalyrosato@cerm.unifi.it

Petra RovoEotvos Lorand UniversityHungarypetrarovo@chem.elte.hu

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Loic SalmonIBS GrenobleFranceloic.salmon@ibs.fr

Brigitte SambainECBelgiumbrigitte.sambain@ec.europa.eu

Ago SamosonTallinn Tech, WarwickEstoniaago.samoson@gmail.com

Guilherme SassakiG. Ronzoni InstituteItalyguilherme.sassaki@gmail.com

Marco SetteUniversity of Rome ”Tor Vergata”Italym77it@yahoo.it

Jurgen SchmidtBiosciences, University of KentUnited Kingdomj.m.schmidt@kent.ac.uk

Einat SchnurWeizmann Institute of ScienceIsraeleinat.schnur@weizmann.ac.il

Harald SchwalbeGoethe University FrankfurtGermanyschwalbe@nmr.uni-frankfurt.de

Svetlana SimovaIOCCP-BASBulgariasds@orgchm.bas.bg

Primoz SketNational Institute of ChemistrySloveniaprimoz.sket@ki.si

Vladimır SklenarNCBR, Masaryk UniversityCzech Republicsklenar@chemi.muni.cz

Roberta SpadacciniUniversity on SannioItalyrspadacc@unisannio.it

Chris SpronkSpronk NMR ConsultancyLithuaniachris@spronknmr.eu

Georgios A. SpyrouliasU of Patras, Dept of PharmacyGreecegspyroul@upatras.gr

Jan StanekUniversity of WarsawPolandjanstanek@chem.uw.edu.pl

Jochen StehleGoethe University FrankfurtGermanystehle@nmr.uni-frankfurt.de

David I StuartUniversity of OxfordUnited Kingdomstuart-pa@strubi.ox.ac.uk

Beata SzaboInstitute of EnzymologyHungaryszabobea@enzim.hu

Richard SteflNCBR, Masaryk UniversityCzech Republicstefl@chemi.muni.cz

Agnes TantosInstitute of EnzymologyHungarytantos@enzim.hu

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Joao M C TeixeiraCERM, University of FlorenceItalyteixeira@cerm.unifi.it

Peter TompaInstiute of EnzymologyHungarytompa@enzim.hu

Zdenek TosnerCharles University in PragueCzech Republictosner@natur.cuni.cz

Lukas TrantırekUtrecht UniversityNetherlandsl.trantirek@uu.nl

Olga TrıskovaNCBR, Masaryk UniversityCzech Republic175607@mail.muni.cz

Iveta UhliarikovaSlovak Academy of ScienceSlovakiaIveta.Uhliarikova@savba.sk

Ilker UnGebze Institute of TechnologyTurkeyilkerun@gyte.edu.tr

Ioannis VakonakisUniversity of OxfordUnited Kingdomioannis.vakonakis@bioch.ox.ac.uk

Barth J van RossumFMP-BerlinGermanybrossum@fmp-berlin.de

Chiara VenturiCERM, University of FlorenceItalyventuri@cerm.unifi.it

Jan VıchaNCBR, Masaryk UniversityCzech Republic151272@mail.muni.cz

Jirı VlachInst. of Chem. Technol., PragueCzech Republicvlachj@vscht.cz

Polytimi Maria G VlachouU of Patras, Dept of PharmacyGreecepha1791@upnet.gr

Jana VyskocilovaNCBR, Masaryk UniversityCzech Republicjanavys@chemi.muni.cz

Louise J WalportUniversity of OxfordUnited Kingdomlouise.walport@chem.ox.ac.uk

Mateus Webba da SilvaUniversity of UlsterUnited Kingdommm.webba-da-silva@ulster.ac.uk

Joern M WernerUniversity of SouthamptonUnited Kingdomjmwe@soton.ac.uk

Hans WienkUtrecht UniversityNetherlandshans@nmr.chem.uu.nl

Michal ZaliberaSlovak Academy of SciencesSlovakiazalibera.m@gmail.com

Andris ZeltinshASLA BiotechLatviaanze@asla-biotech.com

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Igor ZhukovNational Institute of ChemistrySloveniaigor.zhukov@ki.si

Dmitrijs ZulenkovsASLA BiotechLatviadmitry@biomed.lu.lv

Lukas ZıdekNCBR, Masaryk UniversityCzech Republiclzidek@chemi.muni.cz

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