biosoft | biophysics and soft-matter

BioSoftSelected results 2007/2008

Contents Editorial page 3 Introduction page 5 Selected Results page 7

Institute of Solid State Research (IFF) Theoretical Soft Matter and Biophysics page 9 Neutron Scattering page 27 Soft Matter page 43

Institute of Structural Biology and Biophysics (ISB) Cellular Biophysics page 59 Molecular Biophysics page 73 Structural Biochemistry page 89

Institute for Bio- and Nanosystems (IBN) Bioelectronics page 105 Biomechanics page 115

Education & Dissemination page 129 Publications page 139

Impressum BioSoft – Selected Results 2007/2008 Herausgeber: Forschungszentrum Jülich GmbH Stabsstelle Fachstrategie Institut für Festkörperforschung 52425 Jülich Deutschland +49-2461-61-3026 +49-2461-61-3570 www.fz-juelich.de Redaktion: Dr. Wolfgang Speier Kurt Wingerath Druck: Grafische Medien Forschungszentrum Jülich GmbH Juni 2009 © Forschungszentrum Jülich GmbH

Editorial “Physics meets Biology”

Physicists and Biologists worldwide have recognized some time ago that they can benefit a lot from sharing their knowledge and working together to understand the behavior of complex systems. At the Forschungszentrum Jülich, physicists and biologists have started to intensify their collaboration in 2004 by organizing a two-week IFF spring school with the title “Physics meets Biology”. This school was attended by an international audience of about 200 students. We are very happy that Erwin Neher, the Nobel laureate and inventor of the patch-clamp technique (together with Bert Sakmann), was among the lecturers of this school. The success of this spring school made clear to us that a more long-term effort is needed to provide an interdisciplinary graduate education, in order to equip students and young researchers with the necessary techniques and knowledge to address the many exciting scientific challenges at the interface between biology, chemistry and physics. This has lead the foundation of the International Helmholtz Research School on Biophysics and Soft Matter (IHRS BioSoft), together with colleagues from the Universities of Düsseldorf and Köln, supported by a grant from the Helmholtz Association. The school accepted its first students in the fall 2006. The research center CAESAR in Bonn has joined the graduate school recently. In parallel, we have continued to educate a wider range of students by organizing spring schools, in particular in 2008 by a school entitled ``Soft Matter — From Synthetic to Biological Materials". Research in Soft-Matter and Biophysics spans over three institutes on Jülich campus: the Institute of Solid State Research (IFF), the Institute for Bio- and Nanosystems (IBN) and the Institute of Structural Biology and Biophysics (ISB). Scientist from these institutes share many common interests, jointly use modern experimental techniques and instrumentation, and work on several joint research projects. The current brochure presents a selection of reports, which illustrate the research in Soft-Matter and Biophysics in the Forschungszentrum Jülich during the last two years. Gerhard Gompper

Introduction BioSoft – Biophysics & Soft Matter Macromolecules consisting of hundreds to thousands of atoms have many emergent properties, which are not present in small molecules. Prominent examples are linear polymers, which are constructed from a single or a few kinds of building blocks. Even simple synthetic polymers that are typically made from identical monomers allow the design of complex material properties that depend, for example, on their lengths, building blocks, cross linking, and potential combination with other polymers. Highly advanced methods often requiring large-scale facilities are used to investigate their fascinating properties. Also, the molecules of life, nucleic acids and proteins, are linear chain molecules, consisting of four and twenty different building blocks, respectively, in a strictly defined linear sequence. Thus, it is obvious that biological macromolecules have much more complex properties, the investigation of which requires the most advanced and – due to production limits - most sensitive methods available. Many interesting properties of macromolecules are based on their complex interactions among each other. This is especially true for the extremely specific interactions between proteins.

Biophysics and soft matter physics are concerned with the qualitative and quantitative description of structure and dynamics of complex macromolecules and their assemblies at various levels up to living cells.

Biophysics seeks to understand the processes of life by applying advanced physical methods to complex biological systems. This understanding requires exquisitely detailed and quantitative knowledge of the underlying processes at the molecular, cellular, and systems level. The development and fate of every single cell is regulated on the molecular level by a variety of cross-linked mechanisms. Therefore, structural biology aims at obtaining precise geometric and dynamic information about biologically and medically relevant molecules as a basis for an understanding of their molecular functions. On the cellular level, the efficiency of interlinked signal-transduction chains and of information processing is enormously high. By interaction with their environment, cells constantly receive, process, and emit information such as chemical, mechanical and electrical stimuli. Here, a detailed understanding is very important, because malfunctions of cellular information processing caused by endogenous or exogenous factors, e.g., spontaneous mutations, pathogens or environmental influences, can lead to serious diseases or to accelerated ageing processes.

Soft Matter encompasses all kinds of macromolecular assemblies of polymeric, colloidal, or amphiphilic character. The aim of Soft Matter research is to understand the cooperative behaviour of macromolecular systems with many interacting degrees of freedom, and to provide the scientific basis for a rational design of functional nano-scale materials. Thermodynamics drives the self-assembly of macromolecular building blocks into mesoscopic structures with a large variety of rheological, electronic, photonic, magnetic, thermal, or mechanical properties. Due to the weak interactions between building blocks, thermal fluctuations compete with conservative forces arising, e.g., from van der Waals or Coulomb interactions, and with topological constraints. Large mesoscopic length scales imply long time scales, and slow dynamical behavior. Therefore, non-equilibrium behavior is ubiquitous in soft matter systems driven by external fields or hydrodynamic flows. This implies that such fields can be employed to modulate, assist, or direct structure formation, and thereby taylor material properties.

Worldwide, we experience a rapid convergence of physical and biological research areas and possibly also research structures. Soft matter physics and biophysics represent a very dynamic and rapidly growing area of multidisciplinary research at the interface between physics, chemistry and biology. This requires a highly multidisciplinary infrastructure combining medium and large-scale experimental facilities with scientific computing and a high-level laboratory science. There are several compelling reasons for this approach:

A profound understanding of fundamental mechanisms underlying biological function and self-organisation can be fostered by studies of simpler synthetic soft matter systems.

Biological components or biological construction principles can be used or mimicked in the design of new materials.

Advanced physical tools and quantitative methods provide new routes in the investigation of biological molecules and systems.

A profound, quantitative understanding of selected model systems ranging from molecules to cells lays the foundations for modern systems biology.

Knowledge and techniques created by cutting-edge research in this transdisciplinary field is essential for the sustained rapid progress in nanotechnology, especially soft nanoscience, nanobiotechnology, materials sciences, and life and health sciences.

Selected Results Institute of Solid State Research (IFF) Theoretical Soft Matter and Biophysics Neutron Scattering Soft Matter Institute for Structural Biology and Biophysics (ISB) Cellular Biophysics Molecular Biophysics Structural Biochemistry Institute of Bio- and Nanosystems (IBN) Bioelectronics Biomechanics 2007 2008

IFF-2: Theoretical Soft-Matter and Biophysics

Director: Prof. Gerhard Gompper

The main research topic of the Institute “Theoretical Soft Matter and Biophysics“ is the theory of macromolecular systems. Soft matter physics and biophysics are interdisciplinary research areas encompassing statistical physics, materials science, chemistry, and biology. Our systems of interest include polymer solutions and melts, colloidal suspensions, membranes, vesicles and cells, but also composite systems ranging from colloids in polymer solutions to mixtures of surfactants and amphiphilic block copolymers. A major focus is the hydrodynamic behaviour of complex fluids and biological systems, both in equilibrium and under flow conditions.

At IFF-2, a large variety of methods are applied. In fact, a combination of analytical and numerical methods is often required to successfully characterize the properties of these complex systems. In particular, simulation methods (Monte Carlo, molecular dynamics), mesoscale hydrodynamic simulation techniques, field theory, perturbation theory, and exact solutions are employed. Since the building blocks of soft matter systems often contain a large number of molecules, “simplified“ mesoscale modelling is typically required, which is then linked to the molecular architecture.

A characteristic feature of soft-matter research is the fruitful interaction between theory and experiment. IFF-2 closely cooperates with the Institute for Neutron Scattering (Prof. Richter) and the Institute for Soft Condensed Matter (Prof. Dhont) to successfully tackle many of the essential aspects of the systems investigated.

Relevance of Angular MomentumConservation in Fluid SimulationsI. O. Götze , H. Noguchi , G. GompperIFF-2: Theoretical Soft-Matter and Biophysics

The angular momentum is conserved in fluids –with a few exceptions such as ferrofluids in ex-ternal fields. However, it can be violated lo-cally in fluid simulations to reduce computa-tional costs. The effects of this violation are in-vestigated using multi-particle collision dynam-ics, a well-established, highly efficient hydrody-namics simulation technique, where an angular-momentum conserving variant is available. Weshow that there are situations of practical rele-vance, such as multi-phase flows in Couette ge-ometry, where angular momentum conservationis essential to avoid non-physical results.

In simulations of the hydrodynamic behavior of com-plex fluids, one is faced with the challenge of bridg-ing the gap between the mesoscopic length and timescales of the solute and the atomic scales of the sol-vent. As these typically differ by orders of magni-tude, a full treatment on a microscopic level is pro-hibited by the enormous number of involved particlesand the large necessary time range. Moreover, of-ten only the dynamics of the colloidal particles areof particular interest, while the microscopic details ofthe solvent that mediates the hydrodynamic interac-tions are rather unimportant. Thus, a coarse-grainedmesoscopic fluid model is required that is sufficientlysimple to be tractable but still captures the correct hy-drodynamic behavior. By representing a large num-ber of physical solvent molecules by one model fluidparticle at a time, the number of degrees of free-doms can be reduced considerably. Various meso-scopic approaches have been proposed in the lastdecades, which can be categorized in lattice meth-ods or particle-based methods. We focus on particle-based simulations, where particle positions and ve-locities are continuous variables that are updated atdiscrete times. Here, coupling to solute particles aswell as moving boundaries can be treated more eas-ily than in lattice methods and thermal fluctuationsare naturally contained. Moreover, lattice methodsgenerally suffer from the lack of Galilean invariance.

The method used here, multi-particle collision dy-namics (MPC) [1], needs less computational timecompared to other particle based methods such asDPD, thus allowing simulations of larger systems.Here, the fluid is represented by point particles thatundergo subsequent streaming and collision steps.In the streaming step, the particles move ballistically.

Subsequently, they are sorted into collision cells andthe collision step then mimics the simultaneous in-teraction of all particles within each cell by assigningthem new velocities. MPC has been applied to vari-ous systems such as colloids, polymers, membranes,ternary amphiphilic fluids, and chemical reaction sys-tems. Hybrid simulations combining an MPC fluidwith molecular dynamics of solute particles are eas-ily possible. The algorithm is constructed in suchway that mass, energy and translational momentumare locally conserved, which is essential for correcthydrodynamic behavior. However, the angular mo-mentum is not conserved in the most widespreadversion of MPC, which is often called stochastic-rotation dynamics. In order to clarify the effects ofangular-momentum conservation [2], we mainly usethe Andersen-thermostat version of MPC, where an-gular momentum conserving (MPC-AT+a) and non-conserving (MPC-AT−a) algorithms are available [3].

In conventional viscous fluids that do conserve angu-lar momentum, the viscous stress tensor has to besymmetric, i. e. σαβ = σβα. This symmetry is re-quired by the fact that there is no stress expected ina uniformly rotating fluid (rigid body rotation), or alter-natively, by the conservation of angular momentum.On the other hand, for a fluid without conservation ofangular momentum, the above argument is no longervalid and we have to consider in general an asymmet-ric tensor. Then, the viscous stress is given by

σαβ = λ(∇ · v)δαβ (1)

„∂vα∂xβ

+∂vβ∂xα

«+ η

„∂vα∂xβ

− ∂vβ∂xα

where α, β ∈ x, y, z. Here, λ is the second vis-cosity coefficient, and η and η are the symmetricand asymmetric components of the viscosity, respec-tively. The last term in Eq. (1) is linear in the vorticity∇ × v, and does not conserve angular momentum.Thus, the last term vanishes (i. e. η = 0) in angular-momentum-conserving systems. The equation of ve-locity evolution is given by

Dt= −∇P+(λ+η−η)∇(∇·v)+(η+η)∇2v, (2)

where D/Dt is Lagrange’s derivative and P is thepressure. When a fluid is incompressible, this is thenormal Navier-Stokes equation with viscosity η =η + η. Since the equations of continuity and veloc-ity evolution are of the same form, the negligence

of angular-momentum conservation does not modifythe velocity field of fluids when the boundary condi-tions are given by velocities. However, it generatesan additional torque, so that the velocity field canbe changed when the boundary condition is givenby forces. In cylindrical coordinates (r, θ, z), the az-imuthal stress is given by

σrθ = (η + η)r∂(vθ/r)

∂r+ 2η

vθr. (3)

The first term is the stress of the angular-momentum-conserving fluid, which depends on the derivative ofthe angular velocity Ω = vθ/r. The second term isthe additional stress from the negligence of angular-momentum conservation and is proportional to Ω.

We consider a fluid confined between two coaxialcylinders (Couette flow) rotating with the same an-gular frequency Ω. Here, no torque is expected tobe acting on the cylinders in angular-momentum con-serving fluids, as this corresponds to the rotation of arigid body. However, in simulations without angular-momentum conservation, we do observe torques onthe confining cylinders, induced by the stress term ofthe asymmetric viscosity η in Eq. (3). The torque isthe tangential stress 2ηΩ0 multiplied by the circum-ference length 2πR and the radius R. The finite sizeof the collision cells leads to a correction term, so thatthe torques on the confining cylinders are found to be

Tin,out(R) = ±4πηΩR2

„1∓ 3a

«. (4)

In order to verify that the velocity field is not af-fected by the lack of angular-momentum conserva-tion if the boundary condition is given by the veloc-ity, we study fluids confined between eccentric cylin-ders with fixed axes, where the inner one is rotatingat constant angular velocity. As expected, the cor-responding stream lines, which are shown in Fig. 1,are practically identical for both methods. The occur-rence of a back-flow in the large-gap region as wellas the resulting total forces on the inner cylinder arein good agreement with theoretical predictions.

FIG. 1: Stream lines for fluid confined between eccentriccylinders (inner one rotating with constant angular velocity).

A boundary of a fluid does not only exist on solidobjects but also between two fluids or on mem-branes. In order to investigate the fluid-fluid boundary

FIG. 2: Azimuthal velocity vθ of binary fluids in a rotatingcylinder. The fluids with different viscosities are located atr < 5a and 5a < r < 10a, respectively. Symbols representthe simulation results of MPC-AT−a for two different viscos-ity ratios (+,×), and MPC-AT+a (). Solid lines representthe analytical results for MPC-AT−a.

in MPC-AT−a fluids, we consider binary fluids with afixed geometry of the boundary surface, which is im-penetrable to the fluid particles. The inner cylinderof radius R1 of circular Couette flow is replaced by amore viscous fluid, and the outer cylinder with radiusR2 rotates with constant velocity Ω0. This is a sim-plified description of oil and water phase-separateddue to surface tension, or two liquids separated bya membrane. It is assumed that the cylinder rotatesvery slowly, and that the flow stress does not changethe shape of the interface. We choose the fluid in-side (r < R1 = 5a) to have a higher viscosity thanthe fluid outside. The MPC collision performed incells crossing the boundary propagates the momen-tum from one fluid to the other. In MPC-AT+a, bothfluids rotate with Ω0 independent of their viscosities.However, in MPC-AT−a, the inner fluid rotates moreslowly (see Fig. 2). This is caused by the asymmet-ric stress term 2ηΩ. If both fluids rotate at the sameangular velocity, the inner and outer stresses do notcoincide. The theoretical velocity profile is obtainedfrom the stress balance at r = R1 and is in very goodagreement with the numerical results (Fig. 2).

Our main conclusion is that simulations which do notconserve angular momentum can lead to quantita-tively and even qualitatively incorrect results, whenthe boundary conditions on walls are given by forces,fluids with different viscosities are in contact, or finite-sized objects rotate in fluids.

[1] A. Malevanets and R. Kapral, J. Chem. Phys. 110, 8605(1999).

[2] I. O. Götze, H. Noguchi, and G. Gompper, Phys. Rev. E76, 046705 (2007).

[3] H. Noguchi, N. Kikuchi, and G. Gompper, Europhys.Lett. 78, 10005 (2007).

Diffusion and Segmental Dynamics ofDouble-Stranded DNAR. G. Winkler1 , E. P. Petrov2 , T. Ohrt 2 , P. Schwille2

1 IFF-2: Theoretical Soft-Matter and Biophysics2 Institute of Biophysics/BIOTEC, Dresden University of Technology, Dresden

The diffusion and segmental dynamics of double-stranded λ-phage DNA molecules are quantita-tively studied over the transition range from stiffto semiflexible chains. Spectroscopy of fluores-cence fluctuations of single-end fluorescently la-beled monodisperse DNA fragments unambigu-ously shows that double-stranded DNA in thelength range of 102 −2×104 base pairs behavesas a semiflexible polymer with segmental dynam-ics controlled by hydrodynamic interactions.

The dynamic behavior of individual macromoleculesin solution is governed by chain connectivity and hy-drodynamic interactions [1]. Understanding of poly-mer dynamics and quantitative verification of poly-mer theories require detailed information on segmen-tal motion of individual polymer molecules. However,the classical experimental techniques, such as dy-namic light scattering (DLS) or transient electric bire-fringence (TEB), predominantly deliver informationon large-scale shape fluctuations of macromolecules.Fluorescence correlation spectroscopy (FCS) [2] is asingle-molecule technique that can provide more de-tailed information on the macromolecular dynamicsthan the classical ensemble-based methods. By flu-orescent labelling of individual segments [3] or con-tinuous labelling of the whole molecule [4], the dif-fusional motion of segments as well as that of theoverall molecule can be studied at nanomolar con-centration under (quasi)equilibrium conditions in so-lution and cellular systems.

Precise experiments in polymer physics are impossi-ble without well-defined monodisperse polymer sam-ples covering a wide range of molecular weights. Therecent progress in molecular biotechnology resultedin a variety of techniques to produce monodisperseDNA fragments, which stimulated the use of DNAas a model compound in studies of polymer dynam-ics in solution. We employed the technique of poly-merase chain reaction (PCR) to produce monodis-perse samples of DNA fragments with predefined se-quence, structure, and length, fluorescently labeledat the same single end [3]. Double-stranded (ds)DNA is a biopolymer characterized by a large per-sistence length lp ∼ 50 nm [3]. As a result, ds-DNA fragments exhibit rodlike, semiflexible, or evenflexible polymer behavior, depending on their length.Thus, simple generic models of polymer dynamics [1]

are not expected to provide a quantitative descrip-tion of dsDNA behavior, and more advanced models,accounting for the persistence of the polymer chain,e.g., the semiflexible polymer model of Ref. [4], arerequired.Recent FCS studies raised the question whether ds-DNA dynamics in dilute solution is controlled by hy-drodynamic interactions [4] or not [5]. Moreover,no experimental studies were reported previously,where diffusion and intramolecular dynamics of ds-DNA had simultaneously been investigated over thetransition range from stiff to semiflexible chains. OurFCS measurements of lengths L from 102 to 2×104

base pairs (bp) (L/lp ∼ 0.7 − 140) by following theBrownian motion of the labeled ends of single DNAmolecules filled this gap and showed that the exper-imental data can quantitatively be described by thetheory for semiflexible polymer dynamics [3], whichclearly demonstrates that in this length range dsDNAbehaves as a semiflexible polymer with strong hydro-dynamic interactions.In FCS [2], fluctuations of the confocally de-tected fluorescence signal F (t) = 〈F 〉 + δF (t)are studied via the correlation function G (τ) =〈δF (t) δF (t+ τ)〉 / 〈F 〉2. In the absence of addi-tional photophysical processes and chemical reac-tions, the fluorescence signal fluctuates as a re-sult of the Brownian motion of fluorescently la-beled particles through the detection volume whoseshape can be approximated by a 3D Gaussianexp

`−2r2/r2

0 − 2z2/z20

´, where r0 and z0 are the lat-

eral and axial extensions of the detection volume,respectively. With the mean square displacement(MSD)

˙∆r2 (t)

¸of a particle, the FCS correlation

function assumes the form

G (t) =1

〈N〉1„

1 + 23

〈∆r2(t)〉r20

«r1 + 2

〈∆r2(t)〉z20

where 〈N〉 is the effective number of molecules in thedetection volume.Utilizing the Gaussian semiflexible polymer model,analytical calculations yield the MSD˙∆r2 (t)

¸= 6Dt+

∞Xn=1

τnψ2n(L/2)

“1− e−t/τn

”(2)

for the chain end [4], where T is the temperature, kBthe Boltzmann factor, and η the viscosity. ψn (L/2)

FIG. 1: Normalized FCS correlation functions (top) and de-termined mean square displacements (bottom) for λ-DNAfragments of lengths 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 kbp(from left to right). The intramolecular contribution to theMSD is shown by the green lines (bottom). The red linesindicate the power laws

˙∆r2

¸∼ t and t2/3, respectively.

is the value of the n-th eigenfunction of a chain oflength L at its end, τn is the n-th relaxation timein the presence of hydrodynamic interactions and isrelated with the free-draining relaxation time τn viaτn = τn/(1 + 3πηΛHnn). D is the translational dif-fusion coefficient of the macromolecule, and Hnn isthe matrix element of the Rotne-Prager tensor withinthe preaveraging approximation [4]. Here, Λ and ΛDare fit parameters, which are introduced in our quan-titative analysis of the experimental data [3].

For long and flexible molecules, L/lp 1, thetheoretical expression yields the short-time behavior˙∆r2(t)

¸∼ tβ , with the exponents β = 1/2 (free

draining) and 2/3 (nondraining), in agrement withpredictions of the Rouse and Zimm model, respec-tively [1]. For semiflexible polymers, the short-timebehavior is given by β = 3/4 [4]. However, thesepower laws are only valid for times much shorter thanthe longest relaxation time of the polymer, whereas atcomparable and longer times the intramolecular con-tribution to the MSD saturates due to the finite size ofthe polymer coil, as shown in Fig. 1. Thus, direct ap-plication of the power laws to the analysis of the MSDtime dependencies, as has been proposed recentlyin Ref. [5], can be misleading. Indeed, even for thelongest sample the apparent power law in

˙∆r2 (t)

¸observed in the range of 100 − 105 µs is to a largeextent due to the crossover from intramolecular mo-tion to overall polymer diffusion; only for times shorterthan ∼ 103 µs a power law-type behavior can ob-serve in the intramolecular contribution. We wouldlike to point out that such an analysis of the short-time dynamics, still possible in our study for the twolongest DNA fragments, clearly rules out the Rouse-type behavior reported for dsDNA in Ref. [5] andleads to a definitive conclusion on the importance ofintramolecular hydrodynamic interactions in dsDNApolymer dynamics.

Figure 2 displays diffusion coefficients and longestrelaxation times – the upper part is not readily acces-

FIG. 2: Diffusion coefficients (top) and longest relaxationtimes (bottom) of dsDNA. The blue symbols indicate ourFCS data. The other data (black symbols) are taken fromvarious sources [3]. The red lines are predictions of thesemiflexible polymer model [3, 4] with the parameters lp =50 nm, ΛD = 0.9, and Λ = 0.6.

sible by DLS or TEB – which we obtained by fittingof the normalized FCS curves [3]. The experimentalcorrelation functions are very well reproduced for theparameters lp = 51 ± 1 nm, Λ = 0.6, and ΛD = 0.9.Within the range of lengths studied, the diffusion co-efficients and relaxation times exhibit approximatelythe power law behavior D ∼ L−2/3 (note that thereis no excluded volume interaction for short DNAs)and τr ∼ L1.7, and thus do not follow the predic-tions of the Rouse (D ∼ 1/L, τr ∼ L2) nor the Zimm(D ∼ 1/L1/2, τr ∼ L3/2) model. At the same time,our data are in very good agreement with results ob-tained by other experimental techniques not relatedto FCS [3] and closely follow the predictions of thesemiflexible polymer theory [4].

Therefore, our results clearly demonstrate that in therange of the lengths studied, dsDNA behaves as asemiflexible polymer with strong hydrodynamic in-teractions . The Zimm regime with

˙∆r2

¸∼ t2/3,

D ∼ L−1/2, and τr ∼ L3/2 (or corresponding ex-pressions modified to account for excluded-volumeinteractions) [1] can be achieved only for long dsDNAmolecules with lengths exceeding 105 bp, or ∼ 103 lp(Fig. 2).

[1] M. Doi, S. F. Edwards, The Theory of Polymer Dynam-ics (Clarendon Pres, Oxford, 1986)

[2] E. P. Petrov, P. Schwille, State of the Art andNovel Trends in Fuorescence Correlation Spectroscopy(Springer, Berlin, 2007)

[3] E. P. Petrov, T. Ohrt, R. G. Winkler, P. Schwille, Phys.Rev. Lett. 97, 258101 (2006)

[4] R. G. Winkler, S. Keller, J. O. Rädler, Phys. Rev. E 73,041919 (2006)

[5] R. Shusterman, S. Alon, T. Gavrinyov, O. Krichevsky,Phys. Rev. Lett. 92, 048303 (2004)

Biological NanomachinesA. Baumgärtner, J.-F. Gwan, S. Grudinin, M. HaanIFF-2: Theoretical Soft-Matter and Biophysics

Biomolecular machines are large protein com-plexes whose activities are essential for provid-ing “life” to a biological cell. Among the vari-ous known biomolecular devices, ion channels,which reside in cell membranes, are one of thesimplest bionanomachines. We have investi-gated using computer simulations the molecularmechanism of selectivity and transport of ionsthrough a specific potassium ion channel.

FIG. 1: Cartoon representation of the V-ATPase, a biologi-cal nanomachine which acts as a ATP-driven proton pump.

Nanotechnology is perfectly realized in biological sys-tems. Cells are essentially biological assemblersthat build thousands of custom-designed moleculesand construct new assemblers. This view was pio-neered by Richard Feynman’s [1] evocative idea ofa self-replicating assembler building nanoscale de-vices atom by atom. Living cells are made up ofthese complexes, which carry out many of the func-tions essential for their existence, differentiation, andreproduction. In many cases the malfunction ofthese proteins can be a source of disease. Manyof these complexes can be described as “molecu-lar machines” or “molecular motors” or “moleculardevices”, depending on their sizes, complexity andtasks [2]. The essential question in understandingbiomolecular machines is concerned with the expla-nation of the macroscopic phenomenology in terms

of the atomic structures and forces involved. Al-though a complete description is not yet availableeven for the best-characterized system, considerableprogress has been made recently, not only from anexperimental point of view, but also with respect tocomputational and theoretical achievements. One ofthe most fascinating biomolecular machines is theclass of vacuolar H+-ATPases (or V-ATPases), whichare a family of ATP-dependent proton pumps respon-sible for acidification of intracellular compartmentsand proton transport across the cell plasma mem-brane. V-ATPases are multisubunit complexes (Fig.1)composed of a peripheral domain V1 (yellow and or-ange) responsible for ATP hydrolysis and an inte-gral domain V0 (blue and grey) responsible for protontranslocation. The V-ATPases are thought to oper-ate by a rotary mechanism in which ATP hydrolysis inV1 drives rotation of a ring of proteolipid subunits inV0. The V1 and V0 domains are connected by both acentral stalk which is thought to play a crucial role inthe assumed rotary mechanism of ATP-driven protontransport. The molecular details at atomic resolutionare only partly resolved. The theoretical understand-ing are currently beginning to emerge. This molec-ular machine is under investigation using elaboratemodelling and computer simulations.

FIG. 2: Structure of the KcsA potassium channel embed-ded in a water solvated lipid bilayer membrane. Only twomonomers of the tetrameric protein are shown.

A much simpler and much more well characterizedbionanomachine is the KcsA potassium channel [3],which acts as a valve in the plasma membrane ofprokaryotic cells. The cutaway view of the atomicstructure of the protein complex (in ribbon represen-tation) is shown in Fig.2. The figure shows only twomonomers of the tetrameric protein complex embed-ded in a water solvated lipid bilayer membrane. Eachmonomer consists of three helices, one extracellu-lar loop, and as the central part of the channel theselectivity filter containing three potassium ions (de-picted by balls) and one water molecule between.The structural details of the filter are decisive for ionselectivity and transport. The selectivity filter facil-itates the diffusion of potassium ions at rates ap-proaching 108 ions per second under physiologicalelectrochemical gradients. The ability of potassiumchannels to conduct several K+ ions simultaneouslyin a single file through the narrow pore at levels nearthe limit of diffusion is usually described in terms ofconcerted mechanisms. It has been suggested thatto reach a high conduction rate in a long-pore chan-nel, ions must move through the channel pore in amulti-ion fashion : the permeating ions line up in thenarrow channel pore and move in a single file throughthe channel. This is known as “the multi-ion perme-ation process”, and is believed to be a common fea-ture of the ion transportation process in all potassiumchannels. The multi-ion theory has been aceptedover decades, but the molecular mechanism of it re-mained elusive.

FIG. 3: Molecular view of the selectivity filter of the KcsApotassium channel including ions and water molecules.

Employing MD simulations on the basis of the X-ray structure of the KcsA channel, our studies [4,5] have provided useful insights into the structure-conductivity relationship. By pulling out the out-ermost ion from the exit of the pore (Fig.3), weobserved subsequent collective cooperative move-ments of ions, water and carbonyl groups lining thebackbone of the pore. A detailed analysis of thesemovements lead to the development of a simple two-dimensional model of ion-water transport. Based onour molecular dynamics simulations [4, 5] we found

three distinct features of the molecular mechanism.

(1) The movements of neighboring ions and wa-ter molecules are strongly correlated. They mostlymoved in pairs or triples (“permons”). One impor-tant fact for the understanding of the efficiency ofthe correlated movements is that the periodicity ofthe potential in the selectivity filter, ≈ 3 Å, practicallymatches with the distance of the lowest Coulombenergy UKW(∆z) between one ion and one watermolecule. The classical potential energy UKW(∆z)between a single ion and a single water moleculeat distance ∆z, consisting of Coulomb and van derWaals interactions, exhibits energy minima at char-acteristic distances of ∆z ≈ 3 Å (“bound states”).The depth of the potential depends on the orientationof the water molecule with respect to the ion.

(2) Permons are polarized. The absolute minimum ofUKW(∆z) ≈ 30 kBT is at distance ∆z = -2.65 Å andwhen the oxygen of the water dipol is oriented to-wards the ion (“polarized bound state”). When theoutermost ion exits the channel to the periplasmicside, the next neigboring ion hops to this vacancy, theion’s neighboring water molecule prefers to maintainits bound state and follows almost simultaneously thepreceding ion towards the vacancy. This is the move-ment of a permon.

(3) The movements of permons are rectified. An-other interesting observation from our MD simula-tions is that very rarely a water molecule has beenobserved to hop back to the vacancy and join theother ion to form a bound state. The absence of suchan event is explained by the energy barrier imposedby the periodic pore potential on the water molecule.This implies that a water molecule acts as a “pawl”in a ratchet mechanism. One implication is that thetransport of ion-water pairs through an ion channel ismuch more efficient than the transport of ions only.

In summary, the high permeation rate at which ionand water molecules pass through the KcsA ionchannel is based on the cooperative hopping of pairsof ions and water molecules mediated by the flexiblecharged carbonyl groups lining the backbone of thechannel. These observations provide the basis of anatomistic concept of the molecular mechanism of themulti-ion transport mechanism.

[1] R. P. Feynman, in Miniaturisation, page 282-296, ed. byH. D. Gilber, Reinhold, New York, 1961.

[2] A. Baumgaertner, in Handbook of Theoretical andComputational Nanotechnology, ed. by M. Rieth and W.Schommers, Am. Sci. Publ., 2006.

[3] D.A.Doyle, J. Morais-Cabral, R. A. Pfuetzner, A. Kuo,J. M. Gulbis, S .L Cohen, B. T. Chait and R. MacKinnon,Science 280, 69 (1998).

[4] J. F. Gwan and A. Baumgaertner, J. Chem. Phys. 127,045103 (2007).

[5] J. F. Gwan and A. Baumgaertner, J. Compt. Theor.Nanoscience 4, 50 (2007).

Diffusion in a fluid membrane with aflexible cortical cytoskeletonT. Auth1, N. S. Gov2

1 IFF-2: Theoretical Soft-Matter and Biophysics2 The Weizmann Institute of Science, Department of Chemical Physics, P.O. Box 26, Rehovot 76100, Israel

The cytoskeleton hinders protein diffusion in thelipid bilayer of the cell’s plasma membrane. Wecalculate the influence of a flexible network oflong-chain protein filaments, which is sparselyanchored to the bilayer, on protein diffusion. Wedefine a potential landscape for the diffusionbased on the steric repulsion between the cy-tosolic part of the protein and the cytoskeletalfilaments, using the pressure field that the cy-toskeleton exerts on the bilayer. We predict thechanges of the diffusion coefficient upon removalof anchor proteins and for a stretched cytoskele-ton.

Diffusion within the fluid membrane plays an im-portant role for cellular processes, because the cellcommunicates with its surrounding via its lipid bi-layer [1]. For example, diffusion of activated receptormolecules leads to signal amplification and diffusionof adhesion molecules to the contact area is impor-tant for cell adhesion.

The cytoskeleton, which is in close proximity to themembrane, has been identified to act as a strongregulator of the diffusion within the cellular mem-brane. For the red blood cell (RBC), several ex-periments show that the spectrin cytoskeleton slowsdown translational diffusion. Using single-moleculetracking techniques, small compartments have beenfound for the diffusion in the cell membrane [2] thatmay be explained by the cytoskeletal network belowthe bilayer.

Our work focusses on the diffusion in the membraneof cells that have a cortical, two-dimensional cy-toskeleton, which is composed of flexible long-chainproteins [3]. A prominent example of such cells areRBCs, where the long-chain proteins are spectrintetramers. However, a similar cortical cytoskeleton isfound on the plasma membrane of other mammaliancells, and spectrin has been identified in neurons andon membranes of intracellular organelles.

A pressure field on the bilayer is generated by theconformational fluctuations of the cytoskeletal fila-ments to which they are anchored at their ends viaanchor complexes [4] . We calculate the pressurefield using a linear, flexible polymer with bulk ra-dius of gyration Rg that is attached to a hard wallat ρ1 = (x1, y1) and ρ2 = (x2, y2). The pressurediverges close to the anchor points and decreases

FIG. 1: Entropic pressure that a flexible, cytoskeletalprotein that is anchored to a lipid bilayer with its endsexerts on the cell membrane.

exponentially for large distances from the anchors,|ρ| |ρi| (i = 1, 2), see Fig. 1.

The calculation of the pressure is based on the dif-fusion equation to calculate the polymer conforma-tions. This implies an infinite contour length of thepolymer, which is certainly a bad approximation if thedistance d between the anchor points is comparablewith the contour length. This situation is addressedin Ref. [5], where a Gaussian polymer model is com-bined with the condition of a finite contour length.Force-extension relations and end-to-end distributionfunctions have been calculated and the analytic the-ory describes experimental and simulation data verywell.

We use a simple argument to illustrate how a finitecontour length affects the fluctuation pressure: wesubtract from the contour length L = 6R2

g,0/` (where` = L/N is the Kuhn length of the polymer, N thenumber of repeat units in the chain, and Rg,0 the ra-dius of gyration of the free chain) the anchor distanced, because this length of the chain is ’needed to con-nect the anchor points’ and is therefore not availablefor conformation fluctuations.

To calculate the pressure field of the stretched chain,we simply replace Rg in the expression for the pres-sure by the new, effective Rg, which we obtain usingthe effective Kuhn length, `′ = (L − d)/N : R2

g =`′2 N/6 = (`/6)(L − d)2/L. The pressure along theconnection line of the anchor points is large eitherfor very small anchor distances because of the highpressure at the anchor points or for very large an-

FIG. 2: Pressure field for an unstretched RBC cy-toskeleton.

chor distances that are comparable with the countourlength when the polymer conformation is stretchedand therefore everywhere close to the lipid bilayer.

Fig. 2 shows a superposition of the single-filamentpressure fields for an idealized arrangement of spec-trin bonds on a hexagonal lattice, as in the RBC cy-toskeleton. We use a random walk and MetropolisMonte Carlo to simulate the diffusion process in thelipid bilayer. The step length is a = 1 nm, which is thetypical size of the lipid molecules. The influence ofthe cytoskeleton is taken into account by the potentiallandscape that is determined for the pressure field ofthe cytoskeleton multiplied with the effective interac-tion volume for the protein under consideration. Theinteraction volume is determined by the size of the cy-tosolic part of the protein that sterically interacts withthe cytoskeleton. It can be obtained either from elec-tron microscopy data for the spectrin and the diffus-ing protein or phenomenologically as a fit parameterusing measurements of the effective diffusion coeffi-cient.

Once the effective interaction volume is determined,we can predict several aspects of the protein diffusion[3]. For short times, the protein shows normal, fastdiffusion within a single corral that is formed by thecytoskeletal bonds. Also for long times normal diffu-sion is observed, but with a smaller, effective diffusionconstant. Long-time diffusion is hindered becausethe protein needs to hop across the potential barri-ers. The crossover between both regimes can alsobe observed in single-particle tracking experiments.

In healthy red blood cells, the cytoskeletal filamentsare usually attached at their ends but also — with adifferent protein complex — in their middle. In dis-ease, the middle anchor complex can be missing.We predict that the diffusion coefficient in the diseasecase is by a factor 25 larger than for healthy RBCs.

Furthermore, we can predict the influence of com-pression or stretching of the cytoskeleton on pro-tein diffusion. For isotropic stretching, we find a de-crease of the effective diffusion constant because ofthe increased height of the cytoskeletal barriers. Forthe normal RBC, the simulation results are well de-

FIG. 3: Anisotropic diffusion for a cytoskeleton that isstretched by a factor 1.5 and accordingly compressedin the perpendicular direction (for two different orien-tations of the hexagonal network).

scribed by a rescaled analytic expression: the heightof the potential barrier is given by the value in themiddle of the bond where the cytoskeletal pressure islowest. For anisotropic stretching, which constantlyoccurs in vivo when the RBC is deformed in bloodflow, we predict increased diffusion along the direc-tion of the stretch and decreased diffusion perpen-dicular to the stretching.

In Fig. 3, we plot the relative diffusion for ananisotropically stretched network parallel and per-pendicular to the direction of the stretch for differentvalues of the effective interaction volume. For the ef-fective interaction volume v = 6000 nm3 that appliesfor the band-3 protein in RBCs, we find that the dif-fusion is almost completely asymmetric if the RBCcytoskeleton is stretched by a factor 1.5 and accord-ingly compressed in the perpendicular direction (topreserve the total area, which is fixed by the lipid bi-layer).

Our results quantify the interaction between the cy-tosolic part of a transmembrane protein and a cy-toskeleton that consists of a two-dimensional networkof flexible polymers, as for example found in RBCs.We predict the changes in the diffusion due to lackof anchor proteins, such as a missing middle an-chor complex in forms of hereditary spherocytosis.We also predict changes of the diffusion caused by astretched cytoskeleton.

[1] M. J. Saxton, Curr. Top. Membr. 48, 229 (1999).

[2] M. Tomishige, Y. Sako, and A. Kusumi, J. Cell Biol.142, 989 (1998).

[3] T. Auth and N. S. Gov, Biophys. J. 96, 818 (2009).

[4] T. Auth, S. A. Safran, and N. S. Gov, New J. Phys. 9,430 (2007).

[5] R. G. Winkler and P. Reinecker, Macromolecules 25,6891 (1992).

Attractive colloidal rods in shear flowM. Ripoll, R. G. Winkler, G. GompperIFF-2: Theoretical Soft-Matter and Biophysics

Suspensions of rod-like colloids show in equi-librium an isotropic-nematic coexistence region,which depends on the strength of an attractive in-teraction between the rods. By means of hydro-dynamic simulations, we study the behavior ofthis system in shear flow for various interactionstrengths. The shear flow induces alignment inthe initially isotropic phase which generates ad-ditional free volume around each rod and causesthe densification of the isotropic phase at the ex-pense of an erosion of the initially nematic phase.Furthermore, the nematic phase exhibits a collec-tive rotational motion. The density difference be-tween these two regions at different shear rates,allows us to determine the binodal line of thephase diagram. The results are in good agree-ment with experimental observations.

Phase transitions occurring in soft matter systemsare significantly affected by flow. Both the nature andlocation of the phase transition lines are changed dueto the applied flow. The challenge is to find the pa-rameters that determine the non-equilibrium steadystates under flow conditions. Colloidal-rod suspen-sions constitute a particularly interesting system tostudy the effect of flow on their phase behavior, sincerod orientation is strongly coupled to the shear field.Rods in the isotropic (I) phase align with the flow andbecome paranematic (P). This suggests that the tran-sition to the nematic (N) phase, where rods have ori-entational order, is facilitated by shear. On the otherhand, rods in the nematic phase undergo a collectivetumbling motion in the presence of shear flow. Thequestion that then arises is how these two effects willaffect I-N detailed understanding of the flow behaviorof a model system of attractive colloidal rods is usefulfor industrial applications where shear alignment ofelongated objects, such as carbon nanotubes, worm-like micelles, and polymers, play a role.

The non-equilibrium phase diagrams of attractive col-loidal rods in shear flow have been investigated bymesoscale hydrodynamic simulations and comparedto small-angle light-scattering (SALS) and rheologyexperiments [1, 2]. Earlier rheology experiments [3]have studied the non-equilibrium binodal of fd-virusdispersions under shear flow conditions for a single,

FIG. 1: Snapshots of the simulation box with ε = 3.5, (a) atequilibrium, and (b) in a tumbling event at γ = 0.003. Col-ors in (a) and (b) are coding the rod orientation: horizontalis red, vertical is green and perpendicular to plane of viewis blue. Red arrows in (b) denote flow direction. (c) Timeevolution of the normalized density φ/φ0, and (d) of the ori-entational order parameter Sx, along the gradient direction.

fixed strength of attraction. This study showed thatthe P-N transition changes on applying flow. Thesimulations allow for a microscopic understanding ofthe behavior of coexisting phases and their interfaceunder shear, including the possible role of collectivetumbling motion of rods. The attractive rod-rod inter-actions are systematically varied, which affects thephase behavior, interfacial properties of coexistingphases as well as the tumbling behavior.

Molecular dynamic simulations of rod-like colloids ofaspect ratio 20 with attractive interactions (Lennard-Jones potential with a minimum of ε, in units ofthe thermal energy kBT ), are combined with amesoscopic description of the solvent known asmultiparticle-collision dynamics (MPC). This hybridapproach has been shown to account for long-range

FIG. 2: Non-equilibrium phase diagram obtained from sim-ulations for various values of the strength of attraction inter-action, with shear rates normalized by the maximum shearversus the fraction of equilibrium nematic phase. The in-set presents the unscaled data. The dashed lines are theconjectured master curve, which are identical in the actualstudy and in the experimental one [1].

hydrodynamic interactions between rods [4]. Thesimulated system is prepared in equilibrium with co-existing isotropic and nematic phases, where the di-rector of the nematic phase is aligned parallel to theinterface. A snapshot of I-N coexistence is shownin Fig. 1a. We determine the equilibrium phase di-agram as a function of the strength of an attractiveinteraction between the colloidal particles. We find awidening of the two-phase coexistence region, whichis small for weak interactions strengths, and becomesvery pronounced for stronger interactions. This resultvery nicely agrees with recent experimental results inequilibrium [5] in which attraction is induced by a vari-able amount of depleting polymer.

Shear is then applied with the imposed flow direc-tion parallel to the interface, (vx, vy, vz) = (γy, 0, 0),with γ the applied shear rate. Shear flow orients theparticles in the initially isotropic phase. This gener-ates free space around each rod and facilitates thetransfer of rods from the nematic into the parane-matic layer. This mechanism is responsible for thereduction of the density difference between the twophases. Shear flow also induces a rotational mo-tion of individual rods. More interestingly, it leads toa collective rotation motion of large groups of rodsin the nematic phase, as indicated in Fig. 1b. Thisbehavior is characterized by both the local concen-tration and the local orientational order parameterSx(y) ≡

ˆ3 ux ux − 1

˜/2, where ux is the compo-

nent of the unit vector connecting the end-points ofa rod along the flow direction, and the overline in-dicates averaging over the vorticity and flow direc-tions. The time dependence of the density φ andorientational order parameter Sx of rods as a func-tion of the position y along the gradient direction isplotted in Fig. 1c,d. As can be seen from Fig. 1c,the nematic phase has a higher concentration thanthe isotropic phase, as expected. More importantly,Fig. 1d demonstrates the periodic tumbling motion ofrods in the nematic phase. This is seen for all ne-matic domains in coexistence with paranematic re-gions and for all strengths of attractions studied.

Binodals are determined at times where the densityof the paranematic state has reached a stationaryvalue and are plotted in Fig. 2 for different attractions.The inset in Fig. 2 shows that an increasing attrac-tion between rods broadens the coexistence regionand leads to an increase of γmax. The the coexis-tence region widening is the same effect as observedin equilibrium. The factor γmax can be identified withthe maximum of the binodal. Simulations for shearrates just above γmax indicate in fact that the sys-tem evolves into an homogeneous state. In Fig. 2,the concentration is expressed in terms of ϕnem, i.e. the proportion of the nematic phase in the equilib-rium state. The shear rate is scaled by γmax. Withthis normalization, all data points fall onto a mastercurve. Precisely the same master curve was also ex-tracted from experimental results. From this strikingresult we conclude that the effect of attractive inter-actions on the non-equilibrium phase diagram is re-duced to two parameters, γmax and the difference inpacking fractions between the isotropic and nematicphase in equilibrium.

The collective rotational motion induced by shear hasbeen observed only in the coexisting nematic phase.This is in contrast to the paranematic phase that re-mains flow-aligned without any collective motion. Theinterface between the coexisting tumbling and flowaligned states is therefore highly dynamic. The pe-riodic motion of the nematic director during tumblingand kayaking is characterized by a time τ betweensubsequent flow-aligned states. Although this time isnot uniquely defined, a clear trend can be observed.We find that τ decreases linearly with the inverseshear rate, as observed previously well inside thenematic region. Furthermore, we observe that thetumbling time increases with increasing interactionstrength. We believe that this is due to the higherpacking fraction of rods in the nematic phase. Thisimplies that the distance between rods is smaller, andtherefore the hydrodynamic friction for their motionparallel to each other is larger.

We want to emphasize that no nematic tumblingstates have been found in the homogeneously ne-matic phase obtained for rates above the maximum ofthe binodal. This agrees with the experimentally de-termined tumbling-to-aligning phase separation linethat appears at high concentrations and high shearrates and ends at the maximum of the binodal.

[1] M. Ripoll, P. Holmqvist, R. G. Winkler, G. Gompper,J. K. G. Dhont, and M. P. Lettinga. Phys. Rev. Lett.101, 168302 (2008).

[2] M. Ripoll, R. G. Winkler, K. Mussawisade, andG. Gompper, J. Phys.: Cond. Matt. 20, 404208 (2008).

[3] M. P. Lettinga and J. K. G. Dhont. J. Phys.: Cond.Matt. 16, S3929 (2004).

[4] R. G. Winkler, K. Mussawisade, M. Ripoll, andG. Gompper, J. Phys.: Cond. Matt. 16, S3941 (2004).

[5] Z. Dogic, K. R. Purdy, E. Grelet, M. Adams, andS. Fraden, Phys. Rev. E 69, 051702 (2004).

Cooperation of sperm in two dimensions:synchronization and aggregation throughhydrodynamic interactionsY. Yang, E. Elgeti, G. GompperIFF-2: Theoretical Soft-Matter and Biophysics

Sperm swimming at low Reynolds number havestrong hydrodynamic interactions when theirconcentration is high in vivo or near substratesin vitro. The beating tails not only propel thesperm through a fluid, but also create flowfields through which sperm interact with eachother. We study the hydrodynamic interactionand cooperation of sperm embedded in a two-dimensional fluid. Two effects of hydrodynamicinteraction are found, synchronization and at-traction. With these hydrodynamic effects, amulti-sperm system shows swarm behavior witha power-law dependence of the average clustersize on the width of the distribution of beating fre-quencies.

Sperm motility is important for the reproduction of an-imals. Before they find the ova, sperm have to over-come many obstacles in their way. A healthy ma-ture sperm of a higher animal species usually hasa flagellar tail, which beats in a roughly sinusoidalpattern and generates forces that drive fluid motion.The snake-like motion of the tail propels the spermthrough a fluid medium very efficiently. In nature,the local density of sperm is sometimes extremelyhigh. For example, in mammalian reproduction, theaverage number of sperm per ejaculate is tens tohundreds of millions, so that the average distancebetween sperm is on the scale of ten micrometers,comparable to the length of their flagellum. On thisscale, the hydrodynamic interaction and volume ex-clusion are not negligible. In experiments with rodentsperm at high densities [1], motile clusters consistingof hundreds of cells have been found, which showmuch stronger thrust forces than a single sperm. Thesperm seem to take advantage of strong interactionswith neighbor cells of the same species to win thefertilization competition.The higher animal sperm typically have tails with alength of several tens of micrometers. At this lengthscale, viscous forces dominate over inertial forces.Thus, the swimming motion of a sperm correspondsto the regime of low Reynolds number. We describethe motion of the surrounding fluid by using a particle-based mesoscopic simulation method called multi-particle collision dynamics (MPC) [2, 3]. This simu-lation method has been shown to capture the hydro-dynamics and flow behavior of complex fluids overa wide range of Reynolds numbers very well [3]. In

particular, it describes the helical motion of swimmingsperm in three dimensions [4].

Here, we construct a coarse-grained sperm model intwo dimensions. The sperm model consists of parti-cles connected by stiff springs, which form a circularhead and a filament-like tail, see Fig. 1. The beatingmotion is determined by a time-dependent, sponta-neous curvature

cs,tail(x, t) = c0,tail +A sin

»−2πfst+ qx+ ϕs

which generates a propagating, sinusoidal wavealong the tail. The wave number q = 4π/L0 is cho-sen to mimic the tail shape of sea-urchin sperm, andL0 is the tail length. fs is the beating frequency ofthe s-th sperm. The constant c0,tail determines theaverage spontaneous curvature of the tail. ϕs is theinitial phase of the s-th sperm, and A is a constantrelated to the beating amplitude.

We analyzed the hydrodynamic interaction betweentwo sperm and the aggregation behavior of multi-sperm system. In our simulations, sperm clusters arealways seen after the system has reached a station-ary state. We concluded that hydrodynamic interac-tion and volume exclusion play important roles in thecluster formation of healthy and motile sperm. Wealso found that the average cluster size has a power-law dependance on the width of beat frequency dis-tribution [5].

FIG. 1: Head-head distance dh of two cooperating sperm.Simulation data are shown for fixed phase difference (red,). The interpolating (red) line is a linear fit for 0.4π <∆ϕ < 1.5π. The distance dh is also shown as a function oftime t (top axis) in a simulation with a 0.5% difference in thebeat frequencies of the two sperm (solid line).

In two-sperm simulations, we found two effects ofthe hydrodynamic interaction, synchronization andattraction. When two sperm are close in space andswimming parallel, they synchronize their tail beatsby adjusting their relative position. This “synchro-nization" process is accomplished in a very shorttime. Then, the synchronized sperm have a tendencyto get close and form a tight pair. This “attraction"process takes much longer time then synchroniza-tion. Two sperm stay locked in phase and swim to-gether until their phase difference becomes too large.This coordinating behavior decreases the total en-ergy consumption of two sperm. Fig. 1 shows thelinear relation between phase difference and head-head distance of a sperm pair. The cooperation ofour sperm model fails when the phase difference∆ϕ > 1.5π.

With the knowledge of hydrodynamic interaction be-tween two sperm, we study multi-sperm systems.Synchronization and attraction effects help the spermto form large, motile clusters. We give each spermrandom initial position and orientation. Consideringthat in real biological systems the beat frequency isnot necessary the same for all sperm, we performsimulations with Gaussian-distributed beating fre-quencies. δf = 〈(∆f)2〉1/2/〈f〉 denotes the width ofthe Gaussian frequency distribution. Here, 〈(∆f)2〉is the mean square deviation of the frequency distri-bution, and 〈f〉 = 1/120 is the average frequency.

FIG. 2: A snapshot from simulation of 50 sperm with widthsδf = 0.9% of a Gaussian distribution of beating frequen-cies. The red ellipses indicate large sperm clusters. Theblack frames show the simulation boxes. Note that we em-ploy periodic boundary conditions.

The motile sperm embedded in a two-dimensionalfluid aggregate through hydrodynamic interactionsand volume exclusion. Fig. 2 shows a snapshot ofa 50-sperm system with δf = 0.9%. Large clusters ofsynchronized and tightly packed sperm are formed.If δf = 0, once a cluster has formed, it does notdisintegrate without a strong external force. A pos-sible way of break-up is by bumping head-on into an-other cluster. As the cluster size increases, the pos-sibility to bump into another cluster decreases, thusthe rate of break-up decreases. The average cluster

FIG. 3: Dependence of the average stationary clustersize, < nc >, on the width of the frequency distributionδf . Data are shown for a 50-sperm system () and a 25-sperm system (). The lines indicate the power-law decays< nc >= 2.12 δ−0.201

f (upper) and < nc >= 1.55 δ−0.196f

(lower).

size always increases for δf = 0. For δf > 0, how-ever, sperm cells can leave a cluster after sufficientlylong time, since the phase difference to other cells inthe cluster increases in time due to the different beatfrequencies. At the same time, the cluster size cangrow by collecting nearby free sperm or by mergingwith other clusters. Thus, there is a balance betweencluster formation and break-up when δf > 0.

Once a system reaches a stationary state, thecluster-size distribution function obeys a power law.Similar power-law behaviors have been found in sim-ulation studies of swarm behavior of self-propelledparticles [6].

The average cluster size < nc > is plotted in Fig. 3as a function of the width δf of the frequency distribu-tion. Generally, it decreases with decreasing spermdensity or increasing δf . Figure 3 also shows a powerlaw decay,

< nc >∼ δ−γf (1)

with γ = 0.20±0.01. The negative exponent indicatesthat the cluster size diverges when δf → 0.

[1] S. Immler, H. D. M. Moore, W. G. Breed and T. R.Birkhead, PLos One 2, e170 (2007).

[2] A. Malevanets and R. Kapral, J. Chem. Phys. 110,8605 (1999).

[3] G. Gompper, T. Ihle, D. M. Kroll, and R. G. Winkler,Adv. Polym. Sci. 221, 1 (2009).

[4] J. Elgeti and G. Gompper, NIC proceedings, Vol. 39of NIC series, edited by G. Münster, D. Wolf, and M.Kremer, pp. 53-61 (2008).

[5] Y. Yang, J. Elgeti, and G. Gompper, Phys. Rev. E 78,061903 (2008).

[6] C. Huepe and M. Aldana, Phys. Rev. Lett. 92, 168701(2004).

Twist grain boundaries in cubicsurfactant phasesM. Belushkin, G. GompperIFF-2: Theoretical Soft-Matter and Biophysics

Cubic surfactant phases are mesoscale liquid-crystalline structures in which the surfactantmonolayer separating the oil-rich and water-rich domains often has a triply-periodic minimal-surface geometry where the mean curvature Hvanishes on the whole surface. The lattice con-stant which corresponds to the dimensions ofthe fundamental building block - the unit cell -is large, of the order of 10 nm. During the nu-cleation of a cubic phase, many classes of de-fects arise. We have investigated twist grainboundaries in the lamellar, gyroid, diamond andSchwarz P phases. The structure of the mono-layer in the grain boundaries is found to be veryclose to a minimal-surface geometry. The interfa-cial free energy per unit area is found to be verysmall, therefore the density of grain boundariesshould be high in these surfactant phases.

Amphiphilic molecules added to an immiscible oil-water system self-assemble into a large variety ofstructures. Phases with cubic symmetry often fea-ture a triply-periodic minimal surface (TPMS) configu-ration of the surfactant monolayer. Examples of suchphases include the gyroidG, diamondD, Schwarz P ,Schoen I−WP , F −RD, Neovius C(P ) and others,which are encountered in physical systems. Prop-erties of cubic surfactant phases have been a sub-ject of extensive theoretical and experimental inter-est, with applications in biological systems, as tem-plates for mesoporous systems and for the crystal-lization of membrane proteins.

In amphiphilic systems many kinds of interfaces oc-cur: between two ordered phases, between orderedand disordered phases, and between two grains ofthe same ordered phase which differ by their spa-tial orientation. Experiments on block copolymer sys-tems have revealed the structure of many interfaces.For example, twist grain boundaries in the lamellarphase are well described by Scherk’s minimal sur-faces at large twist angles and are helicoid-shaped atsmall twist angles [1, 2], whilst tilt grain boundaries inthe lamellar phase have been shown to be omega-shaped at large tilt angles and chevron-shaped atsmall tilt angles [3].

We report here on the investigation of twist grainboundaries in cubic surfactant phases. The cal-culations are based on a Ginzburg-Landau theory

of ternary amphiphilic systems [4, 5] with a singlescalar order parameter φ(~r) which describes the lo-cal oil-water concentration difference. The geometri-cal properties of the grain boundaries are evaluatedon the isosurface φ(~r) ≡ 0 which defines the posi-tion of the surfactant monolayer. The interfacial freeenergy per unit area of a grain boundary depends onthe angles with respect to the crystalline axes. It isdetermined using the Ginzburg-Landau theory and acomplementary geometrical approach based on theCanham-Helfrich curvature Hamiltonian.

The calculations are performed on a discrete three-dimensional real-space lattice φijk. Initially, half ofeach calculation box is filled with a phase rotated byan angle −α/2 and the other half with a phase ro-tated by an angle +α/2. The free energy is mini-mized using a method based on the gradient descentalgorithm [6].

FIG. 1: Configuration of the surfactant monolayer for thelamellar phase at a twist angle of 53o (left) and the gyroidphase at a twist angle of 90o (right). The grain boundariesare located in the middle and at the top/bottom of each box.

Configurations of the surfactant monolayer in the fullsimulation boxes of the lamellar phase at a twist an-gle of 53o and the gyroid phase at a twist angle of 90o

are shown on Fig. 1.

The locations of the grain boundaries are determinedquantitatively by relating the inital and final simula-tion boxes [6]. In the lamellar, gyroid and diamondphases the thickness of the grain-boundary regionsis about one unit cell of the bulk-phase regions, thusgrain boundaries preserve the typical length scalesof the bulk phases. The bulk-phase regions of theSchwarz P phase are greatly affected by the pres-ence of grain boundaries. The surfactant monolayersin grain-boundary regions of the lamellar (top), gyroid(middle) and diamond (bottom) phases are shown onFig. 2 for twist angles of 53o (left) and 90o (right).

FIG. 2: Twist grain boundary geometries for the lamellar(top), gyroid (middle) and diamond (bottom) phases for twistangles of 53o (left) and 90o (right).

Squared-mean and Gaussian curvature distributionsare calculated for each phase and twist angle.Squared-mean-curvature distributions show that themean curvature of the surfactant monolayer essen-tially vanishes both in the bulk-phase and grain-boundary regions, therefore the configurations ofthe surfactant monolayer are good approximations ofminimal surfaces [6].

FIG. 3: Gaussian-curvature distribution for the G phase ata twist angle α = 27.8o. The blue solid line correspondsto the exact result obtained from the Weierstrass represen-tation. The black vertically-shaded and red slant-shadedhistograms correspond to bulk and grain-boundary regions,respectively.

Gaussian-curvature distributions (Fig. 3) show that [6]

• the Gaussian curvature distributions in the bulk-phase regions are consistent with the exact resultsobtained from the Weierstrass representation

• in the Lα, G and D phases the geometry of thegrain boundaries is significantly different from thegeometry of the bulk phases

Excess free energy per unit area of the grain bound-aries determined from Ginzburg-Landau theory as afunction of the twist angle α is shown on Fig. 4. Geo-metrical approaches based on the Canham-Helfrichcurvature energy Hamiltonian yield similar results [6].The excess free energy of the grain boundaries ex-hibits a non-monotonous dependence on the twistangle α, and the negative values for the P phaseshow it to be unstable with respect to the nucleationof grain boundaries at the investigated point in thephase diagram.

FIG. 4: Grain-boundary excess free energy per unit areaas a function of the twist angle α for the lamellar, gyroid G,diamond D and Schwarz P surfactant phases as extractedfrom Ginzburg-Landau theory.

The free energy of the grain boundaries is very small.The difference in the grain-boundary free energy andthe free energy of bulk-phase regions of equal vol-ume is of the order of 1% at the maxima of Fig. 4.Therefore, the density of grain boundaries should behigh in these surfactant phases.

[1] S. Gido et al., Macromolecules 26, 4506 (1993)

[2] H. Jinnai et al., Macromolecules 39, 5815 (2006)

[3] Y. Cohen et al., Macromolecules 33, 6502 (2000)

[4] G. Gompper, M. Schick, Phys. Rev. Lett. 65, 1116(1990)

[5] U. S. Schwarz, G. Gompper, Phys. Rev. E 59, 5528(1999)

[6] M. Belushkin, G. Gompper, J. Chem. Phys. 130,134712 (2009)

Clustering and alignment of red bloodcells in microcapillariesJ. L. McWhirter, H. Noguchi, G. GompperIFF-2: Theoretical Soft-Matter and Biophysics

The shapes, aggregation, and alignment of redblood cells (RBCs) in cylindrical capillary flow isinvestigated by mesoscopic hydrodynamic simu-lations. We study the collective flow behavior ofmany RBCs, where the capillary diameter is com-parable to the diameter of the RBCs. Two essen-tial control parameters in our study are the vol-ume fraction of RBCs (the tube hematocrit, HT ),and the flow velocity of the RBC suspension. Atvery high flow velocities and very low HT , theparachute-shaped RBCs can be found in clus-ters that are stabilized by the hydrodynamic sol-vent flows. At high HT , the RBCs can exhibitone disordered phase and two distinct orderedphases depending on HT and the flow velocity.Thermal fluctuations, included in the simulationmethod, coupled to hydrodynamic flows are im-portant contributors to the RBC morphology.

In the absence of flow, human RBCs have abiconcave-disc (discocyte) shape whose maximumdiameter and thickness are 7.6 µm and 2 µm withconstant area and volume, Vves. The RBC membraneconsists of a lipid bilayer supported by an attachedspectrin network, which acts as a cytoskeleton andis responsible for the shear elasticity of the mem-brane. The bilayer’s resistance to a bend is controlledby a curvature energy with a bending rigidity, κ, andthe spectrin’s resistance to a shear strain is charac-terized by a shear modulus, µ. At thermal equilib-rium, the discocyte shape of a RBC can be predictedtheoretically by minimizing the membrane’s bendingand stretching energy subject to a fixed surface areaand volume. However, under flowing, nonequilibriumconditions, the shape adopted by the RBCs is deter-mined by the competition between these mechanicalproperties and the external hydrodynamic flow forcesarising from the blood plasma (the solvent) suspend-ing the RBCs.

The spectrin network enables an RBC to remain in-tact while changing its shape in blood flow throughnarrow capillaries with diameters of 0.2 µm to 10µm. At high dilution in fast blood flows through nar-row capillaries, optical microscopy of micro vessels[1] have shown that individual RBCs can adopt aparachute shape; the faster flow in the center ofthe capillary and the slower flow near the walls in-

FIG. 1: Sequential simulation snapshots of six RBCs ina dilute blood suspension (L∗ves = 3.3 or HT = 0.084 =nvesVves/πLzR2

cap = 0.28/L∗ves ) at v∗0 = 7.7 and nves

= 6. The full length of the cylindrical simulation tube is Lz .The critical flow velocity associated with the shape transitionfrom a discocyte at low velocities to a parachute at highervelocities is v∗c ∼ 5. Top panel shows a 6 RBC cluster whilethe bottom two show the break-up of this cluster at a latertime.

duces this shape change. The RBC deformabilityis reduced in blood related diseases, such as dia-betes mellitus and sickle cell anemia; with these dis-eases the resistance to flow is relatively high and theheart must work harder to produce the higher pump-ing pressures needed to ensure normal blood flow.Therefore, the deformability of a RBC is important forthe regulation of oxygen delivery.

We have already studied the shape changes of asingle, isolated RBC in a blood flow at high dilution[2] using a mesoscopic simulation technique whichcombines two methods. The solvent hydrodynamicsis described by an explicit, particle-based dynamicscalled Multi-particle Collision Dynamics (MPC). TheRBC membrane is treated by a discretized mechan-ical model of a two-dimensional elastic membraneparametrized by κ and µ, that is, a dynamically trian-gulated surface model [3]. Our studies showed thatthe shape change of an RBC from a discocyte to aparachute occurs at a critical flow velocity which de-pends on the material parameters κ and µ [2]. Asthe RBC became more rigid, a faster the blood flowwas needed to achieve this shape transition. Thistransition was accompanied by a sudden drop in thepressure needed to drive the flow at a given velocity.

The main focus of the present work is to study thecollective flow behavior of many interacting RBCs atvery low and high HT [4, 5]. We consider a numbernves of vesicles in capillary tube segments of lengthLz and radiusRcap with periodic boundary conditionsin the flow direction, Z. Here L∗ves = Lz/nvesRcap

FIG. 2: Simulation snapshots of nves = 6 RBCs: (a)Disordered-discocyte phase for L∗ves = 0.875 and v∗0 = 2.5;(b) Aligned-parachute phase for L∗ves = 0.875 and v∗0 = 10;and (c) Zigzag-slipper phase for L∗ves = 0.75 and v∗0 = 10.

and v∗0 ≡ v0τ/Rcap, where the characteristic shaperelaxation time is τ = η0R

3cap/κ. A gravitation force

mg is used to generate flow along the Z axis wherev0 = mnsgR

2cap/8η0 and η0 is the viscosity of the

suspending solvent.

At very low HT , parachute shaped RBCs form sta-ble clusters at flow velocities v∗0 much higher thanthe critical flow velocity, v∗c , associated with the disco-cyte to parachute transition. There are two reasonsfor the cluster formation. First, a single RBC sepa-rated from a neighbouring cluster of RBCs is moredeformed by the flow than its neighbours, bendingcloser to the capillary axis or center where the flowis fastest; therefore, this single RBC approaches thecluster, forming a larger cluster. Second, the effec-tive hydrodynamic flow mediated attractions betweenthe RBCs in a cluster stabilize the cluster, increas-ing its lifetime. As v∗0 approaches v∗c , fluctuations inthe RBC shape increase, leading to cluster break-upevents as shown in Fig. 1.

At high HT , three distinct RBC ‘phases’ exist (Fig.2); one is disordered, the disordered-discocyte (D)phase, while the remaining two are ordered, thealigned-parachute (P) and zig-zag slipper (S) phases(Fig. 3). In the D phase at L∗ves & 0.85 and lowv∗0 , the RBCs appear as discocytes with random ori-entations and no significant long-range spatial cor-relations. The P phase was expected at higher v∗0based on the simulations examining the shape tran-sition to a parachute of a single, isolated RBC un-der flow [2]. However, given these earlier simula-tions, the S phase occurred unexpectedly at smallerL∗ves. Here, slipper-shaped RBCs form two regular,interdigitated parallel rows. Curiously, the S phaseproduces a larger pressure drop ∆P ∗

drp, or resis-tance to flow, than the periodic P phase under equiv-alent conditions, as shown in Fig. 3(b). An aligned-parachute conformation at L∗ves . 0.85 is destabi-

FIG. 3: (a) Phase behavior as a function of average vesicledistance L∗ves and mean flow velocity v∗0 , for nves = 6. Thehematocrit varies between HT = 0.22 and HT = 0.45,since HT = 0.28/L∗ves. Symbols represent the disordered-discocyte (?), aligned-parachute (), and zigzag-slipper (•)phases, respectively. (b) Pressure drop ∆P ∗

drp per vesiclefor the aligned-parachute phase (simulations with nves = 1)and the zigzag-slipper phase (simulations with nves = 6) atthe same volume fraction (L∗ves = 0.75, corresponding toHT = 0.37).

lized by the thermal fluctuations that are incorporatedinto the mesoscopic simulation method; these fluctu-ations have the effect of moving an RBC off the cap-illary axis to regions of slower flow. In other theoret-ical approaches, thermal fluctuations are not incor-porated, or their incorporation is not as simple as inMPC.

In summary, we have shown that at very low RBC vol-ume fractions the RBC deformability implies a flow-induced cluster formation at high blood flow veloci-ties. In addition, at high volume fractions, the collec-tive behavior of several RBCs determines their flow-induced morphology and resistance to flow. Futurestudies will examine the dependence of flow proper-ties on channel geometry, polydispersity of the RBCsuspension, and the introduction of more rigid, spher-ical white blood cells.

[1] Y. Suzuki, N. Tateishi, M. Soutani, and N. Maeda, Mi-crocirculation 3, 49 (1996).

[2] H. Noguchi and G. Gompper, Proc. Natl. Acad. Sci.102, 14159 (2005).

[3] H. Noguchi and G. Gompper, Phys. Rev. E 72,011901 (2005).

[4] J.L. McWhirter, H. Noguchi, and G. Gompper, Proc.Natl. Acad. Sci. 106, 6039 (2009).

[5] P. Gaehtgens, C. Duhrssen, K.H. Albrecht, BloodCells 6, 799 (1980).

IFF-5: Neutron Scattering

Director: Prof. Dieter Richter

The Institute for Neutron Scattering is concerned with neutron research placing major emphasis on soft condensed matter, i.e. materials that react strongly to weak forces. Neutron scattering is a valuable tool for these systems because it reveals structure and dynamics of Soft Matter on the relevant length- and timescales.

A major part of the Soft Matter studies is done on polymers. Apart from their structure, we are interested in the dynamics of polymers in melts and solutions (e.g. gels, rubbery networks, aggregates). These polymers often have a complex architecture (copolymers, star-polymers etc.) to tailor them for industrial applications. Another field of interest are complex liquids such as microemulsions or colloid systems.

Biological materials (e.g. proteins) are studied concerning their structure and dynamics. The institute has modern chemical laboratories for the synthesis, characterisation, and modification of Soft Matter. In order to complement neutron scattering experiments several ancillary techniques are used in the institute: rheology, light scattering, calorimetry, x-ray scattering, impedance spectroscopy, and computer simulation.

The Institute for Neutron Scattering is partner in the Jülich Centre for Neutron Science JCNS. In this position it operates several neutron scattering instruments at the research reactor FRM II in Munich, at the Institut Laue-Langevin in Grenoble, and at the Spallation Neutron Source in Oak Ridge, USA. These instruments are available to guest researchers on request. Another focus of research is the development of neutron instrumentation for research reactors and future spallation sources worldwide.

Synthesis of Polyalkylene Oxide Homo- and Block Copolymers J. Allgaier IFF-5: Neutron Scattering The anionic ring-opening polymerization of alkylene oxides like propylene or butylene oxide is accompanied by strong side reactions. This leads to large amounts of by-products. In this work the anionic polymerization of hydrophobic alkylene oxides was investigated at different temperatures, solvents and initiating systems [1]. For polymers synthesized above room temperature significant amounts of by-products were found. With the help of crown ethers the polymerization temperature could be reduced to -23°C. This measure allowed eliminating by-products almost completely in the synthesis of poly(1,2-butylene oxide), poly(1,2-hexylene oxide), and poly(1,2-octylene oxide). Furthermore the method was employed to synthesize amphiphilic block copolymers of these polyalkylene oxides with polyethylene oxide as the hydrophilic moiety. The new synthetic method is generally of interest due to the rheological and dielectric properties of the polymers and the possibility to tune amphiphilicity of the amphiphilic block copolymers.

In contrast to the limited availability of the higher members, the polyalkylene oxide family offers interesting possibilities with respect to their rheological properties due to the systematic variability of the polymer chain diameters. Because of the dipole moment along the chain PAO represents an alternative to the widely used polyisoprene for dielectric measurements. In addition, by combining PEO with the other PAO, the amphiphilicity of the resulting block copolymers can be varied systematically. In contrast to most other block copolymers the hydrophobic moieties in the PAO block copolymers are soluble in a large variety of oils, making them ideal candidates as additives in microemulsion systems. The lowest members of the polyalkylene oxide (PAO) family, polyethylene oxide (PEO) and polypropylene oxide (PPO), are known for a long time. The most widely employed technique for the polymerization of alkylene oxides is anionic ring-opening polymerization using sodium or potassium alcoholates as initiators. This technique allows to polymerize ethylene oxide (EO) basically free of side reactions. For the polymerization of propylene oxide (PO) strong side reactions are present. The reason for the side reactions is the relatively high acidity of the methyl protons of PO leading to

different types of termination and chain transfer reactions. As a result high and low molecular weight by-products are formed [2, 3]. In the older literature, there are reports dealing with the synthesis of the higher and more hydrophobic PAO than PPO. Poly(1,2-butylene oxide) (PBO), poly(1,2-hexylene oxide) (PHO), poly(1,2-octylene oxide) (POO) and even higher PAO were obtained by polymerizing the corresponding monomers with zinc organic catalysts [4-6]. This technique, however, yields polymers with broad MWD and does not allow producing block copolymers. Figure 1 illustrates the chemical structures of some polyalkylene oxides and the corresponding monomers.

FIG. 1: Structures of different alkylene oxide monomers and polymers.

polyoctylene oxide (POO)

1-octylene oxide R=(CH2)5-CH3

polyhexylene oxide (PHO)

1-hexylene oxide R=(CH2)3-CH3

polybutylene oxide (PBO)

1-butylene oxide R=CH2-CH3

polypropylene oxide(PPO)

propylene oxide R=CH3

polyethylene oxide (PEO)

ethylene oxide R=H

OH2C CHR CH2 CH O

monomer polymer

In a first series of experiments butene oxide was polymerized at different temperatures and using different solvents and initiators. The target molecular weight, defined by the amounts of monomer and initiator introduced into the reactor, was 15,000. The SEC trace of sample PBO-1 is given in Figure 2. In this experiment potassium tert.-butanolate (KOt-Bu) was used as initiator at 80oC and toluene was chosen as an inert solvent. Beside the main peak the signal contains a low molecular weight tailing and a high molecular weight shoulder at higher and lower elution volumes. In agreement with this result the molecular weight distribution is elevated and the measured molecular weight is about 25% smaller than the calculated molecular weight, obtained from the amounts of initiator and polymerized

monomer. In further experiments other alkali metal initiators and more polar aprotic solvents were tested in order to reduce the polymerization temperature without reducing reactivity and extending the polymerization time too much. This measure was successful insofar the by-product content could be reduced. However, at 40oC still significant amounts of by-product were present and at lower temperatures polymerization was extremely slow.

FIG. 2: SEC refractive index signals of PBO samples, polymerized without crown ether at 80°C in toluene (PBO-1) and with crown ether at -20°C in toluene (PBO-15).

Therefore a different strategy was chosen based on crown ethers. These additives are strong complexing agents for alkali metal ions. Especially in less polar solvents they increase the degree of ion-pair separation. In case of alkylene oxide polymerization this leads to increasing reactivities and polymerization rates. In our case it was possible to reduce the polymerization temperature considerably below room temperature. Because of the reduced level of by-products already achieved without crown ethers, the target molecular weight was increased from 15,000 to 50,000. This measure generally increases the relative fraction of by-products and helps to get a clearer picture about improvements of the product quality. Again different alkali metal initiators in combination with different crown ethers were tested. It turned out that KOt-Bu together with the crown ether 18C6 was the best combination. Using this system the polymerization temperature could be reduced below -20oC and within experimental error no by-product could be detected (see Figure 2, sample PBO-15). The more hydrophobic monomers hexene oxide and octene oxide could be polymerized under similar conditions as butene oxide. However it was necessary to purify those monomers by careful fractional distillation due to the lower purity of the commercial raw material compared to butene oxide. With this additional measure molecular weight of 50,000 to 100,000 could be obtained without significant by-product content and low molecular weight distributions.

FIG. 3: SEC refractive index signals of a PBO-PEO block copolymer and the corresponding PBO homopolymer with block molecular weights of approximately 10,000.

elution volume 34

PBO-PEO PBO

elution volume

35 40elution volume

PBO-15

A-B block copolymers containing PBO, PHO, and POO as hydrophobic blocks and PEO as hydrophilic block were produced using the polymerization techniques described before. This technique allows to synthesize the block copolymers by successively polymerizing the hydrophobic alkylene oxide and EO in a one-pot-reaction. Within experimental error the calculated compositions and the measured ones were similar. The small values of Mw/Mn indicated narrowly distributed polymers. As an example the SEC trace of a PBO-PEO block copolymer having block molecular weights of 10,000 is given in Figure 3. It shows a symmetrical signal. At higher elution times there is no hint for homopolymer. Even block copolymers having block molecular weights of 50,000 were basically free of homopolymer. This again underlines that the new low-temperature method using crown ethers yields model polymers of high structural quality.

[1] Allgaier, J.; Wilbold, S.; Chang, T. Macromolecules,

2007, 40, 518.

[2] Bailey, F. E., Koleske, J. V. In Alkylene oxides and their polymers, Surfactant Science Series; Schick , M. J., Frederick, M. F., Eds.; Marcel Dekker: New York, 1991; Vol. 35, Chapter 4.

[3] Whitmarsh R. H. In Nonionic Surfactants – Polyoxyalkylene Block Copolymers, Surfactant Science Series; Nace, V. M., Ed.; Marcel Dekker: New York, 1996; Vol. 60, Chapter 1.

[4] Lal, J. J. Polym. Sci. A-1 1966, 4 .

[5] Lal, J. J. Polym. Sci. B 1967, 5, 793.

[6] Booth, C.; Orme, R. Polymer 1970, 11, 626.

Protein Domain Motions observed in

Space and Time by NSE

Ralf Biehl1, Michael Monkenbusch1, Bernd Hoffmann2, Peter Falus3, Sylvain Prévost4, Rudolf Merkel2, Dieter Richter1

1IFF-5: Neutron Scattering

2IBN-4: Biomechanics

3Institut Laue Langevin, Grenoble, France

4 Hahn Meitner Institut, Berlin, Germany

To bind a cofactor to the active center of a protein often a structural change is mandatory. This “induced fit” includes small changes of single bonds but also large domain motions to prepare the active atomic configuration. Neutron spin echo spectroscopy is used to directly observe the domain dynamics of the protein alcohol dehydrogenase [1]. The collective motion of domains as revealed by their coherent formfactor relates to the cleft opening dynamics between the binding and the catalytic domains enabling binding and release of the cofactor to the active center.

Proteins are the molecular machinery of life. As nanomachines of metabolism, they are in every cell of our body tirelessly active, transport, synthesize, divide and transform substances. Many transformation processes are underway in characteristic shaped bags, in which only certain substances fit, as a key fits to the lock. The shape of these pockets is determined by the sequence and three-dimensional arrangement of amino acids. Sometimes the binding of the cofactor or substrate is responsible for the formation of the right shape of the pocket. Through this "induced fit" the lock for a certain key is arranged to fit. There must be movements of atoms, amino acids or entire domains to bring the atoms in the right configuration to get active.

This effect is so far mainly known from structural studies of crystallized proteins, which represent snapshots of different configurations. Internal movements could also be observed by fluorescent labelling of two points on a protein with molecular biological methods to track distance changes [2]. Further information is attainable only by computer simulations.

One of the most studied proteins and one of the key enzymes is Alcohol Dehydrogenase (ADH, Figure 1). It produces ethanol in yeast or converts it back to acetaldehyde in the human liver. A functional important molecule (cofactor) Nicotinamide Adenine Dinucleotide (NAD) and a substrate molecule (ethanol or acetaldehyde) bind to the binding domain in a cleft between active and binding domain. The active domain, with a catalytic Zinc atom in the active centre, opens and closes the cleft by thermal movements. The closing places the active centre in the right

position for the transfer of a hydrogen atom between the substrate and cofactor molecule, and another hydrogen atom is released. Again opened, the cofactor and the product of the reaction will be released.

FIG. 1: Tetrameric Alcohol Dehydrogenase of yeast is build up from two dimers (green-blue and gray). The blue monomer is in the closed conformation with a NAD cofactor (orange) and an ethanol (red) in the cleft between binding domain and the active domain. In the cleft between both domains of the open configuration (green monomer) the catalytic Zinc atom at the catalytic centre is visible. The second dimer (gray) is bound at the backside of the first dimer. The arrow indicates schematically the movement of the active domain here without a bound cofactor.

Neutron scattering provides the possibility to investigate the timescale of these large scale movements in biomolecules. It provides information on the location and movement of atoms, without destroying the sample. The measurements were done in dilute D2O buffer solutions, close to the natural environment of the protein. D2O offers the possibility to focus on the protein scattering, because the D2O is only a weak scatterer for neutrons compared to H2O or the protons in the protein.

Scattering at the atomic nuclei of the protein will produce a characteristic interference pattern.

Movements of the atoms during the scattering process change the speed of the neutrons in the scattered beam. The change in speed for the scattered neutrons is very small. In order to detect it, NSE spectroscopy uses the Larmor precession of the neutron spins in a magnetic field as a stopwatch. The result is a length scale dependent flexibility, which differs from the expected motion of a rigid protein, only showing translation and rotation movements of diffusion. These length scale variations reflect the pattern of the movements due to the underlying dynamics.

FIG. 2: a, Single tetramer diffusion coefficient D0eff after corrections with and without bound cofactor. The black solid line represents the calculated effective diffusion coefficients for the ADH crystal structure, including translational and rotational diffusion of the stiff protein. At low q we observe only translational diffusion. The increase with the peak structure is due to rotational diffusion, which is only visible at larger q. b, Difference of the corrected diffusion coefficients and the calculated translational/rotational diffusion coefficient, reflecting the internal dynamics.

The NSE spectroscopy always measures the collective movement of all protein atoms, so in the evaluation the contribution of translational and rotational diffusion to the observed scattering has to be separated from the internal dynamics (Figure 2). After this we find a remarkable peak in the difference to the rigid body diffusion shown in Figure 2b.

The internal motions of a protein are quite complex. One way to explore possible large scale motions in a protein is the analysis of elastic normal modes. This method uses a simplification, it assumes only elastic forces between a reduced number of atoms representing the main structure of the protein. The method is like building a simplified model of the protein with the same shape made from elastic rubber. By shaking the rubber model it is possible to observe large scale motions with weak parts as flexible hinges between more compact stiffer domains. Analysing

these motions as small variation of the rigid protein structure enables the calculation of the change in the effective diffusion due to overdamped normal mode motions. The change in the effective diffusion of the most prominent normal modes 7 and 11 is shown in Figure 3. Other modes show a smaller contribution. Applying the method to the protein with and without cofactor shows the influence of the cofactor onto these characteristic motional patterns. The peak structure is comparable to the measured peak in figure 2 and shows also the effect of a shift due to the bound cofactor.

FIG. 3: Change of the effective diffusion due to movements along normal mode 7 and 11. Modes below 7 are trivial modes from translation and rotation. Without cofactor mode 7 is a mode which opens the cleft between catalytic domain and binding domain as indicated in figure 1. Mode 11 is a large amplitude mode described as torsion inside of the dimer along their main axis. The normal modes are numbered in sequence starting with the slowest timescale.

We found in our studies that the patterns of the movements observed are mainly due to the movement of the active domain relative to the binding domain at a timescale of 30 ns. Figure 1 illustrates schematically the movement of the active domain, leading to the opening of the gap according to normal mode 7. If the cofactor binds, it stiffens the protein, and the domain movement is significantly reduced. In ADH, we find an average amplitude of about 0.8 nm for the extent of the spatial movements between the two domains. Also we determined a spring constant of about 5 pN/nm for the stiffness of the cleft opening.

[1] Ralf Biehl, Bernd Hoffmann, Michael Monkenbusch, Peter Falus, Sylvain Prévost, Rudolf Merkel and Dieter Richter Phys. Rev. Lett. 101 (2008) 138102

[2] D. W. Pistona, G.-J. Kremers Trends in Biochemical Sciences, 32 (2007) 407

Unexpected power-law stressrelaxation of entangled ring polymersW. Pyckhout-Hintzen1, D. Richter1, D. Vlassopoulos2, M. Rubinstein3

1 IFF-5: Neutron Scattering2 FORTH, Institute of Electronic Structure and Laser, Heraklion, Crete,Greece3 University of North Carolina, Department of Chemistry, Chapel Hill,USA

Linear and long-chain branched polymers relaxby reptation processes out of the confining tubeor via arm retraction respectively. Both mechan-isms are intimately related to the presence ofchain end material. It is of major interest howentangled ring polymers which lack this basic in-gredient of chain ends proceed to relax stress.Self-similar dynamics yielding a power-law stressrelaxation is reported on model ring polymers.Here, the importance of some linear impuritiesbecomes evident. The combination of neutronscattering and rheology for rings is a promisingtool therefore to unravel details of their dynamicsand their difference with linear chains.

The investigation of polymer dynamics is alwaysclosely related to the particular structure or even thearchitecture that the polymers adopt. Fig 1a rep-resent a reptational-like motion through an obstaclefield of chains, which is clearly different from star fluc-tuations in Fig 1b where only arm retraction modescan relax the stress. Rings in Fig 1c and 1d may oc-cur in double-fold shape or even behave like lattice-animals respectively. Latter has a very strong sim-ilarity with a random cayley tree (Fig 1e) for whichthe relaxation time spectrum corresponds to longlogarithmically-spaced time scales for each of the dif-ferent chain sections. These follow a hierarchicalscheme and relax as is well known from former worksfrom the outside-inwards in a sequential way, leadingto typical patterns in the complex relaxation modu-lus. For model-branched architectures, the number ofgenerations is coupled to the number of loss peaks.It is, however, not obvious how a ring-like polymer willperform. Fig f depicts e.g. self-interpenetration andblockage of the rings.In the former considerations the influence of linearcontaminants which are the product of non-perfectlinking chemistry or degradation of closed cycles toopen linear chains, was not yet included. To ver-ify theories which focus on the special structure-dynamics associated with the odd architecture, it isa prerequisite to purify the rings. Recently, develop-ments of liquid chromatography at the critical con-dition have shown to efficiently separate rings andlinear chains via the compensation of entropic sizeexclusion and enthalpic interactions with the pores.The polymers so obtained were analyzed by rheol-ogy and SANS in order to assure their unchanged,

original closed structure.

The linear rheology for the purified rings is very com-patible to the model of self-similar lattice animals ofFig 1(d,e). The associated exponentially decreasingrelaxation modulus G(t) is then given as

G(t) = GN0

«− 25

„− t

τring

FIG. 1: Different correlations of structure with assumeddynamics are summarized and explained in the text. Thecomparison of ring with branched polymers is made clear.

This is distinctly different in linear or branched poly-mers which show an extended entanglement plateau.This expression for the relaxation modulus already in-cludes constraint-release effects for loop rearrange-ment as well as dynamic dilution and contains no fur-ther adjustable parameters.

SANS measurements are shown in Fig 2. From dilutesolution and in a melt of linear chains (not shown),unperturbed chain dimensions as well as the swellingof the ring by excluded-volume statistics could be

confirmed. The experimental slopes in Fig 2 are typ-ical. Very good agreement in both size as well as thescattering amplitudes was obtained. The ring in goodsolvent is swollen by a factor of 1.5 which is in accor-dance with estimates of about 1.4 in literature. Notraces of impurities of linear chain could be spottedfrom SANS.

FIG. 2: SANS experiments on dilute solutions under θ

and good solvent conditions show the characteristic scat-tering vector dependences. q−2 is random-walk statisticson intermediate length scales whereas the q−1.6 is promi-nent for excluded-volume interactions in swollen rings. Thestrong parasitic forward scattering at low q for the cyclohex-ane sample following q−3.1 is due to density fluctuations inthe theta-state.

FIG. 3: Linear chains are percolating through bridging bythe rings at low concentrations.

The effect of the linear chain on the dynamics of aring could be quantified by deliberately mixing in thesample again known amounts of linear chains and re-measure the relaxation modulus. It shows that evenat concentrations of 1/50th of the overlap concentra-tion of the rings, the relaxation time spectrum gets anew long-lived component which grows substantiallywith concentration. The viscosity rises and a plateaushows up again. The understanding of this is impor-tant to relate former experiments in literature on ringswhich were not purified so extensively as here andtherefore always showed a plateau modulus value.

We have forwarded in Fig 3 the molecular picturethat linear chains must be bridged at this extremelylow concentration by the rings to be so effective andtherefore provide considerable increase in the meltviscosity. The entropically driven penetration of ringsby linears leads then to a transient network.

As both natural and synthetic polymers can be foundin this severe cyclic architecture the present resultsare of both fundamental and practical significance.Optimized microprocessing as well as rheology mod-ifications are thus within reach. Rings will be in-vestigated further using adequately isotope-labeledsystems to study differences in segmental dynam-ics and chain fluctuations compared to their linearanalogs. Also the question whether a plateau mod-ulus shows up or power law relaxation persists if therings get considerably larger will be investigated. Ad-ditionally blends with linear homopolymers will be ad-dressed as well to be compared with recent advancesin blends of linear with dendritic polymers. The tech-niques of interest are neutron spin echo (NSE) andsmall angle scattering (SANS) in quenched statewhich focus on the short times i.e. high-temperaturemotion and long times i.e. large-scale dynamics re-spectively. The present work was carried out in theframework of the Joint Programme of Activities ofthe SoftComp Network of Excellence (contract num-ber NMP3-VT-2004-502235) granted under the FP6by the European Commission.

[1] M. Kapnistos, M. Lang, D. Vlassopoulos, W.Pyckhout-Hintzen, D. Richter, D. Cho, T. Chang, M.Rubinstein, Nature Materials, 7, 997-1002(2008)

[2] M. Rubinstein, R. Colby, Polymer Physics, (OxfordUniversity Press, 2003)

[3] M. Rubinstein, Phys. Rev. Lett., 24, 3023-3026(1986)

Nucleation and Growth of CaCO3 Mediated by the Protein Ovalbumin V. Pipich1, M.Balz2, S.E. Wolf2, W. Tremel2, D. Schwahn3 1JCNS: Jülich Centre for Neutron Science 2Institute for Inorganic Chemistry, Johann Gutenberg-University, Mainz 3IFF-5: Neutron Scattering Formation of CaCO3 mineralization in the presence of the egg-white protein ovalbumin was studied by time-resolved small-angle neutron scattering. A particular goal was to achieve a better insight into the early stages of mineralization. This is a relevant goal as the capability of mineral formation by living organism is not yet well understood. Starting from the amorphous phase several stages of mineralization, namely the crystalline polymorphs vaterite and aragonite, were observed. The formation and dissolution of amorphous CaCO3 is accompanied by Ca2+ mediated unfolding and cross linking of about 50 protein monomers showing linear chain characteristic scattering. The protein complexes act as nucleation centers because of their enrichment by Ca2+ ions.

Living organisms are capable of developing inorganic minerals with complex architectures to fulfill important biological functions, such as skeletal support or protection of soft tissues [1,2]. The mineral phase of such materials is intimately associated with organic macromolecules, such as proteins and polysaccharides. However, the role of these proteins in mineralization is not well understood. Small-angle neutron scattering (SANS) is a new technique in biomineralization which allows identification and structural analysis of biomineral composites by the method of contrast variation [2]. With the particular intention of a better insight into the early stages of mineralization, we performed studies of in-situ calcium carbonate formation mediated by the egg-white protein ovalbumin. Details of this work can be found in [3].

A characteristic result after 13 h mineralization by using the gas diffusion technique is shown in Figure 1. The scattering pattern is characterized by two power laws representing μm large mineral particles and nm-sized proteins at small and large scattering vector Q, respectively. The Q-4 power law is a measure of the total mineral surface (Porod’s law: P4·Q-4) whereas the Q-1 power law is caused from the linear chains of ovalbumin. So, the large difference in size of the mineral and protein allows a separate analysis of both constituents. Another important property of SANS is the large variation range of scattering contrast Δρ2 of aqueous solutions by the exchange of H2O and D2O. The

symbol ( )Sρ ρ ρΔ = − describes the difference of

the coherent scattering length density ρ of the mineral or protein and of the solvent ρS. The corresponding values are summarized in the Table together with Φ representing the D2O content when the protein and the mineral polymorphs are matched in water, e.g. 0ρΔ = .

10-3 10-2 10-1

102 2.5mg/ml Ovalbumin

dΣ/dΩ

Q [Å-1] FIG. 1: Scattering pattern after 13 h mineralization in the presence of ovalbumin. The scattering from the mineral and protein are distinct by the two power laws

We performed several mineralization experiments in aqueous solution of different D2O content, in order to identify the mineral polymorphs. Figure 2 shows the time evolution of the amplitude P4 of four relevant contrasts. In all cases a peak becomes visible about 2.5 hours after initiation the mineralization. This scattering was identified from ACC particles as the intercept of 4 0P = occurred at Φ=0.55±0.05 (Table). So, the mineralization started with the formation of an amorphous polymorph, which after reaching a maximum volume fraction transformed to a crystalline polymorph. The pronounced minimum for the Φ=0.74 D2O solvent after 4 h, and the smallest scattering of the Φ=0.82 sample after 5 h provide the interpretation that CaCO3 follows a polymorph sequence of amorphous → vaterite → aragonite (Table).

The scattering patterns in Figure 3 represent the contribution from the protein during mineralization (large Q part in Figure 1). During the first 1.5 h the scattering of the protein is best described by the

0 2 4 6 8 10 120

74% 82%P

4 [10-1

0 cm-1Å

Time [h]

Vaterite

H2O D2O

FIG. 2: Porod amplitude versus mineralization for different scattering contrasts. A peak from the amorphous polymorph (ACC) became visible at ≈ 3h. After 4h the minimum of the 0.74 D2O solution identifies the vaterite polymorph whereas aragonite appears as the stable phase within 12 h.

ρ [1010cm-2] Φ

Calcite 4.69 0.76

Aragonite 5.10 0.81

Vaterite 4.49 0.74

Amorphous 3.29 0.55

Ovalbumin 1.64+1.61Φ 0.41

Water -0.561+6.95Φ ---

TAB.: SANS parameters of the calcium carbonate polymorphs, ovalbumin, and water.

compact structure of the native state. The slight increase of scattering after 90 minutes is the result of a 14.6% increase in the protein scattering length density which can be interpreted as a “loading” of the protein with about 280 Ca2+ ions. After 90 minutes a gradual transition to a Q-1 power law is observed which was complete after nearly 3 h (see also Figure 1); the structural change of the protein was a continuous process during this time interval. The strong increase in intensity at low Q indicated that the process was accompanied by a cross-linking ("salting out") of the protein molecules. In this time interval the formation of the amorphous polymorph proceeded and approached its maximum (Figure 2). After the protein was “salted out”, the amorphous phase started to dissolve as

0 1 2 3 4 5 6 7 80.00

dΣ/dΩ

Q [10-2Å-1]

10-3 10-2 10-110-2

FIG. 3: Protein scattering during mineralization (Β before start, after start of mineralization at 7 15 min , 90 min, and ξ nearly 13h). A gradual aggregation of the proteins is observed. The inset shows ovalbumin in 0.1 M CaCl2 aqueous D2O solution. The enlarged scattering is caused by cross linking of about 52 proteins to an object with the conformation of a Gaussian linear chain as seen by the Q-2 and Q-1 power laws (Radius of gyration ≅600Å).

seen from the reduction of the amplitude P4 in Figure 2. In a separate experiment of ovalbumin in 0.1 M CaCl2 aqueous solution we determined a cross linking of about 50 proteins (inset in Figure 3). This scattering was interpreted in terms of a Gaussian linear chain with relatively large statistical segments.

In summary, we give a first clue concerning the role of protein mediated CaCO3 mineralization. Ovalbumin first acts as a “cation sponge” which locally increases the Ca2+ concentration. These Ca-rich pockets of ovalbumin aggregates seem to build nucleation centers for the incipient calcium carbonate formation. Calcium carbonate nanocrystals are stabilized by surface bound ovalbumin which is eventually occluded between the individual CaCO3 crystallites, thereby forming a mesocrystal, i.e. an inorganic-organic hybrid material [4]. The observed formation from less dense to more dense polymorphs follows the Ostwald-Volmer rule [1].

[1] S. Mann, Biomineralization, Oxford University Press, Oxford 2001.

[2] A. Heiss et al., Biointerphases 2 (2007) 16-20.

[3] V. Pipich et al., J. Am. Chem. Soc. (JACS) 130 (2008) 6879-6892.

[4] D. Schwahn et al., J. Phys. Chem. C111 (2007) 3224-3227.

Kinetics of Micelle Formation L. Willner1, R. Lund2, M. Monkenbusch1, P. Panine3, T. Narayanan3, J. Colmenero2 , D. Richter1 1IFF-5: Neutron Scattering

2Donostia International Physics Center (DIPC), Donostia - San Sebastian, Spain 3European Synchrotron Radiation Facility, Grenoble,France. The route by which amphiphilic molecules self-assemble into nano-scale objects such as micelles is still not fully understood. The formation of block copolymer micelles in selective solvents occurs spontaneously usually in the subsecond range. By means of synchrotron x-ray scattering with millisecond time resolution we got direct structural information in-situ on the birth and growth of block copolymer micelles. Using a quantitative model we showed that the self-assembly process can be viewed as a primary micellization and growth process where the elementary growth mechanism is the exchange of single unimers

A classical example and a model system for self-assembly are amphiphilic diblock copolymers that undergo micellization in aqueous solution [1]. The morphology of such systems has been widely studied during the past, a detailed understanding of the mechanism and kinetic pathways of the self-assembly process has not been reached to date. This is primarily due to the lack of experimental techniques having the correct spatial and temporal resolution with the combination of a suitable well-defined model system. In addition there is a prominent lack of detailed physical modelling of the data. Thus so far, results remain largely inconclusive.

In this work [2] we show that the required nano-scale spatial resolution and millisecond temporal resolution could be achieved for an in situ investigation by using synchrotron x-ray scattering. The self assembly process of a model amphiphilic block copolymer system was triggered by an interfacial tension jump experiment by rapidly changing the solvent quality for one of the blocks. Using a detailed quantitative model we further demonstrate that the kinetic pathway proceeds by unimer exchange where only single chains are added or removed at a time.

As a model system we employed a well-defined poly(ethylene-alt-propylene -poly(ethylene oxide) (PEP1-PEO20, numbers indicate the approximate molecular weight in Kg/mole)block copolymer. This block copolymer forms starlike micelles in water and water/dimethylformamide(DMF)mixtures which are both selective solvents for PEO [3]. A large difference in interfacial tension, γ, with respect to PEPallows an effective tuning of the micellization properties by varying the solvent composition. In pure DMF only single chains (unimers) are present but as soon as some water is added the block

copolymers spontaneously aggregate into micelles. This phenomenon was exploited by rapidly mixing a DMF solution with unimers with water/DMF pure solvent mixture by means of a stopped flow apparatus. The stopped flow set-up was coupled to the small angle x-ray scattering (SAXS) instrument at the high brilliance beamline, ID02, at the European Synchrotron Radiation Facility (ESRF) allowing a synchronization of extremely fast mixing (4.5ms) with rapid data acquisition. We chose an optimized acquisition time of 20 ms.

A typical example of the time evolution of the scattering curves is presented in Figure 1. The data were obtained from a solution containing a total volume fraction of 0.25% block copolymer in a solvent mixture with 90 mole% DMF.

0,01 0,02 0,030

102030405060708090

100 14.5 ms 24.5 ms 194.5 ms 994.5 ms 2914.5 ms 240 s Unimer Reservoir Model fits

dΣ/dΩ

/φ (Q

Q [Å-1]

Fig. 1: Normalized absolute scattering cross sections at different times during the kinetics for the PEP-PEO system at a final polymer concentration of 0.25%. The solid lines display fit results from a standard core/shell model.

The induced self-assembly process causes a strong increase in intensity directly reflecting the growth of the micelles in real time. The scattering data were analysed using a standard core shell model describing the detailed structural features of both the inner PEP core and the outer PEO corona of starlike micelles. The solid lines in Figure 1 represent fit results of the core/shell model. The excellent agreement indicates that at all times a starlike structure is adopted. An important parameter which can be extracted by the model fit is the mean aggregation number, Pmean. The growth of the micelles in terms of an increasing Pmean is shown in Figure 2 on logarithmic time scale for three different concentrations.

Fig. 2: Time dependence of the aggregation number, Pmean, extracted from the fits for three polymer volume fractionson a logarithmical time scale: 0.125% (stars), 0.25% (squares), and 0.5% (triangles). Solid lines represent a fit using the kinetical model described in the text.

Qualitatively the evolution of Pmean can be summarized as follows: At shortest times the data suggest the existence of a fast initial aggregation (t<≈5ms) that cannot be entirely resolved experimentally. This process becomes exhausted at intermediate times leading to a “shoulder” of Pmean that changes with concentration. The terminal relaxation towards a common equilibrium slows down with time. The overall rate increases with concentration.

In order to quantitatively discuss the experimental data we have derived a kinetic model that involves simple unimer exchange as the single elementary growth step:

UM P + 1+PMk+

k- with MP as the number of micelles of size P and U the number of unimers. k+/k- denote the insertion and expulsion rate constants which both depend on P. Within the context of classical nucleation and growth theories [4,5] the formation and growth of the polymeric micelles is governed by the micellization potential, G(P,φ1) (φ1=unimer volume fraction),which in our case was taken as the difference of the free energy of a starlike micelle and an equivalent amount of unimers taking properly into account the translational entropy. Following Neu et al. [5] we assume the validity of the “detailed balance” or “microscopic reversibility” principle, which gives a net creation rate in terms of the concentration, φP+1, and flux, jP+1:

( ) ( )( )[ ]11111 ,,1exp)( φφφφφ PGPGPkj PPP −+−= +++ which is only determined by the insertion rate constant k+ and the potential G(P,φ1). The whole micellar evolution can then be calculated by solving a system of differential equations which was done numerically by using standard routines and by fitting to the experimental data. Fit results are shown as solid lines in Figure 2. All concentrations could almost perfectly be described by a consistent set of parameters. The parameters nicely compare with

macroscopically determined quantities, e.g. the interfacial tension, γ, between PEP and water/DMF was found to be 19mNm compared to 12mNm obtained by pendant drop tensiometry.

The kinetic pathway from unimers to the final micelle is schematically depicted in Figure 3. The initial free unimers are rapidly consumed in a primary micellization event as shown in region leading to classical overnucleation. Consequently, metastable micelles with a broader size distribution are obtained in region II corresponding to the shoulder in Pmean at intermediate times in Figure 2. A further growth of the micelles requires that some of them, particularly the smaller ones, disassemble to provide unimers. At later times in region III the equilibrium is approached with a narrow size distribution of the final micellar entity.

IIIIII

fast slow

IIIIII

fast slow

Figure 3: Schematic view of the kinetic pathway in the formation of polymeric micelles.

In summary, the kinetics of formation of block copolymer micelles have been directly observed in situ by synchrotron small angle X-ray scattering with millisecond time resolution. Applying a quantitative model, we see that the formation and growth of micelles can accurately be described by a primary micellization and growth process governed by single unimer exchange mechanism. The contribution of other more complicated mechanisms, like fusion and fission, cannot entirely be excluded, however, the experimental data are sufficiently explained by insertion/expulsion of a single chain at a time.

[1] Hamley , I. W. The Physics of Block Copolymers, Oxford University Press, Oxford , UK (1998).

[2] Lund, R. et al. submitted to Phys. Rev. Lett..

[3] Lund, R. et al. Macromolecules, 39,4566 (2006).

[4] «Nucleation Theory and Applications» (J.W.P. Schmelzer ed.), Wiley-VCH Verlag GmbH & Co., KGaA, Weinheim (2005).

[5] Neu, J.C. et al. Phys. Rev. E, 66, 061406 (2002).

Polymer Chain Dynamics in a RandomEnvironmentK. Niedzwiedz1, A. Wischnewski1, M. Monkenbusch1, D. Richter1, A.-C. Genix2, A. Arbe3,J. Colmenero2,3, M. Strauch4, E. Straube4

1FZJ, Institute of Solid State Research - Neutron Scattering, Julich, Germany2Donostia International Physics Center, Paseo Manuel de Lardizabal 4, 20018 San Sebastian, Spain3Centro de Fısica de Materiales CSIC-UPV/EHU, Apartado 1072, 20080 San Sebastian, Spain4Martin-Luther-Universitat Halle-Wittenberg, Fachbereich Physik, D-06099 Halle, Germany

The question of what determines the behaviourof two polymers which are perfectly miscible onthe one hand, but very different in their dynam-ical properties on the other hand, is at presentmatter of controversial discussion. We presenta neutron scattering study on the misciblepolymer blend poly(ethylene oxide)/ poly(methylmethacrylate) (PEO/PMMA) which due to the verydifferent time scales of motion is a perfect can-didate to address this topic. Only exploring dif-ferent time and length scales allowed to identifythe mechanisms of dynamic heterogeneity on amolecular scale.

The dynamics of a polymer chain in the melt is welldescribed by the Rouse model [1]. Here, the relax-ation of thermally activated fluctuations is balancedby entropic and viscous forces (friction). The rele-vant parameter in this model is the friction of the ob-served chain in a heat bath representing the adja-cent chains. One important consequence of the fact,that the segments of a chain are linked to each other(chain-connectivity), is that segments exhibit a meansquared displacement proportional to the square rootof time different to the displacement for a Fickian dif-fusion which is increasing linearly in time.At present the investigation of dynamic miscibility ina blend of two different polymers is an active area ofresearch. One of the important questions is how thesegmental friction arises if a segment is surroundedby a chemically heterogeneous environment. Thesystem PEO / PMMA is in particular interesting be-cause of the significantly different glass transitiontemperatures (T PEO

g ≈ 200 K, T PMMAg ≈ 400 K).

In such a situation, for temperatures around and be-low T PMMA

g , PEO moves in the random environmentcreated by the frozen PMMA component [2].Starry-eyed, one could assume that in a perfectlymiscible polymer blend the friction of a given segmentis determined by the average friction of the blendcomponents.We have investigated the PEO dynamics in PMMAby quasielastic neutron scattering. This has beenrealized by preparing a mixture of protonated PEOchains in deuterated PMMA. Samples with differentconcentrations (25%, 35% and 50% of PEO) havebeen measured at different temperatures between350 K and 400 K. By neutron backscattering spec-troscopy we have followed the mean square displace-

ment (msd) of the PEO segments, the experimentswere performed at the instrument BSS at the FZJ inJulich.

FIG. 1: Mean square displacements obtained frombackscattering spectra from the 35% PEO/PMMA sampleat different Q and temperatures.

Fig. 1 shows the result for a sample with 35% proto-nated PEO in 65% deuterated PMMA at two differ-ent momentum transfers Q. The data follow nicelystraight lines when plotted against the square root oftime in good agreement with the Rouse prediction.Depending on temperature, the maximum displace-ment of a PEO segment observed during about 1 nsamounts to less than 1 nm. The related values for thefriction can be obtained from the slope of these lines.They show a temperature dependence very similarto the one found in pure PEO, however the friction isabout one order of magnitude larger than in the puresystem.So far we may state that the dynamics of PEO ina matrix of PMMA at temperatures where PMMA isnearly frozen is in good agreement with Rouse dy-namics characterized by an average friciton. Fromthis one could conclude that the effect of blendingis not more than imposing a different friction to agiven segment, and therefore that the above men-tioned naive assumption is true.Fig. 2 displays neutron-spin-echo (NSE) measure-ments of the single chain dynamic structure factorof PEO chains in the PMMA matrix measured at theIN15 (ILL, Grenoble) up to about 80 ns. In order toachieve this result a deuterated PMMA matrix wasused, where a mixture of deuterated and protonatedPEO was immersed. The dotted lines in Fig. 2 are

a result of a Rouse description based on the aver-age Rouse friction obtained from the backscatteringdata in fig. 1. Obviously this description fails grosslypredicting a by far too fast decay. Apparently the mo-tion is strongly slowed down towards longer times orlarger length scales. We may roughly quantify thisslowing down in fitting an effective smaller Rouserate. This reveals retardations by factors 4 to 20(depending on PEO-content and temperature) com-pared to the 1 ns scale and gives clear evidence foran additional effect of blending which has not beenseen in the BSS data.Finally, fig.3 shows Fourier transformed time-of-flight(TOF) data on the same blend measured at the FO-CUS instrument (PSI, Villigen) at T=400 K. For theblend also the backscattering data are included. Athigh Q, quasielastic neutron scattering observes lo-cal processes. Compared to the spectrum from purePEO, the blend data are strongly broadened demon-strating a broad distribution of local relaxation pro-cesses in the PMMA environment. From the de-cay we realize that a shift of the characteristic timefrom 1 ps in pure PEO to about 6 ps for PEO/PMMAis found –in excellent agreement with NMR data [3]and also with the ratio of the corresponding Rouserates as determined by the backscattering experi-ment. However, the question remains where the ex-tremely broad distribution comes from.

FIG. 2: Single chain dynamic structure factor of the35% PEO/PMMA system at T=400 K. Q-values are 1 nm−1,1.5 nm−1, 2 nm−1 and 3 nm−1 from top to bottom. Thedashed base represents the elastic contribution to the scat-tering. The dotted lines illustrate the Rouse theory withRouse relaxation rate obtained from the backscatteringdata. The solid lines show the results of the model withrandomly distributed friction.

In the following we demonstrate that the contradic-tory experimental findings can be explained assum-ing that the mobile PEO chain experiences a het-erogeneous environment imposed by the rather stiffPMMA matrix. This picture can be rationalized bya chain where the beads undergo a random frictionwith a distribution of friction coefficients.The dynamic structure factor for such a chain hasbeen calculated by starting with the Langevin equa-tion for a polymer chain and introducing a set ofbead mobilities following a log-normal distributioncentered around the average mobility obtained from

the backscattering data, the only adjustable param-eter being its width. The solid lines in Fig. 2 showthe best result which could be achieved for the 35 %PEO/PMMA sample. The three lowest Q values arein very good agreement with the experimental data,however, the highest Q data are reproduced at veryshort times only. The origin of this significant devi-ation at the highest Q and longer times is still un-clear, though it might have to be related to additionalconfinement effects imposed by the matrix. The val-ues of the width obtained by applying the same pro-cedure to the other compositions and temperaturesshow the expected behaviour: the width of the distri-bution function increases with increasing PMMA con-centration (in the limit of zero PMMA concentration itshould go to zero) and within one blend it increaseswith decreasing temperature.Having in mind that at high Q we observe directlythe distribution of relaxation times, we now can tryto describe the TOF spectra of the blend displayedin Fig. 3. Starting from the pure PEO spectrum weshift the relaxation rate by a factor obtained from thebackscattering data for the blend. In a second stepwe convolve the thus shifted PEO spectra with thelog-normal distribution function using the result fromthe NSE data. With this we obtain the solid line inFig. 3 in perfect agreement with the observed spec-trum. Having in mind that all parameters have beenobtained from other measurements, the width fromthe NSE-data and the shift of the relaxation rate fromthe BSS data, this agreement which combines dataover 5 orders of magnitude in time is remarkable.

FIG. 3: Fourier transformed TOF data of pure PEO and35% PEO/PMMA at T = 400 K. For the blend system alsobackscattering data are included. The solid line throughthe PEO data describes a fit with a stretched exponential(β = 0.5). Line through the blend data: see text.

[1] P. E. Rouse, J. Chem. Phys. 21, 1272 (1953)

[2] A.-C. Genix, A. Arbe,F. Alvarez, J. Colmenero, L.Willner and D. Richter, Phys. Rev. E 72, 031808(2005)

[3] T. R. Lutz, Y. He, M. D. Ediger, H. Cao, G. Lin andA. A. Jones, Macromolecules 36, 1724 (2003)

JCNS InstrumentationT. Gutberlet 1

1 JCNS: Jülich Centre for Neutron Science

On May 2nd, 2006 the FRJ-2 research reactor atForschungszentrum Jülich GmbH was shut downending a successful scientific work of more thanfour decades. The end of this research facil-ity with neutrons marked at the same time thestart of a new era for research with neutrons atForschungszentrum Jülich GmbH with the timelyestablishment of the Jülich Centre for NeutronSciences (JCNS) on February 16th, 2006. At thenational and international leading sources FRM II,ILL and SNS JCNS operates state-of-the art neu-tron instruments under a common scientific ob-jective. Instruments operated by JCNS are opento scientists from Germany, Europe and acrossthe globe for the benefit of their scientific re-search goal. Access is organized by the JCNSUser Office.

The JCNS reflects the unique role of Forschungszen-trum Jülich GmbH within the German neutron scat-tering landscape based on a compelling in-housecondensed matter research program focussing onneutron scattering in the areas soft condensed andbiological matter and nanomagnetism and highly cor-related electron systems. Some of the best instru-ments from the closed DIDO reactor were transferredto the new national neutron source FRM II in Garch-ing close to Munich (Fig. 1). Besides the necessaryadaptations of the instruments, a rigorous upgradeand renewal programme was embarked, which com-prised of up to 13 instruments. A new branch lab with32 scientists and technicians has been established atthe new research reactor FRM II in Garching since2006.

Transferred instruments included the powerful smallangle neutron scattering units KWS-1 and KWS-2,the novel focussing VSANS machine KWS-3, theJülich neutron spin echo spectrometer J-NSE, the dif-fuse polarized neutron spectrometer DNS and the re-flectometer HADAS. HADAS was transferred to FRMII to serve as test and development instrument TR-EFF. DNS, KWS-2, and J-NSE have been in oper-ation for users since autumn 2007 now. The secondSANS KWS-1 and the focusing SANS KWS-3 will be-come available for users soon. KWS-3 has been re-build and up-dated including a brand new mirror coat-ing and will be available for users till end of 2009.

FIG. 1: View of neutron guide hall of FRM II.

In addition to the relocated instruments the secondgeneration backscattering spectrometer with phasespace transformer SPHERES was built at FRM IIin the frame of the "Verbundforschung" funding ofthe BMBF. Regular operation and access or externalusers started in March 2008. At current SPHERESoffers an energy resolution of 0.65 µeV with a signalto noise ratio of better as 600:1.

JCNS is further constructing four more brand-new in-struments at FRM II: together with the Technical Uni-versity of Munich, a diffractometer for biological struc-ture determination BIODIFF is being built till 2010.A brand-new high intensity magnetism reflectome-ter MARIA is in construction to be operational in2009. The new thermal time-of-flight spectrometerTOPAS, replacing the former SV-29 of FRJ-2, and thenew time-of-flight powder and texture diffractometerPOWTEX will be installed at the new guide hall eastof FRM II. Both instruments are expected to be op-erational in 2011. POWTEX is realised and financedin the frame of the BMBF "Verbundforschung" andbuild in a collaboration of RWTH Aachen Universityand the Forschungszentrum Jülich within the JülichAachen Research Alliance JARA with the GöttingenUniversity as further partner. This instrument is de-signed to serve the need of the solid state chemistryand geoscience communities.

In addition of developing, constructing and runninghigh class neutron instruments at FRM II JCNS alsooffers users specialized and unique sample environ-ment and laboratory facilities on-side. Modern chem-istry and biology labs can be used for sample char-acterization and sample preparation.

In addition to the FRM II location, JCNS operates out-stations at the first MW spallation source SNS in Oak

FIG. 2: Inside of the spin echo spectrometer at SNS.

Ridge, USA and the highest flux neutron researchreactor of the Institute Laue-Langevin ILL in Greno-ble, France. At SNS, the erection of the ultra highresolution neutron spin echo spectrometer is nearingcompletion (Fig. 2). This next generation spin echospectrometer will offer unprecedented resolution anddynamical range. This investment in the instrumen-tation of the SNS gives German users access to twofurther high class instruments: the backscatteringspectrometer BASIS and the high resolution powderdiffractometer POWGEN3.

Within a CRG contract with ILL concerning the coldtriple axis spectrometer IN12 is operated in a consor-tium together with the CEA Grenoble. This engage-ment also provides German users additional accessto the thermal triple axis spectrometer IN22 and thelifting counter diffractometer D23. In the framework ofthe ILL Millennium programme, IN12 will be relocatedto a new guide end position and JCNS has commit-ted itself to a complete overhaul of the instrument,which will lead to significant improved performance.An overview of JCNS operated instruments is givenin Table 1.

JCNS instruments are open to scientists from Ger-many, Europe and across the globe for the benefitof their scientific research goals. JCNS gratefully ac-knowledges complementary funding support from theEU within the 6th Framework Programme through theKey Action: Strengthening the European ResearchArea, Research Infrastructures in the project NMI3- Integrated Infrastructure Initiative for Neutron Scat-tering and Muon Spectroscopy. 2/3 of the availablebeam time at JCNS instruments is distributed to ex-ternal users by application for beam time reviewed byan international Scientific Review Committee twice ayear.

Figure 3 shows the distribution of external propos-als received in the four proposal rounds in 2007 and2008 for the instruments of JCNS. Concerning thenumber of proposals the small-angle scattering ma-chine KWS-2 clearly stands out.

With the various instruments at FRM II, ILL and SNSJCNS provides external users unique access to worldclass instruments under standardized conditions atthe neutron source best suited for their respective ex-

Instrument Facility ParametersBIODIF FRM II cold neutrons

protein crystallo-graphy

DNS FRM II cold neutronspolarisation analysis

J-NSE FRM II Fourier times2 ps (4.5 Å) < τ< 350 ns (16 Å)

KWS-1 FRM II high resolutionGISANSpolarisation analysis

KWS-2 FRM II high intensityKWS-3 FRM II 10−4 < q <

10−3 Å−1

MARIA FRM II high intensitypolarisation analysis

POWTEX FRM II middle resolutiontexture diffractometer

SPHERES FRM II energy resolution0.65 µeVq = 0.16 to 1.84 Å−1

TOPAS FRM II energy transfer up to100 meVpolarisation analysis

TREFF FRM II polarisation analysisIN12 ILL polarisation analysisSNS-NSE SNS Fourier time up to

1 µsec

TAB. 1: JCNS instruments.

FIG. 3: Number of proposals submitted in the year 2007and 2008 on the neutron scattering instruments of JCNS.

periments.

Further information on the instruments of JCNS andCall for proposals for beam time can be found on theJCNS web pages at www.jcns.info.

IFF-7: Soft Condensed Matter Director: Prof. Jan K. G. Dhont

The Soft Condensed Matter group investigates the chemistry and physics of colloidal systems. Colloidal systems can be regarded as solutions of very large molecules which exhibit phase transitions and show non-equilibrium phenomena that are also found for simple molecular systems. Due to the slow dynamics of colloids and the tuneable interactions between the colloidal particles, however, there are many transitions and non-equilibrium phenomena that do not occur in simple molecular systems, like gellation and shear-band formation. The aim is to understand structure, dynamics and non-equilibrium phenomena on a microscopic basis with an open eye for possible technological applications.

The main topics that are studied include,

the phase behaviour, pattern formation, phase separation kinetics and dynamics of suspensions of spherical and rod-like colloids under shear flow,

mass transport induced by temperature gradients,

dynamics and micro-structural properties of colloidal systems near walls and interfaces,

the effects of pressure on interactions, the location of phase transition lines and gellation transitions and the dynamics of colloids and polymers,

response of colloids to external electric fields, the equilibrium phase behaviour of mixtures of

colloids and polymer-like systems, dynamics of various types of colloidal systems in

equilibrium, and the synthesis of new colloidal model particles,

with specific surface properties, interaction potentials and particle geometries.

Tracer-Sphere Diffusion inRod-NetworksK. Kang1 , A. Patkowski 2 , J. K. G. Dhont11 IFF-7: Soft Condensed Matter2 A. Mickiewics University, Poznan, Poland

Mass transport of colloidal-like particles throughnetworks is relevant for a number of separation-,purification- and characterization techniques ofmacromolecular mixtures and might play a role indiffusive transport (of proteins) through crowdedenvironments in cells. To gain in understand-ing on the physics that underlies the trans-port characteristics through such confining me-dia, translational diffusion of tracer spheres inisotropic and nematic networks formed by longand thin colloidal rods is investigated as a func-tion of ionic strength and rod concentration. Inparticular, the hydrodynamic screening lengthof isotropic and nematic rod-networks is deter-mined from a newly developed theory and exper-imental tracer-diffusion data obtained with Fluo-rescence Correlation Spectroscopy (FCS).

The majority of reported tracer-diffusion experimentsof spherical particles in rod networks focus on pro-teins in suspensions of F-actin, which are relevantfor mass transport in cells. Recently, experimentson tracer diffusion of spheres in host suspensionsof slender particles, other than F-actin, have beenreported : nucleosome core particles in dispersionsof DNA [1], colloidal spheres in solutions of "livingpolymers" [2] and colloidal spheres in dispersions ofxanthan [3]. The host networks in these referencesexhibit a quite complicated dynamics by themselves.As far as we know, there are no experimental dataavailable on tracer diffusion of colloidal spheres inthe much more simple quasi-static networks consist-ing of relatively stiff, very long and thin rods, asidefrom three earlier papers of the present authors [4]-[6]. Besides experimental work, an attempt has beenmade in these papers to develop a microscopic the-ory for tracer diffusion of spheres through networks ofstiff, long and thin rods, where both (screened) hydro-dynamic interactions and direct interactions betweenthe tracer sphere and the rod network are explicitlyaccounted for.

We use fd virus as a rod-like colloidal host parti-cle, which is indeed a very long and thin, ratherstiff rod (length 880 nm, thickness 7 nm and per-sistence length 2500 nm), and does not exhibitpolymerization/de-polymerization nor possible bun-dle formation (like F-actin, wormlike micelles and

FIG. 1: The two extreme cases where (a) the tracer sphereis large compared to the mesh size of the network, and (b)where the sphere is small compared to the mesh size.

EHUT). Moreover, due to the very small van derWaals attractions between the fd-virus particles, therod networks are stable over a wide range of salt con-centrations, which offers the possibility to study diffu-sion in these networks as a function of the range ofelectrostatic interactions.

There are fundamentally different mechanisms lead-ing to slowing down of diffusion of a tracer spheredue to the presence of a rod network, depending onwhether the tracer sphere is large or small in compar-ison to the mesh size of the network. For large tracerspheres, translational motion of the tracer sphere isonly possible when the network structure is severelydistorted (see Fig.1a). In this case, direct interactionsbetween the sphere and the rods is much more pro-nounced than hydrodynamic interactions. Tracer dif-fusion can then be described with the neglect of hy-drodynamic interactions [4]. For small tracer spheres,where the sphere can move through the voids of thenetwork without distorting the structure of the net-work (see Fig.1b), hydrodynamic interactions are im-portant. Due to entanglement of the rods in the net-work, hydrodynamic interactions between the tracersphere and the rods are screened. This screening isquantified by the so-called hydrodynamic screeninglength κ−1, which measures the penetration depthof shear waves into the network. For the rod net-works considered here, there is no theory availableyet for the hydrodynamic screening length. Despite

the fact that the network structure remains essen-tially unchanged during the diffusive motion of sucha small sphere, there is nevertheless a distortionof the pair-correlation function between the sphereand the rods. This distortion is due to the fact that,when the sphere moves past a rod, the probabilityto find the sphere on the side-of-approach of the rodis larger than that on the "shadow side" of the rod.The rod-sphere pair-correlation function is thereforedistorted, giving rise to a force on the sphere thataffects its diffusive motion. We referred to the distor-tion of the pair-correlation function in ref.[5] as "theshadowing effect". The shadowing contribution is notonly determined by hard-core interactions, but alsoby charge-charge interactions in case the rods andthe sphere carry surface charges. The importance ofthe shadowing effect relative to hydrodynamic inter-actions can thus be varied systematically by chang-ing the electrostatic screening length through varia-tion of the ionic strength of the rod suspensions [6].

FIG. 2: Long-time self diffusion coefficients of apoferritinin fd-virus particle suspensions for two ionic strengths. (a)TRIS-buffer concentration of 0.16 mM (from ref.[6]) and (b)20 mM plus 100 mM added NaCl (from ref.[5]). The ver-tical grey bars indicate the two-phase, isotropic-nematic co-existence region.

Tracer diffusion constants of apoferritin in fd-virussuspensions were measured with Fluorescence Cor-relation Spectroscopy (FCS). Since fd-virus particlesfluoresce to some extent, the resulting contribution tothe correlation function must be subtracted [5]. Thecreation of mono-domain nematics through align-ment in a magnetic field and the measurement of ori-entational order parameters of the nematic samplesis discussed in that reference.

In Fig.2 the measured diffusion coefficients are plot-ted as a function of fd-concentration for two analyt-ical TRIS/HCl-buffer concentrations that are used(0.16 mM , and 20.0 mM plus 100 mM NaCl). Ascan be seen, there is a strong effect of the ionicstrength on the diffusive behaviour of apoferritin.

The way to extract hydrodynamic screening lengthsfrom the diffusion data, based on the newly devel-oped theory, is extensively discussed in ref.[6].

The hydrodynamic screening length as a function ofthe fd-concentration for isotropic networks is shown

FIG. 3: (a) The hydrodynamic screening length κ−1 in unitsof the rod length L for the isotropic state versus the fd-concentration. The symbols refer to the different buffer con-centrations : cT = 0.16 mM ♦, 0.80 mM 9, 4.00 mM 4,20.0 mM ©, 20 mM +100 mM NaCl . (b) The screen-ing length for the highest buffer concentration and 100 mM

added salt in the isotropic and the nematic phase versus thefd-concentration.

in Fig.3a. The screening length κ−1 for variousionic strengths scale onto a single master curve, anddecreases with increasing fd-concentration, as ex-pected. The hydrodynamic screening lengths for thenematic phase are given in Fig.3b. Here, the distinc-tion between directions perpendicular and parallel tothe nematic director should be made. Surprisingly,the hydrodynamic screening length increases with in-creasing fd-concentration in the nematic state. Thisis probably due to the increasing degree of alignmentof the rods with increasing concentration. It thusseems that the hydrodynamic properties of nematicnetworks is highly sensitive on the orientational de-gree of order. This is confirmed from measurementsof κ−1 at a fixed fd-concentration but different orien-tational order parameter through the variation of ionicstrength [6].

[1] S. Mangenot, S. Keller, J. Rädler, Biophys. J. 85, 1817(2003).

[2] J. van der Gucht, N.A.M. Besseling, W. Knoben, L.Bouteiller, M.A. Cohen Stuart, Phys. Rev. E 67, 051106(2003).

[3] G.H. Koenderink, S. Sacanna, D.G.A. Aarts, A.P.Philipse, Phys. Rev. E 69 (2004) 021804.

[4] K. Kang, J. Gapinski, M.P. Lettinga, J. Buitenhuis, G.Meier, M. Ratajczyk, J.K.G. Dhont, A. Patkowski, J.Chem. Phys. 122, 044905 (2005).

[5] K. Kang, A. Wilk, J. Buitenhuis, A. Patkowski, J.K.G.Dhont, J. Chem. Phys. 124, 044907 (2006).

[6] K. Kang, A. Wilk, A. Patkowski, J.K.G. Dhont, J. Chem.Phys. 126, 214501 (2007).

Enhanced Slowing Down of theColloidal Near Wall Dynamics in aSuspension of RodsP. Holmqvist, D. Kleshchanok, P. R. LangIFF-7: Soft Condensed Matter

In this report, we will show the influence of rod-like particles, fd-virus, on the diffusion of spher-ical polystyrene colloids close to a wall. Thesphere diffusivity normal to the wall, < D⊥ >,is strongly affected by the presence of the rods,while the effect on the parallel diffusivity, <D‖ >, is less pronounced except in the imme-diate vicinity of the wall [1].

The slowing down and the anisotropy of Brownianmotion close to a wall due to hydrodynamic dragforces has been theoretically predicted [2] and re-cently experimentally verified [3]. So far, only thecase of particles interacting by excluded volume witha wall has been considered. The effect of depletionon the dynamics close to a wall has received very lit-tle or no attention, neither theoretical nor experimen-tal.

FIG. 1: Schematic picture of the sphere/rod system underevanescent illumination close to the surface

We chose to apply fd–virus as a depletant, which arerodlike mono-disperse particles and nonadsorbingneither on glass nor on polystyrene latex particles.They have a contour length of 880 nm which is com-parable to the maximum penetration depth applicablein an EWDLS experiment. In other words, the de-pletion potential mediated by fd–virus is expected tobe effective throughout the entire scattering volume.Further, we performed EWDLS measurements withour tipple axis setup, with which we can determinethe parallel and normal component of the diffusivity

independently [3]. The depletion potential mediatedby rodlike particles has been calculated by Mao et al.to third order in rod number density [5]. In the first or-der approximation, there is a simple closed analyticalform, which can be introduced in the expressions forthe initial relaxation rate Γ of the time auto correlationfunction of the scattered intensity. Our experimentalTIRM data for the depletion potential mediated by fd-virus is in quantitative agreement with these predic-tions. Therefore, we used the first order approxima-tion to estimate the lowest rod concentration at whichthe depletion potential would cause a notable effecton the near wall dynamics of spherical colloids witha radius of R = 85 nm. We note that the Derjaguinapproximation is not valid at this radius/rod length ra-tio, but it will give a lower boundary for the requiredrod number density, because the strength of the de-pletion potential decreases with decreasing R/L atconstant density. From this, we chose as a startingpoint a rod concentration of twice the overlap con-centration which would give a contact potential of ap-proximately 0.5kBT . Further, at this rod concentra-tion, the solvent viscosity will be changed only by afew percent and the fd–virus contribution to the scat-tering is essentially negligible, which keeps the datatreatment on a tractable level. To our surprise, wefound a much larger effect on the near wall particledynamics than expected from these considerations.

Aqueous buffer solutions (20 mM TRIS) of PS latexspheres with a radius of R = 85 nm were investi-gated with two different fd–virus concentrations, thatis, 0.05 and 0.17 g/L, above and below the overlapconcentration, c∗ = 0.075 g/L. In bulk solutions withthe same fd–virus content, the diffusion of the col-loids is reduced by less than 20% [6]. The PS la-tex spheres are charge stabilized by sulfonate sur-face groups, and they were diluted from their stocksolutions to a volume fraction of 2 × 10−4. At theseconditions, the particles may be regarded as hardspheres, since the Debye screening length is in therange of 3 nm, while the mean interparticle distanceis of the order of several thousand nanometers. Aschematic picture of the system close to the surfaceunder evanescent illumination can be seen in figure1. To illustrate the effect of the fd-viruses on the col-loidal diffusion close to the wall correlation functionsg1(t) are presented in figure 2 at a constant pene-tration depth of 2/κ = 270 nm and a total scattering

FIG. 2: Initial decay ln(g1(t)) for 2 × 10−4 volume frac-tion PS latex spheres (R = 85nm) in cfd = 0.17 g/L atQtot = 0.0157 nm−1 and 2/κ = 270 nm for bulk (opensquares) and for two different combinations of Q‖ and Q⊥as follows: Q‖ = 0.0136 nm−1 and Q⊥ = 0.00785 nm−1

(open circles ), and Q‖ = 0.00785 nm−1 and Q⊥ = 0.0136

nm−1 (open triangles). The lines are the expected ln(g1(t))for the same system without fd–viruses for bulk (black), forQ‖ = 0.0136 nm−1 and Q⊥ = 0.00785 nm−1 (red), and forQ‖ = 0.00785 nm−1 and Q⊥ = 0.0136 nm−1 (green).

vector, Qtot = 0.0157 nm−1, for two different com-binations of Q‖ and Q⊥ together with the bulk g1(t)for a fd–virus concentration of 0.17 g/L. The EWDLSg1(t)) show a much smaller slope than that of the bulkcurve. Further, the slope of the g1(t) at Q⊥ > Q‖ issignificantly smaller than that at Q⊥ < Q‖, which in-dicates a strong anisotropy of the near wall diffusion.If these data are compared to the expected g1(t) forthe same system without depletant [3] (lines in fig-ure 2), it is clear that the sphere near wall dynam-ics is affected much more than the bulk dynamicsby the presence of the rods. In bulk, the g1(t) val-ues with and without the fd–virus are almost identi-cal (the 20% reduction is hardly measurable), whilea large difference is obvious for the EWDLS correla-tion functions. Not only do the near wall dynamicsslow down significantly, but the anisotropy increases.This slowing down of the diffusion close to the sur-face cannot be explained solely by the small reduc-tion of the bulk diffusion. To investigate this in de-tail, systematic measurements of the diffusivity par-allel and normal to the surface at different penetra-tion depths were performed. Using the same pro-cedure described in our recent papers [3] the meandiffusivities for the parallel, < D‖ >, and the nor-mal diffusion, < D⊥ >, is determined for the two dif-ferent fd–virus concentrations. To get a more com-plete picture of the effect of fd–virus on the colloidaldynamics close to the wall, the procedure describedabove was performed for a series of six penetrationdepths. In figure 3, the extracted mean diffusivitiesnormalized to the bulk diffusion constant, D0, areplotted against the reduced penetration depth, 2/κR(where R is the colloidal radius). The anisotropy ofthe diffusion can nicely be seen as the separationbetween < D‖ > (black squares) and < D⊥ > (redcircles) for both concentrations, 0.05 g/L (solid sym-

FIG. 3: < D‖ > (squares) and < D⊥ > (circles) nor-malized to the bulk diffusion, D0, are plotted against thereduced penetration depth, 2/κR, for cfd = 0.05 g/L (solidsymbols) and cfd = 0.17 g/L (open symbols). The lines arethe expected curves for the colloid solution without deple-tant.

bols) and 0.17 g/L (open symbols). For comparison,we show the expected curves for the colloid solu-tion without fd–virus as solid lines. It is notable thatthe anisotropy is always larger for the high fd–virusconcentration, while for the low fd-virus concentra-tion the anisotropy is close to what can be expectedfor a system without depletant. If one compares thedata with the expected mean diffusivities without de-pletant (solid lines), no effect on < D‖ > can be seenat large penetration depths for both fd–virus concen-trations. However, at penetration depths below about450 nm, that is, 2/κR . 5, a pronounced effect canbe seen for both fd–virus concentrations. < D‖ > isdecreasing much more with decreasing penetrationdepth than was observed for the system where no de-pletant was present. This effect is more pronouncedin the solution with the high fd–virus concentration. Ifone looks at the normal diffusivity, < D⊥ >, a sig-nificant reduction can be seen as compared to thenondepletant system also at large penetration depths(distances) for both fd–virus concentrations. As for< D‖ >, < D⊥ > decreases with decreasing pene-tration depth, and for the high fd–virus concentrationsystem < D‖,⊥ > drop more strongly at low penetra-tion depths.

[1] Holmqvist P.; Kleshchanok, D.; Lang, P. R. Langmuir23, 12010 (2007).

[2] Brenner, H. Chem. Eng. Sci. 16, 242 (1961).

[3] Holmqvist, P.; Dhont, J. K. G.; Lang, P. R. Phys. Rev. E74, 021402 (2006).

[4] Kihm, K. D.; Banerjee, A.; Choi, C. K.; Tagaki, T. Exp.Fluids 37, 811 (2004).

[5] Mao, Y.; Cates, M. E.; Lekkerkerker, H. N. W. J. Chem.Phys. 106, 3721 (1997).

[6] Kang, K.; Gapinski, J.; Lettinga, M. P.; Buitenhuis, J.;Meier, G.; Ratajczyk, M.; Dhont, J. K. G.; Patkowski, A.J. Chem. Phys. 122, 044905 (2005).

Dynamics in colloidal suspensions: fromneutral to charged particlesG. Nägele1, A.J. Banchio2

1 IFF-7: Soft Condensed Matter2 Universidad Nacional de Córdoba, Argentina

We have made a comprehensive study on the dy-namics of suspensions of charge-stabilized andneutral colloidal spheres. Numerous short-timedynamic properties including diffusion functions,translational and rotational self-diffusion coef-ficients and the high-frequency viscosity havebeen computed by means of a powerful accel-erated Stokesian Dynamics simulation method.The results of this study were used, in particu-lar, to explore the validity of generalized Stokes-Einstein relations, and the possibility of measur-ing self-diffusion in a scattering experiment at anexperimentally accessible wavenumber.

The dynamics of suspensions of charged colloidalparticles is of fundamental interest in soft matter sci-ence, surface chemistry and food science. The scopefor these systems has been broadened even furtherthrough the increasing importance of biophysical re-search dealing with charged biomolecules such asproteins and DNA [1]. Many of the theoretical andcomputer simulation methods developed in colloidphysics are applicable as well to biological moleculesand cells. In addition, suitably modified methodsfrom colloidal physics can also be applied to dis-persions of very small, i.e., nanosized particles notmuch bigger than the solvent molecules. Chargedcolloidal particles interact with each other directlyby means of a screened electrosteric repulsion, andthrough solvent-mediated hydrodynamic interactions(HIs). These interactions cause challenging prob-lems in the theory and computer simulation of thecolloid dynamics.

In this communication, we report on extensive Stoke-sian Dynamics (SD) simulations where the influ-ence of electrosteric and hydrodynamic interactionshas been studied for numerous short-time proper-ties including the high-frequency viscosity η∞, thewavenumber-dependent diffusion function D(q) de-termined in a scattering experiment, the so-calledhydrodynamic function H(q), and the rotational andtranslational short-time self-diffusion coefficients Dr

andDs, respectively. The simulations have been per-formed, with a full account of the many-body HIs,in the framework of the model of dressed sphericalmacroions interacting by an effective pair potential ofscreened Coulomb type [2, 3]. A large variety of sys-

FIG. 1: Simulation test of short-time GSE relations relatingthe rotational and translational self-diffusion coefficients Ds

and Dr , and the cage diffusion coefficient, D(qm), respec-tively, to the high-frequency shear viscosity η∞. All depictedquantities are normalized by their zero concentration valuesmarked by the subindex 0. Symbols: SD simulation data.Lines: theoretical results. (a) Charged particles in salt-freesolvent (water). (b) Suspension of neutral colloidal spheres.From [2].

tems with differing particle concentrations, chargesand added salt concentrations have been consid-ered, spanning the range from hard-sphere systemsto low-salinity suspensions where the charged parti-cles repel each other over long distances. Throughcomparison with the simulation data, the applicabil-ity and the level of accuracy of analytical methods

FIG. 2: SD results for the static structure factor, S(q), nor-malized short-time diffusion function, D(q), and hydrody-namic function H(q) = S(q) ×D(q)/D0 of a salt-free sus-pension of charged particles, as a function of the wavenum-ber q times the particle radius a. The dynamic quantitiesD(q) and H(q) are scaled by the respective short-time self-diffusion coefficient Ds. The dashed vertical lines mark thewavenumber qs where S(qs) = 1. From [3].

and ad-hoc concepts has been tested, which are fre-quently applied in the description of the colloid dy-namics.

As an important application, the SD simulationscheme has been used to scrutinize the validity ofshort-time generalized Stokes-Einstein (GSE) rela-tions that provide an approximate link between dif-fusion coefficients and the high-frequency viscosity.GSE relations are fundamental to a growing numberof microrheological experiments. The assessment ofthe quality of these relations is a necessary prereq-uisite for the experimentalist interested in deducingrheological information from dynamic scattering ex-periments. Our simulation results show that the ac-curacy of a GSE relation is strongly dependent onthe range of the interaction potential, and the par-ticle concentration. See Fig. 1 for an illustration ofthis important point where simulation results for thestatic structure factor S(q) at various volume fractionsare compared to corresponding results for D(q) andH(q). The function H(q) is a direct measure of theHIs. Without HI, H(q) would be a constant equal toone, independent of the scattering wavenumber.

A strictly valid GSE relation would be represented inFig. 1 by a horizontal line of height equal to one, in-dependent of the colloid volume fraction φ. As shownin this figure, the GSE relation for the cage diffusioncoefficient, D(qm), related to the position, qm, of thestatic structure factor peak, applies reasonably wellto neutral hard spheres and to high-salinity systems(see Fig. 1b). However, it is strongly violated in thecase of salt-free suspensions of charged particles(see Fig. 1a). Our simulations show further that rota-tional self-diffusion, and to a lesser extent also trans-lational self-diffusion, are faster than predicted by the

corresponding GSE relations. The general trends inthe concentration and salt-dependence of the trans-port coefficients predicted in our SD simulations arein agreement with experimental findings on charge-stabilized suspensions and neutral particle systems.

It has been suggested that the (short-time) self-diffusion coefficient can be probed in a dynamic lightscattering (DLS) experiment at a specific wavenum-ber qs located to the right of the principal peak inS(q), where S(qs) = 1. The assumption madehere is that the dynamic scattering function at sucha wavenumber is essentially determined by self-diffusion, without noticeable collective diffusion con-tributions. Indeed, if this assumption is valid at leaston an approximate level, Ds ≈ D(qs), where D(qs) isthe diffusion function measured at qs. Fig. 2 includesSD simulation results for S(q), D(q) and the hydro-dynamic function H(q) for a salt-free suspension ofcharged particles. All dynamic quantities are normal-ized by the self-diffusion coefficient Ds. As can benoticed, Ds ≈ D(qs) is obeyed indeed within rea-sonable accuracy. For all systems examined in ourSD study, we find the difference between D(qs) tobe less than ten percent, both for charged and neu-tral particles. Thus, DLS at the specific point qs canbe used to obtain a decent estimate for the value ofDs. This finding is of relevance in numerous scatter-ing experiments on colloidal systems where the largewavenumber regime is not accessible experimentally,and where alternative techniques to measure Ds di-rectly are not applicable or unavailable.

Colloidal hard spheres have a common static andhydrodynamic length scale set by the particle ra-dius a. This leads to the occurrence of an isobesticwavenumber qa ≈ 4.02 where both S(q) andD(q)/Ds are equal to one, independent of the vol-ume fraction. In low-salinity suspensions, the ge-ometric mean particle distance becomes anotherlength scale of physical relevance. Consequently, inthese systems there is no isobestic point for S(q)and H(q). The non-existence of a φ-independentisobestic point in salt-free suspensions is exemplifiedin Fig. 2.

In summary, our comprehensive SD simulation studyof short-time transport properties forms a usefuldatabase for researchers interested in information onthe suspension dynamics of globular colloidal par-ticles, from systems of large, micron-sized colloidsdown to proteins in the nanometer range such as ly-zozyme and apoferritin. A simulation analysis of thelong-time dynamics of charged colloidal spheres witha full account of the HIs remains as a major challengewhich we plan to address in future work.

[1] M.G. McPhie and G. Nägele, Phys. Rev. E 78, 78,060401(R) (2008).

[2] A.J. Banchio and G. Nägele, J. Chem. Phys. 128,104903 (2008).

[3] A.J. Banchio, M.G. McPhie and G. Nägele, J. Phys.:Condens. Matter 20, 404213 (2008).

Synthesis of silica rods, wires and bundlesusing filamentous fd virus as templateJ. Buitenhuis, Z. ZhangIFF-7: Soft Condensed Matter

We explored fd as a template to direct the for-mation of silica nanomaterials with different mor-phologies through simple sol-gel chemistry[1].Depending on the conditions silica nanowirescan be formed, which seem to accurately tran-script the bending conformation and the lengthof the fd viruses in solution. But also surpris-ingly straight silica rods may be formed, and un-der other conditions bow-tie-shaped bundles ofrods are formed, which have a remarkably welldefined shape and dimension.

One dimensional anisotropic inorganic nanostruc-tures such as tubes, rods, wires, fibers, etc. arein the focus of research interests due to their po-tential applications, for example in optical, electronicand mechanical devices, sensors and catalysis[2, 3].The synthesis of these anisotropic nanostructures isa big challenge, because most inorganic materialsdo not form the desired structure by themselves. Incontrast to inorganic systems, biological and organicmaterials, especially supramolecular systems, usu-ally have a well defined structure down to the nano-scale. Using (bio)organic materials as a templateto build up anisotropic inorganic nanostructures hastherefore emerged as a highly attractive method in re-cent years. The results described in the present pa-per may serve as a basis for the further developmentof the synthesis of inorganic materials using biopoly-mers as a template.

In this paper, the filamentous fd virus is used as atemplate to regulate the formation of silica nanomate-rials with well-defined morphologies. Fd viruses havea length of 880 nm and a diameter of 6.6 nm. M13, avirus which is almost identical to fd, differing only inone amino acid per coating protein, has been inten-sively explored by Belcher, Hammond and co-workeras a template in the synthesis of metallic and othermagnetic and semiconducting nanowires[4]. Theirstrategy is to modify the coat protein of M13 via ge-netic engineering specifically for each metal or oxide,so that the coat protein can selectively induce precip-itation or assembly of that specific metal or oxide onthe surface of the virus. However, as far as we know,there is no report concerning the application of fd orM13 as a template for silica precipitation.

In contrast to the complicated genetic engineering

FIG. 1: TEM images of typical rods. (A) rods with a uniformsilica layer and semi-spherical ends, an assembly of threerods is indicated by the arrow; (B) a rod with a clear core-shell structure; (C) a slightly curved rod where no core-shellstructure is visible; (D) EDAX analysis of the rod shown inC.

route used with the M13 virus, we show here thatwild-type fd virus can also be used as a templatein the synthesis of inorganic materials using sim-ple sol-gel chemistry. Under different conditions, us-ing acid-catalyzed hydrolyzation and condensation oftetraethoxysilane as silica precursor, three kinds ofmorphologies are observed: 1) silica nanorods witha diameter of 20 nm and a homogeneous silica layer,2) nanowires with a curved shape and 3) bow-tie-shaped bundles with well-defined shape and hierar-chy. As far as we know, we are the first to use fd asa template for material synthesis and have observedseveral interesting structures.

Single silica rods with high uniformity, aggregatedsilica-virus hybrid nanorods as well as pronouncedgranular silica are observed for sample type 1, seefig. 1. Along the axis of the rods, the diameter isconstant and the silica layer is homogeneous. Thesurface of these rods is smooth under the maximum

FIG. 2: TEM of nanowires. A broken wire can be seen inthe inset as indicated by the arrow.

resolution of the TEM we used and the shape of theends of the rods is semi-spherical. Some of the rodsshow a clear core-shell structure with a low contrastpart along the center of the whole rod (fig. 1b). Thelow contrast part might be fd. However, some rods donot show such low contrast part and look like pure sil-ica rods (fig. 1c). The "pure" silica rod shown in fig. 1cwas subjected to EDAX analysis. Apart from siliconand oxygen, nitrogen and phosphorus are detected(fig. 1d). The nitrogen and phosphorus can only beattributed to fd, given that no agents containing nitro-gen or phosphorus were used during the synthesis.Although fd is semi-flexible and somewhat curved indispersion, most of the rods are straight and only afew slightly curved rods are seen (fig. 1c). From asingle rod point of view, the silica coating is highlyuniform. Also the diameters of different rods are allclose to the average value of about 20 nm. However,large differences in length are seen for different rods.Long rods with a length comparable to the length ofintact fd are observed along with short rods, whichmight form from the silica coating of the fragments ofdecomposed fd.

At somewhat different reaction conditions, long,curved wires are observed entangled with each other(sample type 2, fig. 2). The surface of these wires isless smooth than that of the straight rods describedbefore, and the diameter of these wires shows aless sharp distribution with an average diameter ofabout 23 nm. The contour length of these wires is inthe range of that of intact fd, while long wires witha length twice that of fd are also observed. Thelonger wires seem to consist of two viruses stickingtogether by partial parallel overlap. These results im-ply that most fd remains intact during wire formation(an exception is the broken wire shown in the insetof fig. 2 by the white arrow), in contrast to the caseof the straight rods described above, where manyrods much shorter than fd are observed. The curvedshape of these wires probably originates from thebending configurations of the semi-flexible fd virus inaqueous media. Therefore, these hybrid silica wiresshow an example of a quite precise transcription ofthe template, here, semi-flexible fd. Whether or notthe silica coating solidifies the fd virus completely so

FIG. 3: TEM image of bow-tie-shaped bundles dispersedin the background of granular silica. A possible subunit ofthe bundles, a silica fan is indicated by the arrow. Inset:schematic drawings of possible structures of the bundles.

that the flexibility is lost is not clear.

Bow-tie-shaped bundles of silica rods are formed(fig. 3) if the aqueous straight rod sample of type 1 ismixed with a methanol/ammonia mixture. This mor-phology is remarkable because as far as we know, nosimilar morphology has been reported for any othervirus or organic template in the past. The bundlesall have similar dimensions. The maximum lengthalong the y axis of the well-defined bundles is about2000 nm, comparable to the total length of two in-tact fd viruses joined with each other head-to-tail (seethe cartoon in fig. 3). The formation of the bow-tie-shaped bundles seems to originate from an aggrega-tion of (silica coated) fd viruses (and granular silica)after addition of the methanol/ammonia mixture, butthe exact reason for the shape and size of the bun-dles remains unclear.

We demonstrated the capability of fd viruses to beused as a template for the formation of 1D silicananomaterials. Three nanostructures with distinctmorphologies have been observed under differentsol-gel conditions using TEOS as silica precursor:silica rods, wires and bow-tie-shaped bundles. Sil-ica wires seem to transcript the bending conforma-tion and length of intact semi-flexible fd, but undersomewhat different reaction conditions also remark-ably straight silica rods are formed that have a highuniformity in terms of the thickness and homogene-ity of the silica layer. Work devoted to further under-standing the results obtained in this paper and ex-ploring the above problems is ongoing.

[1] Z. Zhang, J. Buitenhuis, Small 2007, 3, 424.

[2] Y. N. Xia, P. D. Yang, Y. G. Sun, Y. Y. Wu, B. Mayers,B. Gates, Y. D. Yin, F. Kim, Y. Q. Yan, in AdvancedMaterials, 2003, 15, 353.

[3] G. R. Patzke, F. Krumeich, R. Nesper, Angew. Chem.-Int. Edit. 2002, 41, 2446.

[4] C. B. Mao, D. J. Solis, B. D. Reiss, S. T. Kottmann, R.Y. Sweeney, A. Hayhurst, G. Georgiou, B. Iverson, A.M. Belcher, Science 2004, 303, 213.

Self-diffusion of Rod-like Viruses ThroughSmectic Layers.M. P. Lettinga 1, E. Grelet 2

1 IFF-7: Soft Condensed Matter2 Centre de Recherche Paul Pascal, CNRS-Université Bordeaux 1, 115 Avenue Schweitzer, 33600 Pessac, France

We report the direct visualization at the scaleof single particles of mass transport betweensmectic layers, also called permeation, in a sus-pension of rod-like viruses. Self-diffusion takesplace preferentially in the direction normal to thesmectic layers, and occurs by quasi-quantizedsteps of one rod length. The diffusion rate corre-sponds with the rate calculated from the diffusionin the nematic state with a lamellar periodic or-dering potential that is obtained experimentally.

Since the pioneering work of Onsager on the entropydriven phase transition to a liquid crystalline state[1], the structure and the phase behavior of complexfluids containing anisotropic particles with hard coreinteractions has been a subject of considerable in-terest. Understanding of the particle mobility in thedifferent liquid crystalline phases is more recent. Inexperiments various methods have been applied toobtain the ensemble averaged self-diffusion coeffi-cients. Only a few studies have been done wheredynamical phenomena are probed at the scale of asingle anisotropic particle. The self-diffusion in a ne-matic phase formed by rod-like viruses [2] representa recent examples, where the diffusion parallel (D‖)and perpendicular (D⊥) to the average rod orienta-tion (the director) has been measured. Knowledgeof the dynamics at the single particle level is funda-mental for understanding the physics of mesophaseswith spatial order like the smectic (lamellar) phase ofrod-like particles. In this mesophase the particle den-sity is periodic in one dimension parallel to the longaxis of the rods, while the interparticle correlationsperpendicular to this axis are short-ranged (fluid-like order). For parallel diffusion to take place, therods need to jump between adjacent smectic layers,overcoming an energy barrier known as the smec-tic order parameter. This process of interlayer diffu-sion, or permeation, was first predicted by Helfrich[3], but has never been verified experimentally forlyotropic systems. Here we use video fluorescencemicroscopy to monitor the dynamics of individual la-beled colloidal rods in the background of a smecticmesophase formed by identical but unlabeled rods.In this way we have directly observed permeation ofsingle rods in adjacent layers.The system of rods used in this work consists of fil-amentous bacteriophages fd, which are semi-rigidpolyelectrolytes with a contour length of 0.88 µm,

FIG. 1: (a) Overlay of DIC and fluorescence images,showing smectic and two fluorescently labeled parti-cles. The cartoon shows the jump of rod-like particlebetween adjacent smectic layers. The layer spacing isL ' 0.9 µm. (b) Displacement of a given particlein the direction parallel (red line) and perpendicular(black line) to the normal of the smectic layers. Thegreen lines indicate the time for which one particle staysin a given layer.

a diameter of 6.6 nm, and a persistence length of2.2 µm. Suspensions of fd rods in aqueous solu-tion (20 mM Tris, pH 8.2) form several lyotropic liq-uid crystalline phases, in particular the chiral nematic(cholesteric) phase and the smectic phase. The ex-istence of a smectic phase in suspensions of hardrods is an evidence of the high monodispersity andtherefore of the model system character of such fila-mentous viruses. The colloidal scale of the fd bacte-riophage facilitates the imaging of individual rods byfluorescence microscopy, as well as smectic layersby differential interference contrast (DIC) microscopy.Fig. 1(a) shows a sequence of images of a singleregion where both techniques are combined. A com-parison of the images shows that some rods jump be-tween two layers while others remain within a givenlayer. The trajectory of one of the rods is plotted inFig. 1(b) in the direction parallel (z) and perpendic-ular (x) to the director. This figure summarizes thekey observation: the diffusion throughout the smecticlayers takes place in quasi-quantized steps of one rod

FIG. 2: The mean ordering potential in the z-directionobtained by applying the Boltzmann on the probabilitydensity function of the center of mass with respect tothe average position within the layer.

FIG. 3: (a) Evolution of the self Van Hove function atdifferent times. The functions are normalised to one,the z-axis is scaled by the smectic layer thickness L.

length. Moreover, it shows that the diffusion withinthe smectic player is extremely slow. The “hopping-type” diffusion is the consequence of the underly-ing ordering potential imposed by the smectic layers,which can be determined experimentally from the dis-tribution of particle positions with respect to the mid-dle of a layer parallel to the director. The smectic or-dering potential is then deduced from the Boltzmannfactor for the probability of finding a particle at po-sition z, as shown in Fig. 2. The potential can bebest fitted with a sinusoidal, giving an amplitude ofU0 = 1.36 kBT .The use of such a potential is verycommon due to its simplicity [4], but this orderingpotential nor its height had never been directly ob-served.

The ordering strongly influences the diffusion of theparticles. This is exemplified by the self-van Hovefunction G(z, t), which is the probability for a dis-placement particle to a position z after a time t. Sincesingle particles are experimentally identified, G(z, t)can be directly obtained from the histogram of par-ticle positions after a time t, as plotted in Fig. 3.A smooth gaussian distribution that smears out overtime is expected for G(z, t) for a fluid made of Brown-ian particles. In contrast,G(z, t) shows distinct peaksexactly at integer multiples of the particle length (andtherefore of the layer thickness, see Fig. 3(a)). Thisimplies that the permeation is a function of positionz, due to the ordering potential. The effect of the or-dering potential is also obvious with respect to theoverall mean square displacement (MSD). The timeevolution of the MSD given by 〈∆r2(t)〉 ∼ tγ pro-vides the diffusion exponent γ: γ < 1 is character-

istic of a sub-diffusive behavior, while γ > 1 is re-ferred to as super-diffusion. The parallel motion isclose to be diffusive (γ ' 1) in the nematic phase(γ = 0.95) and reduces in the smectic phase toγ = 0.81, i.e. where the discrete peaks in the self-van Hove function are observed. The perpendicu-lar motion is in all cases strongly subdiffusive: afterthe nematic-smectic (N-Sm) transition it reduces from0.68 to 0.46. This anomalous subdiffusive behaviorsuggests that a “cage escape” mechanism is at handfor both parallel and perpendicular diffusion. For par-allel diffusion the cage is formed by the energy barrierimposed by the smectic layers. Perpendicular diffu-sion at high volume fractions is only possible througha subdiffusive reptationlike motion along the long axisto escape the local excluded volume and thus alsohindered by the ordering potential. The anisotropy inthe total diffusion, D‖/D⊥, which is about 20 in thenematic phase [2], increases in the smectic phase.Therefore the diffusion in the smectic phase can beeffectively considered as a one-dimensional diffusionof a Brownian particle in a periodic potential in thehigh friction limit. The diffusion coefficient in thesmectic phase can then be calculated taking D0 asthe diffusion coefficient in the nematic phase closeto the N-Sm transition. Thus the diffusion coefficientis predicted to decrease by a factor of 0.44. Indeed,using this factor the MSD in the smectic phase is ob-tained from the MSD in the nematic phase.

In conclusion, we have for the first time visualizedthe process of permeation in the smectic phase atthe scale of single particles for a system of chargedrods. This allowed us to give a full and coherentdescription of the diffusion process without any as-sumptions on the system. The diffusion is stronglyanisotropic in the direction normal to the smectic lay-ers and quasi-discontinuous due to the presence ofthe layers. The diffusion rate complies with the ratein the nematic phase, taking into account the or-dering potential, which is obtained directly from ourmeasurements. Since the diffusion within the layeris glasslike, we conclude that the smectic phase ofsemi-flexible hard rods consists of layers of glassyrods rather than fluid layers. Thus permeation can bedescribed in terms of Brownian particles diffusing ina one-dimensional periodic potential.

[1] L. Onsager, Ann. N.Y. Acad. Sci. 1949, 51, 627.

[2] M. P. Lettinga et al., Europhys. Lett., 2005, 71, 692.

[3] W. Helfrich, Phys. Rev. Lett., 1969, 23, 372.

[4] B. Mulder, Phys. Rev. A, 1987, 35, 3059.

[5] M.P. Lettinga and E. Grelet, Phys. Rev. Lett., 2007,99, 197802.

Electric Phase/ State Diagrams of Charged Fibrous Viruses (fd) K. Kang and Jan K. G. Dhont IFF-7: Soft Condensed Matter We explore phase/state transitions in suspensions of charged fibrous viruses (fd) at low ionic strengths, induced by external electric fields at low frequencies where double layers are polarized. On the basis of the different observed optical morphologies, phase/state diagrams are constructed in the applied field amplitude versus frequency plane. The various phases/states and the characterization of transition lines have been investigated by pitch measurements, video correlation spectroscopy, electric birefringence and small angle dynamic light scattering.

The phase/state transitions that are considered here are the result of interactions between external field-induced double layers. The phase/state diagram in the field-amplitude versus frequency plane is given in Fig.1 (see also Ref.[1]). Without an applied electric field, the equilibrium state is a nematic in coexistence with an isotropic phase. In existing literature, the nematic phase of fd-virus suspensions is found-to be chiral-nematic (in the absence of an electric field). This is the result of the chiral structure of the core of fd-virus particles. In the current study, a much lower TRIS/HCL buffer concentration is used, where the relatively large Debye length screens the chiral nature of the core of fd-particles to an extent that renders the nematic phase non-chiral. At these low buffer concentrations, the ionic strength is significantly affected by carbon dioxide that dissolves from the air (this is discussed in detail in Ref.[2]).

At low applied field amplitudes (smaller than about 1.0 V/mm), coexistence between a non-chiral nematic and an isotropic state is (see the first depolarized microscopy image in Fig.1). This phase is referred to as the N-phase. On increasing the field amplitude, a chiral nematic *N -phase is induced, as can be seen from the striped patterns in the second image in Fig.1. There is a gradual transition to the *DN -phase at higher amplitudes at low frequencies, where the N-domains become smaller and disconnected and smaller sizes (see the third image in Fig.1). On further increasing the field amplitude, the chiral texture disappears and the small N-domains melt and reform. In the dynamical sD -state, the dynamics of melting and forming is slow, while the characteristic time for melting and forming is fast (of the order of a

second) in the fD -state (where the subscripts “s” and “f” stand for “slow” and “fast”, respectively). The fourth image in Fig.1 is a snap shot of a dynamic state.

At high frequencies, larger than a few kHz, the depolarized image is uniform. Birefringence measurement shown that in this H-phase the rods are “homeotropically” aligned [3].

100 101 102 1030

[V/mm]

ν [Hz]

[fd]=2.0 mg/ml

100 101 102 1030

[V/mm]

ν [Hz]

[fd]=2.0 mg/ml

FIG. 1: The electric phase/state diagram of fd-virus with a concentration of 2.0 mg/ml. Several phases/states are induced at frequencies below a few kHz, while a uniform aligned phase is observed at higher frequencies..

An electric cell is used to characterize several phases and states by various optical techniques: Electric birefringence is used to detect alignment and the degree of orientational order, especially in the H-phase. These measurements reveal that the rods are aligned along the electric field, with a degree of orientational order that is essentially independent on the field amplitude and frequency. [3].

The optical-pitch variation is measured from optical images up to the *N - *DN -transition line on variation of the field amplitude. For low amplitudes, the measured chiral π -pitch shows a quite large spread, which decreases with increasing amplitude to a saturation value of about 12 mμ . Since the 2D projection of the optical pitch is measured, the largest measured optical π -pitch is the true pitch. The large spread near the *N -

*DN -transition line indicates that the director attains arbitrary values, while at higher field amplitudes the director tends to be perpendicular to the external field. The pitch is observed to diverge at the *N - *DN -transition line.

On further increasing a field amplitude at low frequency, dynamical states appear: The dynamics of melting and forming of N-domains in the sD - and fD -states is characterized by means

of video correlation functions VC , defined from the transmitted light intensity time traces as recorded by a CCD camera (a snap shot is given in the fourth image in Fig.1),

[ ][ ][ ]2

( ) ( ) (0) (0)( )

(0) (0)V

I t I t I IC t

− < > − < >=

− < >

Here, I is the transmitted intensity at a given pixel and the brackets <…> denote averaging over all pixels. The left plot in Fig.2 shows a few correlation functions together with fits to a single-stretched exponential. The plot on the right in Fig.2 shows the time constant for melting and forming as a function of the field amplitude. As can be seen, the time constant diverges on approach of the *DN -to- sD transition line. Within the fD -state the time constant is essentially independent of the field amplitude.

Interestingly, the microscopic dynamics as probed with small angle dynamic light scattering is discontinuous at the *DN -to- sD transition line This is probably due to the fundamentally different microstructural ordering in the *DN -phase and

the sD -state, resulting in different mobilities of the fd-viruses

At the point in the phase-state diagram in Fig.1 where several transition lines meet, not only the time constant for melting and forming of N domains diverges, but also the domain size

0 10 20 300.0

time [s]

3 4 5 6 70

τ [ s ]

E0 [ v/mm ]

FIG. 2: Left: Video correlation functions VC for four field amplitude at a fixed low frequency (the numbers are in V/mm). The solid lines are fits to a single stretched exponent. Right: The characteristic time constant of melting and forming of small N-domains as a function of the applied field amplitude.

diverges. There thus seems to be an analogous “critical behaviour” as in equilibrium systems.

Inter-colloidal interactions of fd-virus particles through polarized, thick electric double-layers are probably responsible for the observed phases and states. So far there is no theory that could explain the observed phase/state behaviour. It is even an open question whether polarization of condensed ions may play a role (about 85 % of the bare charge on fd-virus is screened by condensed ions).

The phase/state diagram in Fig.1 is not corrected for electrode polarization. Electrode polarization arises from the formation of double layers at the electrodes that partly screen the applied electric field. In Ref.[3] we derived an expression for the attenuation factor (the ratio of the bulk-value of the electric field and the applied field amplitude). This expression shows that electrode polarization can be neglected for our electrical cell for frequencies above 100 Hz. The corrected phase/state diagrams can be obtained from this electrode-polarization theory [3].

[1] K. Kang, J.K.G. Dhont, Eur. Phys. Lett. 84, 14005, 2008

[2] K. Kang, A. Wilk, A. Patkowski, J.K.G. Dhont, J. Chem. Phys. 126, 214501, 2007

[3] K. Kang, J.K.G. Dhont, Submitted to Phys. Rev. E. 2008

Thermal diffusion behavior of hardsphere suspensionsH. Ning, J. Buitenhuis, S. WiegandIFF-7: Soft Condensed Matter

The molecular origin of the thermal diffusion pro-cess or Ludwig-Soret effect is one of the un-solved problems. It relates to our poor under-standing of non-equilibrium statistical mechan-ics pointing out our incapability of obtaining, insome cases, even qualitative predictions, whichare of practical importance in separation pro-cesses (Thermal field flow fractionation of poly-mers and colloids, isotope separation), charac-terization of geochemical processes (Salton Seageotherm, oil reservoir composition) and com-bustion. Recent advancements open up allur-ing perspectives to exploit thermophoresis as anovel tool in microfluidic manipulation, and se-lective tuning of colloidal structures. One of ourstrategies to tackle this problem is the investiga-tion of a spherical colloidal model system with ashort range repulsive interaction potential. Westudied a colloidal dispersion in the intermediateconcentration range (volume fraction φ < 10 %)and found that the interactive part of the Soretcoefficient agrees with an analytical theory. Athigher concentration the Soret coefficient followsa power law. The temperature dependence of theSoret coefficient is mainly determined by singleparticle contributions and agrees to some extentwith the temperature dependence of the surfacecoating material, octadecane, in toluene.

Colloidal particles are small enough to exhibit ther-mal motion commonly referred to as Brownion mo-tion. Being just very large molecules in a solvent,colloidal particles show many physical phenomenathat are also found in ordinary molecular systems.Consequently, colloids have been used frequently tostudy fundamental questions in physics. Therefore, itis expected that they are also a suitable model sys-tem to illuminate the microscopic mechanism under-lying the Ludwig-Soret effect, which was discoveredalready 150 years ago. This effect, also known asthermal diffusion, describes the diffusive mass trans-port induced by a temperature gradient in a multi-component system. The scenario for a binary mix-ture of particles is sketched in figure 1.

A number of recent studies show that interactionsplay an important role for the thermal diffusion behav-ior, where long ranged repulsion between chargedmicelles and colloids has been considered [1].

Conceptually, thermal diffusive behavior of highly di-luted and concentrated solutions can be differenti-ated. In dilute solutions, where colloid-colloid inter-actions can be neglected, the thermal diffusion co-efficient of the colloids is determined by the natureof the interactions between single colloidal particlesand solvent molecules (and possibly other soluteslike ions that form a double layer around the colloids)[2]. Structural changes of the surrounding solvationlayer due to temperature changes and/or changes ofthe solvent composition may induce a sign changeof the thermal diffusive behavior of single colloidalparticles. Usually, the mechanism leading to a signchange is system dependent. Although, for severalaqueous mixtures with and without solutes such aspolymers and colloids, we found the sign change con-centration is almost system independent and stronglycorrelated with the breakdown of the hydrogen-bondnetwork [3]. Also the temperature dependence ofST for a large class of macromolecules and colloidsin water shows a distinctive universal characteristic[1]. A pronounced concentration dependence of theSoret coefficient has been found in experiments [4, 1]and is predicted by theory [5].

FIG. 1: Schematic illustration of the thermal diusion pro-cess in a binary mixture in a temperature gradient. Thesmall and big particles accumulate at the hot and cold side,respectively.

In recent years, modern optical techniques havebeen developed which allow the investigation of com-plex fluids with slow dynamics such as polymer solu-tions and blends, micellar solutions, colloidal disper-

sions and bio-molecules. The main issues of interestwere the derivation of scaling laws and to understandthe sign change of the Soret coefficient for macro-molecular and colloidal systems on the basis of ex-isting theories for molecular fluids.

In the past few years several theoretical conceptshave been proposed to understand single particleand colloid-colloid interaction contributions to thethermophoretic motion of colloidal particles [1]. Whilethe majority of the theoretical approaches give ex-pressions for the single particle contribution, the workby Dhont gives explicit expressions for the contribu-tion of colloid-colloid interactions to the thermal diffu-sion coefficient DT. These interaction contributionslead to a concentration dependence of the thermaldiffusion coefficient. According to this theory, a signchange of the Soret coefficient as a function of tem-perature and concentration is possible for appropriateinteraction parameters.

FIG. 2: Concentration dependence of the Soret coecientat dierent temperatures. The solid line represents a t ofthe data according to the theory by Dhont [5].

We studied spherical silica particles, sterically sta-bilized by octadecane, with a radius of a = 27 nmdispersed in toluene. The thermal diffusion behav-ior was studied in a concentration range between 1%and 30% by volume fraction and in a temperaturerange from 15-50 C [6]. Fig. 2 shows the Soret coef-ficient as function of the volume fraction φ for differenttemperatures. At low temperature the colloids moveto the warm side, while at high temperatures the col-loids move to the cold side. For intermediate temper-atures a sign change of the Soret coefficient occursfor higher concentration. Typically, the movement ofthe solute particles to the warm side is an indicationfor poor solvent conditions, while under good solventconditions the particles move to the cold side. Forthe investigated system the vicinty of a gel line at lowtemperatures is probably responsible for movementof the colloids to the warm side. In the intermediateconcentration range the concentration dependenceof the Soret coefficient can be described by the the-ory for hard spheres [5]. At high concentrations ST

follows a power law as it also has been found for poly-mers approaching the glass-transition [7]. Althoughthe exponent is two orders of magnitude smaller.

FIG. 3: The measured Soret coecient (•) versus temper-ature for a colloidal suspension with a volume fraction ofφ=10%. According to the theoretical approach by Dhontthe single (

⊙) and the collective part (

⊕) of ST can be

separated. The dashed lines are guides to the eye. The insetshows ST of octadecane in toluene versus temperature fora octadecane concentration of w=5 wt%.

A separation of the Soret coefficient into a singleparticle contribution and an interactive part, shows,that the interactive part is almost temperature inde-pendent, while the single part shows a strong tem-perature dependence, which has the same tendencyas the Soret coefficient of octadecane in toluene (cf.Fig. 3). But for the low molecular weight system nosign change occurs in the investigated temperaturerange. This might be an indication that also the sil-ica core of the particles influences the thermal dif-fusion behavior. Also the fact that octadecane isbound to a surface might influence the thermal dif-fusion behavior. In general one can expect, that ifthe particle coating changes from " organophilic" to"organophobic" a sign change could be expected, butunder which conditions this is the case needs to beclarified in further investigations.

[1] R. Piazza. A. Parola, J. Phys-Cond. Matt 20, 153102(2008).

[2] J.K.G. Dhont, Eur. Phys. J. E 25, 61 (2008).

[3] S. Wiegand, H. Ning, and R. Kita, J. Non-Equilib.Thermodyn., 32, 193 (2007).

[4] H. Ning, R. Kita, H. Kriegs, J. Luettmer-Strathmann,and S. Wiegand, J. Phys. Chem. B. 110, 10746(2006).

[5] J. K. G. Dhont, J. Chem. Phys. 120, 1632 (2004) and1642 (2004).

[6] H. Ning, J. Buitenhuis, J. K. G. Dhont, and S. Wie-gand, J. Chem. Phys. 125, 204911 (2006).

[7] J. Rauch and W. Köhler, Macromolecules 38, 3571(2005).

ISB-1: Cellular Biophysics

Acting Director: Prof. Frank Müller

The Institute of Structural Biology and Biophysics, Cellular Biophysics ISB-1 (formerly INB-1 or IBI-1) is dedicated to research in molecular and cellular neurobiology, signal transduction, information processing, and cell bio-physics.

The cell is the smallest living unit. In every cell, chemical and physical stimuli evoke characteristic physiological responses. Signal transduction usually begins with receptor proteins that register a stimulus and start a cascade of biochemical reactions which often ends with the opening or closure of ion channels that changes the electrical properties of the cell membrane. Moreover, in order to adapt to changes in the environment each cell must constantly modulate these signalling processes. In ISB-1, signal transduction and information processing are studied at different levels of complexity: on the molecular level we study the properties of receptor proteins and ion channels. On the cellular level, G-Protein-mediated signalling as well as mechanisms of excitation and adaptation of sensory cells and neurons are investigated. On the network level, we study information processing in small well-characterized neuronal circuits, such as the retina or the olfactory bulb.

A strength of the ISB-1 has always been the tight co-operation of biologists, chemists, and physicists. Our technical repertoire reaches from the analysis of proteins using molecular biological and biochemical methods to physiological studies in vitro and in vivo in normal and in transgenic animals. Fluorescence-based optical methods and imaging play an important role in the research of ISB-1. To monitor the concentration of intracellular messengers like calcium or cAMP, imaging techniques have been estab-lished, including two-photon fluorescence-lifetime imaging microscopy, single molecule fluorescence spectroscopy, and camera-based ratiometric imaging setups. Both synthetic fluorescent dyes as well as genetically encoded sensors based on fluorescent proteins are employed in the institute. The analysis of cellular processes with high spatial and temporal resolution provides the basis for an in-depth understanding of the “physics of the cell”.

The Role of HCN4 channels in Cardiac Pacemaking D. Harzheim1, R. Seifert1, K.H. Pfeiffer1, L. Fabritz2, E. Kremmer3, T. Buch4, A. Waisman5, P. Kirchhof2, U. B. Kaupp1 1ISB-1: Cellular Biophysics; 2Med. Klin. & Poliklinik,Univ. Münster und IZKF Münster, D-48129 Münster 3Helmholtz Zentrum München, D-81377 München; 4Exp. Immunologie, Univ. Zürich, CH-8057 Zürich 5I. Med. und Poliklinik, Univ. Mainz, D-55131 Mainz Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated ion channels. The depolarizing current flowing through HCN channels, designated as Ih or If, plays an important role in controlling cardiac rhythmicity. Moreover, HCN channels are important targets for adrenergic stimulation. Upon activation of adrenergic receptors, cAMP levels increase, leading to a higher open probability of HCN4 channels. We studied the role of If in mice, in which binding of cAMP to HCN4 channels was abolished by a single amino-acid exchange (R669Q) [1]. Homozygous HCN4R669Q/R669Q mice die during embryonic development. Prior to E12, homozygous and heterozygous embryos display reduced heart rates and show no or attenuated responses to catecholaminergic stimulation. Adult heterozygous mice display normal heart rates at rest and during exercise. However, following β-adrenergic stimulation, hearts exhibit pauses and sino-atrial node block. These results suggest that in the embryo, HCN4 is the principal cardiac pacemaker and persistent elevation of the heart rate by cAMP is essential for viability. In adult mice, HCN4 channels take no longer part in heart rate regulation, but prevent sinus pauses during and after stress. Thus, the mechanism of pacemaking may switch during development and HCN4 may serve two different functions that critically rely on the presence of cAMP.

Spontaneous activity of the mammalian heart is generated in the sino-atrial node (SAN). The question how pacemaker activity is generated in SAN cells and how pacemaking is regulated by the autonomouos nervous system is still a matter of debate. Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are thought to play a major role in cardiac pacemaking. Within the superfamily of voltage-gated channels, HCN channels are unique in that they are activated upon hyperpolarization rather than by depolarization of the membrane potential. In addition, voltage-dependent opening of these channels is regulated by the direct binding of cAMP to the polypeptide. Native HCN channels consist of four subunits. In mammals four genes (HCN 1 – 4) have been identified to encode individual subunits. The If current, mediated by HCN channels, is activated at

negative membrane potentials. If leads to excitation of the cell and spontaneous firing of action potentials and is, therefore, thought to be one of the primary ionic mechanisms generating cardiac spontaneous rhythmic activity.

In this study, we decided to investigate the precise role of HCN4 in cardiac pacemaking of mice. We generated knock-in mice that harbour a single amino-acid exchange (R669Q) in the cyclic nucleotide-binding domain of the HCN4 channel that abolishes cAMP binding to the protein. The arginine residue is crucial for the binding of cAMP, because it interacts with the negatively charged phosphate group. To ascertain that the mutation abolishes regulation by cAMP, we analyzed the wild-type and the HCN4R669Q mutant after heterologous expression in a cell line. The voltage-dependent activation of HCN4R669Q mutant and wild-type channels is similar. Cyclic AMP shifted the activation curve of the wild-type HCN4 channel by +25 mV, whereas no shift was observed for the HCN4R669Q mutant (Fig. 1).

Fig. 1: Electrophysiological characterization of HCN4 and HCN4R669Q channels. Voltage-dependent activation of heterologously expressed HCN4 (circles) and HCN4R669Q channels (squares). Voltages of half-maximal activation (± SD) in the absence (open symbols) and presence (filled symbols) of cAMP (100 µM) were -79.3 ± 3.7 (n = 8) and -55.6 ± 2.7 mV (n = 3) for HCN4, and -84.5 ± 3.4 (n = 7) and -81.3 ± 7.1 mV (n = 5) for HCN4R669Q, respectively.

We analyzed the spontaneous beat frequency of hearts isolated from HCN4+/+, HCN4+/R669Q

HCN4R669Q/R669Q embryos prior to E11.5. Under basal conditions, hearts from heterozygous and homozygous embryos beat regularly without obvious arrhythmias; however, the heart rate was significantly slower compared to hearts from wild-type embryos (Fig. 2). Furthermore, in wild-type embryos, the heart rate increased during E9 to E11 of embryonic development (Fig. 2). This increase was no longer present in HCN4+/R669Q embryos whereas in HCN4R669Q/R669Q embryos, the heart rate is significantly reduced (Fig. 2). Notably, the beat frequency of hearts from HCN4R669Q/R669Q and HCN4-/- embryos at E9.5 was identical, demonstrating that the point mutation in the HCN4 channel has the same effect on the basal heart rate as the complete loss of the channel [2].

Fig. 2: The HCN4R669Q channel alters the embryonic heart beat. Basal heart rate during embryonic development (E9.5-11, HCN4+/+: red, HCN4+/R669Q: green, HCN4R669Q/R669Q: blue). The number of embryos analyzed is indicated.

Isoproterenol superfusion increased the rate of isolated hearts from HCN4+/+ and HCN4+/R669Q

embryos (~ 37.4 % and 19.8 %, respectively), whereas no increase was observed in hearts from HCN4R669Q/R669Q embryos (2.8 %). Similarly, superfusion with NKH477, an activator of membrane-bound adenylyl cyclases, increased the heart rate from HCN4+/+ and HCN4+/R669Q embryos, but not from HCN4R669Q/R669Q embryos. These results demonstrate that HCN4 is the principal target for cAMP during the embryonic stages of mouse development.

The differences in basal heart rate and in the activation of If between wild-type and heterozygous embryos prompted us to study the heart rate in freely moving adult HCN4+/+ and HCN4+/R669Q mice by telemetric recording of ECG. Neither the heart rate at rest nor during exercise or mental stress was different between genotypes. The heart rate of sedated animals and the intrinsic rate of the isolated heart was also not different between genotypes, nor was the electrophysiology of the atrio-ventricular node and the ventricle. After exercise, HCN4+/R669Q mice developed pauses and sino-atrial block more often than their wild-type littermates (Fig. 3, HCN4+/R669Q: 9 ± 7 pauses per 55 minutes, HCN4+/+: 6.5 ± 5 pauses per 55 minutes). Sino-atrial block and sinus pauses were also found in spontaneously beating, Langendorff-

perfused hearts, mostly during washout of the β-adrenoceptor agonist orciprenaline (Fig. 3; pauses in 4/10 HCN4+/R669Q hearts vs. 1/9 HCN4+/+ hearts; during orciprenaline infusion: pauses in 5/10 HCN4+/R669Q vs. 1/9 HCN4+/+ hearts; during washout of orciprenaline: pauses in 8/10 HCN4+/R669Q vs. 0/9 HCN4+/+ hearts).

Fig. 3: HCN+/R669Q mice display a sino-atrial block. a, Representative examples of telemetric ECG recordings in freely moving mice carrying an implanted ECG transmitter. All recordings were obtained during the 55 minute recovery period after stress tests (air jets or swimming). Animal numbers are indicated. b, Representative recording of spontaneous rhythm in isolated, Langendorff-perfused hearts. Shown is a ventricular monophasic action potential (MAP), a right atrial electrogram (RA), and lead II of the tissue bath ECG (ECG). The WT heart shows a constant sinus rhythm, the HCN4+/R669Q heart shows three pauses with doubling of cycle length, consistent with sino-atrial block.

Our results show that only in embryonic mice, HCN4 channels serve as pacemaker controlling the heart beating frequency. Notably, this activity is severely affected when cAMP modulation of the channel is impaired. In adult mice, however, the contribution of HCN4 channels is less pronounced. Here, the channels may serve a back-up mechanism that maintains a stable heart beat in situations during and after stress and, HCN4 channels are probably no longer involved in sympathetic stimulation of the heart rate.

[1] D. Harzheim, K.H. Pfeiffer, L. Fabritz, E. Kremmer, T. Buch, A. Waisman, P.Kirchhof, U.B. Kaupp and R. Seifert, EMBO J. 27, 692 (2008).

[2] J. Stieber, S. Herrmann, S. Feil, J. Löster, R. Feil, M. Biel, F. Hofmann and A. Ludwig, Proc. Natl. Acad. Sci. USA 100, 15235 (2003)

Light Responses in the Mouse Retina are Prolonged Upon Targeted Deletion of the HCN1 Channel Gene F. Müller1, G.Knop1, M.W. Seeliger2, F. Thiel1, A. Mataruga1, U.B. Kaupp1, C. Friedburg3, N. Tanimoto2 1ISB-1: Cellular Biophysics 2Inst. Ophthalmic Res. Univ. Tübingen, D-72076 Tübingen 3Dept. Ophthalmology, Univ. Giessen and Marburg, D-35390 Giessen In the mammalian retina, the light response of photoreceptors is processed by an elaborate neuronal network. The photoreceptors, rods and cones, are depolarized in the dark, but hyperpolarize upon illumination. At least ten types of cone bipolar cells and one type of rod bipolar cell provide the pathways for the signal flow from photoreceptors to ganglion cells, the output neurons of the retina. Along the way from photoreceptors to ganglion cells, cellular signals are shaped by two principal mechanisms: 1. Lateral inhibition within the neuronal network involving feedforward and feedback mechanisms and 2. Shaping of voltage responses by the kinetic properties of the ion channels involved in the generation and propagation of electrical signals. One family of voltage-gated channels present in the retina are the HCN channels, which are activated by hyperpolarization and gated by cyclic nucleotides. In mammals, four channel genes (HCN1-4) have been identified. Here, we show that HCN1 is particularly strong expressed in both rod and cone photo-receptors. By recording scotopic and photopic light responses in normal mice and in HCN1 knock-out mice, we show that HCN1 channels shorten retinal light responses in both rod and cone pathways [1]. HCN channels co-determine the resting potential and membrane conductance and, thereby, play an important role in the integrative behaviour of neurons and the sensitivity to synaptic input. HCN channels affect the cable properties of dendrites and shape the time course and propagation of excitatory and inhibitory postsynaptic potentials. We have previously shown that HCN channel isoforms are differentially expressed in the retina [2, 3]. In rod and cone photoreceptors, HCN channels were suggested to shape the light response. HCN channels become activated during hyperpolarization in bright light and depolarize the cell toward the dark membrane potential, making the light response transient.

HCN1 is strongly expressed in the mouse retina

HCN1 is expressed throughout the retina. In the outer retina, rod photoreceptors are strongly HCN1-immunoreactive. Cone photoreceptors that can be labelled with antibodies against the calcium-binding protein CabP5 are also positive for HCN1.

Fig. 1: Immunohistochemical localization of HCN1 in the mouse retina. (A) HCN1-specific antibody labels photoreceptor inner segments, somata, axons and axon terminals in the outer retina. Somata of certain bipolar cells (long arrow) and amacrine cells (short arrow) are labelled in the INL, corresponding bands of bipolar cell axon terminals (long horizontal arrow) and amacrine cell dendrites (arrowhead) are visible in the IPL. Some ganglion cell somata are also HCN1-positive in the GCL. (B) No HCN1 immunoreactivity is observed in retinal sections obtained from HCN1 knock-out animals. (C) CabP5-immunoreactivity is detected in a subset of cone photoreceptors. (D) Double staining against CabP5 (left) and HCN1 (right) shows that HCN1 is present in the plasma membrane of cones (arrow). OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Light responses are prolonged in HCN1 knock-out mice

The murine Ganzfeld ERG is a measure of the overall retinal function. We performed single-flash ERG recordings under scotopic and photopic adaptation conditions and with different stimulus intensities. Wild-type and HCN1 knock-out animals were littermates (six weeks of age). Representative records of a wild-type mouse are

shown in the left columns of Fig. 2A (scotopic) and B (photopic). Typically, ERG waveforms consist of an early negative-going component best visible at high light intensities, the a-wave, followed by a large positive-going component, the b-wave. Whereas at least the initial portion of the a-wave reflects the primary light response in photoreceptors, the b-wave is dominated by the activity of ON-bipolar cells. Riding on top of the b-wave are the oscillatory potentials, small wavelets that probably involve inner retinal circuitry. In the scotopic regime, the knock-out of HCN1 channels had no substantial effect on the onset of the ERG response or the amplitudes of the a- and b-waves (Fig. 2A, C). In the statistical analysis (Fig. 2D, n= 4 for both wild-type and HCN1 knock-out animals), the scotopic b-wave amplitude data recorded from HCN1 knock-out animals (box plots) fall well within the black lines that limit the 5% to 95% normal range of the wild-type data. In contrast, the photopic (light-adapted) responses are reduced and their oscillatory components also diminished (Fig. 2B,C). The most prominent effect of HCN1 knock-out concerned the duration of light responses. Under both scotopic and photopic conditions, the b-wave was considerably prolonged in HCN1 knock-out animals at higher light intensities.

Fig. 2: Effect of HCN1-deficiency on retinal function under scotopic and photopic conditions. Direct comparison of flash ERG recordings in dark-adapted (A) and light-adapted (B) wild-type (Wt) and knock-out mice (HCN1-/-). (C) Superposition of recordings from wild-type and knock-out animals reveals that light responses are considerably prolonged in knock-out mice. (D) Statistical evaluation of the corresponding ERG b-wave data. Boxes indicate 25 % and 75 % quantile range, whiskers the 5 % and 95 % quantiles and the asteriks the median of the HCN1-/- data. The normal range is delimited by black lines indicating the 5 % and 95 % quantile of the wild-type data.

Flicker detection is compromized in HCN1 knock-out mice

As single flash responses are prolonged, it is tempting to speculate that already at the level of the retina the flicker fusion frequency, i.e. the ability to respond to repetitive stimulation, should be reduced. The following figure compares ERG recordings in wildtype and HCN1 knock-out animals to repetitive light stimuli at different frequencies and different light intensities. At low flash intensities, light responses in wildtype and knock-out animals were similar, presumably because the hyperpolarization of the photo-receptors was too small to activate HCN1 channels. Indeed, at 10 mcd*s/m², the flicker data were rather similar. In contrast, at higher flash intensities like 300 mcd*s/m² that caused a manifest prolongation of the responses, there was a remarkable reduction of the ability to follow higher frequency flicker in the knock-out animals.

Fig. 3: Effect of HCN1-deficiency on scotopic ERG flicker fusion frequency. (A) At low flash intensities like 0.01 cd*s/m², there were no substantial differences between wt and knock-out mice. (B) At higher flash intensities like 0.3 cd*s/m², the apparent prolongation of the waveform led to a marked reduction in the ability to follow high frequency stimulation.

Our results show that already in the first cell of our visual system, i.e. the photoreceptor, light responses are significantly modulated.

[1] G.C. Knop, M.W. Seeliger, F. Thiel, A. Mataruga, U.B. Kaupp, C. Friedburg, N. Tanimoto and F. Müller, Eur. J. Neurosci. 28, 2221 (2008)

[2] E. Ivanova and F. Müller, Vis. Neurosci. 23, 143 (2006).

[3] F. Müller, A. Scholten, E. Ivanova, S. Haverkamp, E. Kremmer and U.B. Kaupp, Eur. J. Neurosci. 17, 2084 (2003).

Molecular and Functional Properties of Native and Heterologously Expressed Adenylyl Cyclases A. Baumann1, S. Wachten1, N. Fuss1, R. Gauss1, J. Schlenstedt2, S. Mujagic3, J. Erber3 1ISB-1: Cellular Biophysics 2Institute of Biochemistry and Biology, Univ. Potsdam, D-14476 Golm 3Institute of Ecology, TU Berlin, D-10587 Berlin Cyclic AMP serves as an important intracellular messenger in virtually all organisms. This small organic compound participates e.g. in sensory signal trans-duction, cardiac myocyte regulation, as well as in learning and memory. Production of cAMP is based on the activity of adenylyl cyclases (ACs). A variety of factors can modulate the properties of these enzymes and lead to dynamic changes of the intracellular cAMP concentration. Recently, we cloned the first gene from the honeybee (Apis mellifera) encoding a membrane-bound AC (Amac3) [1]. Molecularly, this gene is orthologous to the mammalian olfactory AC type 3. The enzymatic properties of AmAC3 were determined after heterologous expression of the gene in a cell line. We have now compared the biochemical and pharmacological properties of hetero-logously expressed AmAC3 with native AC-activity in identified subregions of the honeybee brain [2]. Values for half-maximal activation with a water-soluble analogue of forskolin (NKH477) of both, cloned and native ACs, were in the low micromolar range. Biosynthesis of cAMP was specifically blocked by P-site inhibitors. The same rank order of inhibitory potency was shared between AmAC3 and ACs in the antennal lobes of honeybee brain. Our results suggest a role for AmAC3 in sensory, motor and higher-order information processing in the honeybee brain.

Membrane-bound ACs (tmACs) share a common and characteristic topology with two large hydrophobic membrane domains (M1 and M2) within which the amino-acid chain spans the plasma membrane six times. The catalytic domains of the enzyme reside in the cytosolic loop connecting the M1 and M2 domains and in the cytoplasmic C-terminus, following the M2 domain. All tmACs are activated by α subunits of stimulatory G-proteins (Gsα). Most tmACs are also activated by the diterpene forskolin or NKH477 (s.a. [3]). As a result of AC-activity, the concentration of the intracellular messenger cAMP increases. This messenger regulates and modulates the activity of protein kinases, ion

channels as well as transcription factors. Therefore, changes of the cAMP concentration can have a significant impact on cellular signaling behavior.

After cloning the Amac3 gene from honeybee brain, we performed a phylogenetic analysis of the derived amino acid sequence to a variety of vertebrate and invertebrate AC sequences. A dendrogram was constructed (Fig. 1) that showed the closest relationship between AmAC3 and AC sequences from rat olfactory neurons (RnAC3; [4]) and the Drosophila protein (DmAC39E; [5]).

Fig. 1: Phylogenetic comparison of AmAC3 with mammalian, Drosophila, and C. elegans ACs. The amino-acid sequences were obtained from NCBI databases RnAC1 , AC1 from Rattus norvegicus (Rn); MmAC2, AC2 from Mus musculus (Mm); RnAC3; RnAC4; RnAC5; RnAC6; MmAC7; MmAC8; MmAC9; DmACrutabaga; AC1 from Drosophila melanogaster (Dm); DmAC78C; DmAC76E; DmAC35C; DmAC39E); DmAC10B; CeACY1; AC1 from Caenorhabditis elegans (Ce); CeACY2; CeACY3; CeACY4. The sequence of the soluble MmAC10 was used as out-group. The numbers at the nodes represent the per cent bootstrap support for each branching. The scale bar allows the conversion of branch lengths in the dendrogram to genetic distance between clades (0.1 = 10% genetic distance).

In order to determine the enzymatic properties of AmAC3, a cell line was generated (Fig. 2) that constitutively expresses AmAC3 and a cAMP-gated ion channel (CNG). This CNG channel is permeable to Ca2+ ions. Therefore, an increase in AmAC3 activity can be monitored by Ca2+-imaging due to the subsequent activation of the CNG channel. We used this cell line as well as a cell line that only expresses the CNG channel to determine the dose-response curve of AmAC3-activation by forskolin, a specific agonist of tm-ACs (Fig. 3). In addition, the cell lines were used to monitor the G-protein coupled receptor mediated activation of AmAC3. In both cell lines ß-adrenergic receptors are expressed endo-genously. The EC50 values for activation of AmAC3 by forskolin and norepinephrine were 15.2 µM and 3.1 µM (Fig. 3).

Norepinephrine [µM]

0.01 0.1 1 10 100

flpTMflpAC3

Fig. 2: Immunocytochemical detection of heterologous expressed AmAC3. HA-tagged AmAc3 was visualized using a rat anti-HA-antibody and a fluorescently labeled secondary antibody (goat-anti-rat ALEXA 568). (Left) Light-microscopic photograph of a cell. (Right) The same cell viewed under fluorescence-microscopic conditions. The AmAC3 protein is located in the plasma membrane (arrows).

Fig. 3: Effects of AC activation on Ca2+-dependent Fluo-4 signals in AmAC3-expressing (flpAC3) and parental (flpTM) cells. Data are mean values of two independent experiments performed in duplicate. Error bars indicate the standard deviation. Relative fluorescence intensities are given in counts / mg total protein. Above: Fluo-4 signals of flpTM- and flpAC3 cells evoked by AC stimulation with increasing forskolin concentrations (0.1-150 µM). Below: Fluo-4 signals in flpTM- and flpAC3 cells evoked by AC stimulation with increasing norepinephrine concentrations (0.01-30 µM).

The distribution of Amac3 transcripts and protein in honeybee brain was examined by in situ hybridization and immunological staining [1, 2]. Using protein preparations of subregions of the honeybee brain and from the AmAC3-expressing cell line, we analysed the biochemical and pharmacological properties of native and cloned enzymes. Adenylyl cyclases expressed in the antennal lobes of honeybees and AmAC3 share very similar, if not identical properties. These results have been submitted for publication recently [2]. The prominent expression of AmAC3 in the antennal lobes of honeybee brain suggests AmAC3 to participate in the processing of olfactory signals. Therefore, AmAC3 may serve in vivo to translate GPCR- and/or Ca2+-mediated signals into changes of the intracellular cAMP concentration and thus to participate in sensory, motor and even higher brain functions.

15 µm

[1] S. Wachten, J. Schlenstedt, R. Gauss and A. Baumann, J. Neurochem. 96, 1580 (2006).

[2] N. Fuss, S. Mujagic, J. Erber, S. Wachten and A. Baumann, submitted.

Forskolin [µM]

0,1 1 10 100

8x106flpTMflpAC3

[3] Y. Chern, Cell. Signal. 12, 195 (2000).

[4] H.A. Bakalyar and R.R. Reed, Science, 250, 1403 (1990).

[5] V. Iourgenko and L.R. Levin, Biochim. Biophys. Acta, 1495, 125 (2000).

Functional Studies of a Prokaryotic Cyclic Nucleotide-Gated Channel A. Cukkemane, B. Grüter, K. Novak, T. Gensch, W. Bönigk, R. Seifert, U. B. Kaupp ISB-1: Cellular Biophysics Cyclic nucleotide-gated (CNG) channels play a fundamental role in signal transduction of sensory neurons. Upon binding of cyclic nucleotides, CNG channels open and thereby cause changes in membrane potential. Although the physiological role of CNG channels has been extensively studied, the molecular events that relay ligand binding to channel activation are not well understood. The molecular interactions that tune affinity and selectivity of cyclic nucleotide binding are not fully understood, because ligand affinity has not been measured directly but rather has been inferred from electrophysiological studies. As binding and gating are intimately coupled, it is difficult to separate one from the other. Here, we examined the ligand-binding properties of a K+-selective CNG channel that was cloned from the bacterium Mesorhizobium loti (mlCNG) [1]. Ligand binding studies were performed on heterologously expressed and purified tetrameric mlCNG protein as well as on its isolated cyclic nucleotide-binding domain (CNBD). Both, the mlCNG protein and the CNBD bind cAMP in a non-cooperative manner with similar affinity. These results indicate that the binding and gating properties of the bacterial channel are distinctively different from those of mammalian CNG channels.

In order to perform binding studies on purified protein, the mlCNG was expressed as a fusion protein with a hexa-histidine tag at its C-terminal end. This allowed purifying large amounts of the protein using a Co2+ affinity column.

A fluorescent cAMP-analogue, i.e. 8-NBD-cAMP, was used to study ligand binding to mlCNG. The NBD-group is a polarity sensitive molecule. It is largely non-fluorescent in aqueous solution and becomes fluorescent in a hydrophobic environment. This property of the NBD group has been widely utilized in biophysical studies on lipids as well as on proteins, like epithelial exchange factor (EPAC). We observed a pronounced fluorescence signal upon binding of 8-NBD-cAMP to purified mlCNG. This allowed determining quantitatively the binding affinity of the channel for this ligand (Fig. 1A). The data were analyzed assuming a simple binding model. The mean KD value for 8-NBD-cAMP was 15.9 ± 2.7 nM. To determine the KD values of physiological cyclic nucleotides, i.e. cAMP and cGMP, the binding of

8-NBD-cAMP to the mlCNG protein was competed with non-labelled cyclic nucleotides (Fig. 1B). The KD value was 81.6 ± 17.5 nM for cAMP and 320.7 ± 25.5 nM for cGMP.

Next we determined whether the binding properties of the tetrameric channel differed from the isolated CNBD. Previously, the wildtype CNBD and a mutated version (R348A), that has a much lower affinity, have been expressed and purified as GST-fusion proteins to determine their crystal structures either in the ligand-bound or ligand-free (R348A) state [2]. We expressed both variants of the CNBD and purified the proteins on a glutathione affinity column. The presence of a thrombin cleavage site allowed us, to purify the CNBD without its GST fusion partner. We observed that cAMP co-purifies with wildtype CNBD. In contrast, R348A has no cAMP bound. To perform ligand binding experiments with the wildtype CNBD, it was necessary to unfold and refold the CNBD to obtain cAMP-free protein.

Fig. 1: Ligand binding of the full-length mlCNG protein. (A) Normalized increase of fluorescence of 8-NBD-cAMP on binding to mlCNG protein (0.5 µM). 8-NBD-cAMP fluorescence in the absence of the mlCNG protein was subtracted. The solid line represents non-linear least-squares fit. The KD value was 17.3 nM. (B) Competition between 8-NBD-cAMP (0.5 µM) and cAMP (closed circles) or cGMP (open circles) for binding to the mlCNG protein (0.3 µM). The solid lines represent non-linear least-squares fit. The KD values were 71.3 nM (cAMP) and 363.9 nM (cGMP).

Ligand binding to purified CNBDs was studied using the same assay as for the intact channel (Fig. 2). The KD values were 67.8 ± 8.7 nM for cAMP and 300.4 ± 15.4 nM for cGMP for the wildtype CNBD. The KD values obtained for the R348A mutant were 18.5 ± 4.3 µM for cAMP, and 22.3 ± 5.7 µM for cGMP.

Fig. 2: Ligand binding to the CNBD protein. (A) Increase of 8-NBD-cAMP fluorescence on binding to the CNBD protein (0.5 µM). 8-NBD-cAMP fluorescence in the absence of the CNBD protein was subtracted. The solid line represents nonlinear least-squares fit. The KD value was 17.6 nM. Inset: emission spectrum of 8-NBD-cAMP (1 µM) in the absence (black) and presence of CNBD protein (1 µM) (red). (B) Competition between cAMP (closed circles) or cGMP (open circles) and 8-NBD-cAMP (1 µM) for binding to CNBD (1 µM). The KD values were 73.5 nM (cAMP) and 296.9 nM (cGMP). (C) Increase of fluorescence of 8-NBD-cAMP (0.5 µM) on binding to increasing concentrations of the mutant CNBD (R348A). The KD value was 7.3 µM. (D) Competition between cAMP and cGMP with 8-NBD-cAMP for binding to mutant CNBD (R348A) (3 µM). The KD values were 22.9 µM (cAMP) and 27.7 µM (cGMP).

These results show that the binding affinity is very similar for the tetrameric channel and the wildtype CNBD. However, the affinity for cAMP-binding is 4 to 5-fold higher than for cGMP-binding suggesting that physiologically, mlCNG represents a cAMP-gated channel. Comparing our results obtained from ligand-binding experiments with those of channel activation [3] reveal that the KD of cAMP-binding coincides with the K1/2 of channel activation by cAMP. The KD of cGMP binding is 2 fold lower than K1/2 of cGMP-dependent channel activation. These findings strongly suggest that binding and gating in the mlCNG channel is a non-cooperative process. A corollary is that the binding event is not affected by intersubunit contact and that binding sites in the tetrameric channel act independently of each other. In contrast to mlCNG, activation of CNG channels expressed in photoreceptors or olfactory neurons is a cooperative process.

Although we could determine the KD values for cAMP and cGMP binding to the mlCNG channel in quantitative terms, the molecular basis that determines ligand affinity and ligand selectivity of CNG channels still is not well understood. Several residues in the CNBD as well as at sites distant from the CNBD affect cyclic nucleotide binding [4]. We observed a pronounced change of the binding affinity in the mutated CNBD (R348A). The mutation of this arginine residue results in a CNBD that could be purified in cAMP-free form. This

residue is also a crucial determinant of cNMP-binding to the CNBD of the hyperpolarization-activated and cyclic nucleotide-gated (HCN) 2 channel [5]. The arginine residue interacts directly with cAMP. The CNBD in CNG channels is lacking this particular residue. This indicates that the arginine residue may be one of the determinants of ligand selectivity and sensitivity. The KD values derived for R348A are in the micromolar range, i.e. ~ 400 fold lower than for the wild-type CNBD or mlCNG channel. Interestingly, the mutated CNBD has similar binding affinities for cAMP and cGMP. These results clearly indicate that both, ligand selectivity and sensitivity are affected by this single amino acid exchange and thereby foster the molecular understanding of the CNG channel activation process.

[1] A. Cukkemane, B. Grueter, K. Novak, T. Gensch, W. Boenigk, R. Seifert, U. B. Kaupp EMBO Rep. 8, 749 (2007).

[2] G.M. Clayton, W.R. Silverman, L. Heginbotham and J.H. Morais-Cabral, Cell 119, 615 (2004).

[3] C.M. Nimigean, T. Shane and C. Miller, J. Gen. Physiol. 124, 203 (2004).

[4] U.B. Kaupp and R. Seifert, Physiol. Rev. 82, 769 (2002).

[5] W.N. Zagotta, N.B. Olivier, K.D. Black, E.C. Young, R. Olson and E. Gouaux, Nature 425, 200 (2003).

Parallel Information Processing Starts at the First Synapse in the Visual System F. Müller1, E. Ivanova1, A. Mataruga1, C. Puller2, S. Haverkamp2, H. Wässle2 1ISB-1: Cellular Biophysics 2MPI Brain Research, D-60528 Frankfurt In the mammalian retina, the light response of rod and cone photoreceptors is processed by an elaborate neuronal network (for review see [1], [2]). At least ten cone bipolar cell types and one rod bipolar cell type provide the direct pathways from the photoreceptors to the ganglion cells that relay the information to the brain. Most importantly, these bipolar cell types form the basis for parallel processing of visual signals. We, therefore, investigated the connectivity and the functional properties of all bipolar cell types. Each cone feeds its signal into every bipolar cell type. Patch-clamp recordings revealed that each morphologically identified bipolar cell type expresses a unique inventory of voltage-gated ion channels. These results suggest that bipolar cell types differ in their functional properties, each one being specialized to filter certain aspects from the visual information. Finally, we identified bipolar cells that provide for the recently proposed “alternative rod pathway”. Bipolar cells establish parallel pathways

In the mammalian retina the bipolar cells provide parallel pathways to relay the information from the photoreceptors to the ganglion cells. Cones feed into ON- and OFF-cone bipolar cells that excite ON- and OFF-ganglion cells, respectively. In the “classical” rod pathway, rods feed into rod bipolar cells that provide input into both the ON- and the OFF-pathway via AII amacrine cells. In a collaboration with the MPI for Brain Research in Frankfurt, we extended our studies on the identification of bipolar cell marker proteins, bipolar cell densities, connectivity to photoreceptors, and bipolar cell axonal tiling in the mouse retina. We found that the dendritic trees and axon terminals of each bipolar cell type tile the retina without much overlap. Each and all cones are connected to at least one member of any given type of cone bipolar cell [3]. In a highly complex synaptic formation, the cone pedicle provides output onto at least ten different postsynaptic bipolar cells. Thus, parallel information processing starts at the very first synapse in the visual system.

Each bipolar cell type expresses a unique inventory of ion channels

The light response of bipolar cells is shaped by two different means: by the precise timing of excitatory and inhibitory input and by the kinetics of the receptors and ion channels involved in the generation of the input and in the propagation of the electrical signal. Using the patch-clamp technique in the whole cell configuration we studied the expression of voltage-gated ion channels in bipolar cells in rat retinal slices. While recording, cells were filled with Lucifer yellow to reveal their morphology. Figure 1 shows all bipolar cell types revealed in our study. Note that the axons of different bipolar cell types stratify at different levels of the inner plexiform layer (IPL). The traces show representative current recordings of type 4, type 5, and type 6a bipolar cells. From the holding potential of -60 mV, the membrane potential was clamped to values from -65 to -125 mV in -15 mV increments in a family of 500 ms long hyperpolarizing voltage steps. In type 5 bipolar cells, these voltage steps elicit pronounced slowly activating inward currents (seen as downward deflections). Upon depolarization to 0 mV, large sustained outward currents are measured (seen as upward deflection at the end of the trace). These membrane currents are conducted by different ion channel types. For example, the inward currents are carried by hyperpolarization-activated and cyclic nucleotide-gated channels (HCN channels), while outward currents are conducted by outwardly rectifying potassium channels. Current traces recorded from other bipolar cell types look different, indicating differences in the expression of ion channels. For example, type 4 bipolar cells show no inward currents and, therefore, do not express HCN channels. Type 6a bipolar cells show inward currents; however, their shape differs from those found in type 5 bipolar cells. In type 6a bipolar cells, currents carried by calcium-activated chloride channels are superimposed on HCN channel currents. We found that each morphologically identified bipolar cell type expresses a unique inventory of voltage-gated ion channels suggesting that different bipolar cell types differ in their functional properties. The

different repertoires of currents can serve as “finger prints” to identify the bipolar cell types. Moreover, our combined electrophysiological and morphological approach allowed us to describe a new type of rat ON-cone bipolar cell (type 6b) that, based on morphological criteria alone, would not have been classified as a distinct cell type [4].

Fig. 1: Top: Different bipolar cells in the retina stratify at different levels of the inner plexiform layer (IPL). Bottom: Representative current recordings of type 4, type 5, and type 6a bipolar cells (for details see text). Current traces of different cell types show characteristic differences, indicating differences in ion channel expression.

An alternative pathway for rod photoreceptor signals

The retina functions over a wide range of light intensities. The very sensitive rod photoreceptors are suitable for vision at low (scotopic) light levels whereas the less sensitive cones provide for daylight (photopic) and colour vision. The cones are connected to the cone bipolar cells that synapse onto the ganglion cells.

For a long time it was thought that rod signals are only relayed by one type of bipolar cell to the inner retina, the rod bipolar cell. Recent evidence suggested an alternative route in which rods directly contact some types of OFF-cone bipolar cells. We performed an immunohistochemical analysis on the level of light and electron microscopy to identify the bipolar cells and ganglion cells that are involved in this “alternative rod pathway” of the mouse retina. We identified two types of OFF-bipolar cells, one of which was hitherto unknown, that form contacts at both cone pedicles and rod spherules. The axon terminals of these bipolar cells co-stratify with the dendrites of a large, putatively postsynaptic OFF-ganglion cell. These newly identified cell types represent the basis of a neuronal circuit in the mammalian retina that could provide for an alternative fast rod pathway [5].

Fig. 2: Electronmicrograph. Dendrites of one type of OFF-cone bipolar cell labeled by antibodies against protein kinase A (arrows) form basal contacts at a rod spherule (rs). M, mitochondrium, dots, horizontal cell processes, asterisks, rod bipolar cell dendrites.

[1] H. Wässle and B.B. Boycott, Physiol. Rev. 71, 447 (1991).

[2] R.H. Masland, Curr. Opinion Neurobiol. 11, 431 (2001).

[3] H. Wässle, C. Puller, F. Müller and S. Haverkamp, J. Neurosci. 29, 106 (2009).

[4] E. Ivanova and F. Müller, Vis. Neurosci. 23, 143 (2006).

[5] A. Mataruga, E. Kremmer and F. Müller, J. Comp. Neurol. 502, 1123 (2007).

Maturation and Modulation of Neuronal Chloride Homeostasis T. Gensch1, D. Gilbert2, A. Woitecki2, C. Franjic-Würtz2, K. Funk2, S. Frings2, F. Möhrlen2

1ISB-1: Cellular Biophysics 2Institute of Zoology, Univ. Heidelberg, D-69120 Heidelberg In most neurons of the adult CNS, the internal chloride concentration [Cl-] is low. Hence, opening of GABA- or glycine-activated ionotropic receptors mediate Cl- influx, i.e. lead to inhibition of the cell. Excitatory Cl- currents occur in neurons which accumulate Cl-, i.e. in immature neurons of the CNS, in olfactory sensory neurons, and in neurons challenged by ischemia, inflammation, or neurological disorders. The balance between inhibitory and excitatory Cl- effects is determined by Cl- uptake and Cl- extrusion pathways in the cell. A Na+-K+-2Cl- co-transporter NKCC1 provides the main route for Cl- uptake, whereas KCC2 couples Cl- extrusion to K+ efflux. We have determined the intracellular Cl- concentration [Cl-] of somatosensory neurons using two-photon fluorescence-lifetime imaging microscopy (2P-FLIM) with the Cl--sensitive dye MQAE [1] and monitored the expression of NKCC1 and KCC2. In a second approach, we studied the effects of inflammatory mediators on Cl- homeostasis in dorsal root ganglion (DRG) neurons [2]. Our results show, that DRG neurons undergo a developmental transition of chloride homeostasis during the first three postnatal weeks. In contrast to CNS neurons, DRG neurons display a heterogeneous pattern of Cl- concentration. Inflammatory mediators raise the intracellular Cl- concentration, most likely due to post-translational modification of NKCC1 and KCC2 and thereby change the excitability of DRG neurons. This may be the basis for increased pain perception during inflammation.

Neuronal activity can be strongly influenced by Cl- currents. Whether the opening of Cl- selective ion channels leads to excitatory or inhibitory currents, is controlled by the membrane potential and by the intracellular Cl- concentration. Cation-coupled Cl--cotransporters control the [Cl-] in neurons. Cl- uptake is mainly mediated by NKCC1 [3] activity, whereas the efflux of Cl- ions is controlled by KCC2 [4]. Regulatory mechanisms that control the activity of Cl- transporters include phosphorylation as well as dimerization. Changing the balance between Cl- import and export can have profound effects on the Nernst-potential for chloride ECl and thereby determine whether the opening of Cl- channels causes Cl- efflux, leading to depolarization and excitation of the cell, or to Cl-

influx that causes hyperpolarization and inhibition of the neuron.

We have studied the effect of inflammatory mediators (i.e. CGRP or substance P) on the [Cl-] as well as on the expression of NKCC1 and KCC2 in DRG neurons. To monitor changes of the Cl- concentration during exposure to inflammatory mediators, we loaded DRG neurons with the Cl--sensitive fluorescent dye MQAE. The MQAE fluorescence is quenched by Cl- and is well suited to monitor time-dependent changes of the intracellular Cl- level. As an experimental parameter we analyzed the fluorescence lifetime of MQAE, which is independent of the intracellular dye concentration. The recorded fluorescence lifetime was colour-coded for microcopic images such that warmer colours represent higher Cl- concentrations (Fig. 1).

Fig. 1: Monitoring intracellular Cl- concentration in DRG neurons. (A) Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intact DRGs loaded with MQAE. The colour code indicates the fluorescence lifetime (τ) which is inversely proportional to the Cl- concentration. Warm colours represent high intracellular Cl- (small τ values). (B) Quantitative analysis of 2P-FLIM data show a highly significant increase of the inverse lifetime (LT = 1/τ) two hours after starting the treatment with inflammatory mediators.

The data indicate that inflammatory mediators raise intracellular [Cl-] significantly within 2 hr of treatment with inflammatory mediators, e.g. substance P and calcitonin-gene related peptide (CGRP). Notably, the 2P-FLIM measurements revealed that virtually all DRG neurons visible in the image increase their [Cl-] in response to the inflammatory stimulus. This effect coincided with

enhanced phosphorylation of NKCC1. Immunological staining of DRG sections with antibodies specifically binding to phosphorylated NKCC1 showed an increase of 18% and 45% of the phosphorylated form of NKCC1 after 1 and 2 hrs of inflammatory treatment. These results suggest that phosphorylation of NKCC1 in DRG neurons causes the early (< 3 hr) phase of enhanced Cl- accumulation observed by 2P-FLIM. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl- importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment.

In a second study we examined the maturation of Cl- homeostasis after birth (Fig. 2). Somatosensory neurons in the dorsal root ganglia undergo a transition of Cl- homeostasis during the first three weeks of postnatal development. This process parallels the developmental “chloride switch” in the CNS. However, while most CNS neurons achieve homogeneously low Cl- levels, somatosensory neurons maintain a heterogeneous pattern of Cl- values. Somatosensory neurons have a high internal [Cl-] after birth (Fig. 2). However, within postnatal weeks 1 – 3, roughly a third of the cells maintain the high Cl- levels of the early postnatal days, whereas the majority of somatosensory neurons contain either intermediate or low levels of Cl-. This results, as displayed in Fig. 2 with 2P-FLIM measurements of MQAE, in a reatehr heterogeneous distribution of Cl- levels in a DRG.

Fig. 2: Determination of Cl- in somatosensory neurons by 2P-FLIM. (a) Comparison of fluorescence intensity (left) and lifetime (right) images from the same DRG. In the 2P-FLIM image, the fluorescence lifetime, τ, is colour-coded as in Fig.1. (b) Calibration of 2P-FLIM signals in isolated DRG neurons with [Cl-] set to the indicated values using ionophores. The colour scale illustrates the false-colour representation of [Cl-] in the following images. (c) 2P-FLIM images illustrating [Cl-] levels in somatosensory neurons from newborn (P1-P4) and adult (≥3rd week) mice. Newborn neurons show almost uniformly high [Cl-] (≥70 mM). During maturation, most somatosensory neurons decrease their [Cl-] to some extent, resulting in a heterogeneous mosaic of [Cl-] levels in the ganglia of mature animals.

To unravel the molecular basis for the maturation-dependent transition of the Cl- concentration, we investigated the expression level of several Cl- cotransporters. Surprisingly, we did not detect changes in the transcript level during maturation in any of these genes. Therefore, it is highly unlikely that transcriptional regulation underlies the maturational change in Cl- homeostasis in somatosensory neurons.

Our studies demonstrate that Cl- homeostasis in somatosensory neurons develops during postnatal maturation into a state where the [Cl-] can be regulated individually in each neuron. Rather than on changes of the expression level of electroneutral cation-chloride cotransporters, this regulation may be achieved by postranslational modification of existing proteins. This notion is supported by our results on DRG neurons treated with inflammatory mediators in which Cl- accumulation coincides with phosphorylation of the NKCC1 transporter. Taken together, the efficiency of Cl- accumulation sets the level of [Cl-] and, hence, may control the sensitivity of adult sensory neurons.

[1] D. Gilbert, C. Franjic-Würtz, K. Funk, T. Gensch, S. Frings and F. Möhrlen, Int. J. Dev. Neurosci. 25, 479 (2007).

[2] K. Funk, A. Woitecki, C. Franjic-Würtz, T. Gensch, F. Möhrlen and S. Frings, Mol. Pain 4, 32 (2008).

[3] B.B. Pond, K. Berglund, T. Kuner, G. Feng, G.J. Augustine and R.D. Schwartz-Bloom. J. Neurosci. 26, 1396 (2006).

[4] V. Stein, I. Hermanns-Borgmeyer and T.H. Jentsch. J. Comp. Neurol. 468, 57 (2004).

ISB-2: Molecular Biophysics Director: Prof. Georg Büldt

One ultimate goal in biophysics is to understand the functions of proteins and macromolecular complexes as well as whole cellular processes like signal transduction pathways in their cellular environment. For most biophysical techniques it is difficult or in many cases impossible to apply physical methods to living cells. ISB-2 follows several approaches to achieve this goal:

• Proteins or complexes are isolated, crystallized and high-resolution X-ray structures are determined from ground and intermediate states of their working cycles to obtain a nearly complete time-resolved image of their mechanisms.

• Protein unfolding and refolding as well as the interactions between proteins are studied in aqueous solution and in membranes.

• Cell-free transcription and translation systems in combination with fluorescence single molecule spectroscopy allow us to accompany protein synthesis and folding at the ribosomal machinery.

FKBP42, an immunophilin modulatingABC transporter activityO. H. Weiergräber1, R. Batra-Safferling1

, J. Granzin1

1 Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2)

FKBP42 is a type-II membrane protein whichplays an important role in the regulation of polarauxin transport in higher plants. These effectsare mediated by direct interaction with auxin car-riers belonging to the ABC transporter superfam-ily. We have determined the X-ray structure ofthe soluble portion (aa 1-339) of FKBP42. TheN-terminal domain displays an FKBP-type foldbelonging to the αβ-class. The C-terminal seg-ment, on the other hand, represents a helicalbundle comprising three TPR motifs. Based onsequence conservation, we predict that the in-teraction of FKBP42 with the heat shock proteinHSP90 will occur in a similar way as found forother immunophilins. Finally, we have built amodel for the complex of FKBP42 with the C-terminal nucleotide binding domain of the auxintransporter PGP1. We propose that FKBP42 mayenhance auxin transporter activity by stabilizinga conformation of the nucleotide binding domainwhich is competent for ATP loading and/or hy-drolysis.

The FK506 binding proteins (FKBPs) represent aubiquitous protein family named after the role ofseveral members as primary targets of FK506-typeimmunosuppressants in animal and human cells.Based on this activity, the FKBPs, together withthe family of cyclophilins, have also been termed"immunophilins". Another feature shared by manyFKBPs is the ability to act as peptidylprolyl cis-transisomerases (PPIases), which implicates these pro-teins in peptide folding and chaperoning processes.Multi-domain FKBPs are characterized by additionalprotein modules, typically a tetratricopeptide repeat(TPR) domain, connected to one or more FKBP do-mains.Members of the FKBP family have also been iden-tified in plants. FKBP42 from Arabidopsis thaliana,also termed TWISTED DWARF1 (TWD1) due to thereduced height and disoriented growth of null mu-tants, is a type-II membrane protein comprising 365amino acids. In addition to a single FKBP-type do-main, it contains a tripartite TPR motif and a hy-drophobic C-terminal membrane anchor [1]. Likemany other multi-domain immunophilins, the pro-tein interacts with HSP90, which has been impli-cated in plant development and response to envi-ronmental stress. Intriguingly, FKBP42 is devoid

FIG. 1: Ribbon representation of the FKBP42 structure.Mean B factors are indicated by a colour gradient from white(B < 35 Å2) to red (B > 90 Å2). Side chains establishinginter-domain contacts are indicated.

of PPIase activity and does not display measurableaffinity for FK506. The FKBP domain of FKBP42has been demonstrated to physically interact withplasma membrane-localized ABC transporters PGP1and PGP19, whereas the TPR domain appears tobe responsible for functional association with vacuo-lar transporters MRP1 and MRP2. Phytohormonesof the auxin family represent essential regulatorsof plant growth and development. The predomi-nant auxin, indole 3-acetic acid, is synthesized atthe shoot apex and undergoes a basipetal transportwhich is crucial for the establishment of plant polar-ity. Recently, PGP1 and PGP19 have been shown todirectly mediate cellular auxin efflux, and this activityis regulated by FKBP42 [2].

We have crystallized fragments of FKBP42 coveringthe N-terminal domain (aa 1–180) and the entire sol-uble portion (aa 1–339), respectively [3]. The X-raystructure of the former construct reveals a canoni-cal FKBP-type fold, consisting of a five-stranded an-tiparallel β-sheet wrapped around a short α-helix.

FIG. 2: Model of the FKBP42-PGP1 complex, side view.

The absence of detectable PPIase activity as wellas FK506 affinity could be explained by the activesite being partly occluded by protein side chains[4]. The crystal structure of the larger constructis shown in Figure 1. The N-terminal part of themolecule (left) displays the expected FKBP-type fold.The C-terminal segment, on the other hand, repre-sents a helical bundle (right), as anticipated from theearly sequence-based classification as a TPR do-main. The domain interface of FKBP42 is composedof a hydrophobic network surrounded by hydrogenbonds and electrostatic contacts [5].

Recent evidence has indicated that many of theprotein-protein interactions in the HSP90 chaper-one complex, which had originally been investi-gated in animal systems, are conserved in plants.These include the association with multi-domain im-munophilins. Based on sequence conservation, wepropose that the interaction of HSP90 family mem-bers with FKBP42 is mediated by the invariant C-terminal pentapeptide MEEVD engaging a TPR do-main of the binding partner via a "two-carboxylateclamp" mechanism.

FKBP42 is unique among large immunophilins in thatboth the FKBP and TPR domains have been shownto interact with ABC transporters in vivo. Specifically,the C-terminal portions of PGP1 and PGP19 contain-ing the second nucleotide-binding domain (NBD2)bind to the FKBP fold, whereas the equivalent do-mains in MRP1 and MRP2 associate with the TPRmodule. In order to improve our understanding ofthese novel protein-protein interactions, we have de-veloped homology models for the NBD2 of PGP1 andMRP1 representing the two classes of ABC trans-porters considered here. The resulting models weresubjected to an in silico docking procedure togetherwith the appropriate domains of our FKBP42 struc-ture. Figures 2 and 3 present an updated modelillustrating the interaction of FKBP42 with PGP1 (un-published data). To visualize the overall architectureof the complexes and the orientation of the com-ponents with respect to the membrane, the NBD2model (blue) has been incorporated into the crystal

FIG. 3: Model of the FKBP42-PGP1 complex, bottom view.

structure of Sav1866 from S. aureus. The crystalstructure of FKBP42 described here represents thefirst three-dimensional structure of a multi-domain im-munophilin from plants. While the overall architectureof the two domains matches the known characteris-tics of the FKBP and TPR folds, respectively, theirarrangement is unique and may have important func-tional implications. Experimental evidence indicatesthat regulation of ABC transporters by members ofthe FKBP family may not be restricted to plant cells.Our models featuring the complexes of FKBP42 withPGP1 and MRP1 may serve as working hypothe-ses stimulating further investigation of these novelprotein-protein interactions.

We are currently striving for experimental valida-tion of our models by obtaining X-ray structures ofFKBP42-ABC transporter complexes. To this end,we are establishing expression and purification ofthe C-terminal nucleotide-binding domains of PGP1and PGP19, either alone or as fusion constructs withtheir respective N-terminal counterparts. In the con-text of our current efforts towards improving crystal-lization of membrane proteins, investigation of a full-length ABC transporter, with or without an associatedFKBP42, may ultimately be feasible.

[1] T. Kamphausen, J. Fanghänel, D. Neumann, B.Schulz and J. U. Rahfeld, Plant J. 32, 263–276 (2002).

[2] R. Bouchard, A. Bailly, J. J. Blakeslee, S. C. Oehring,V. Vincenzetti, O. R. Lee, I. Paponov, K. Palme, S.Mancuso, A. S. Murphy, B. Schulz and M. Geisler,J. Biol. Chem. 281, 30603–30612 (2006).

[3] A. Eckhoff, J. Granzin, T. Kamphausen, G. Büldt, B.Schulz and O. H. Weiergräber, Acta Cryst. F61, 363–365 (2005).

[4] O. H. Weiergräber, A. Eckhoff and J. Granzin, FEBSLett. 580, 251–255 (2006).

[5] J. Granzin, A. Eckhoff and O. H. Weiergräber,J. Mol. Biol. 364, 799–809 (2006).

Visual signal transduction studied by a variety of biophysical methods R. Schlesinger1, B. König2, R. Batra-Safferling1, G. Büldt1, G. Dasara Raju1, J.Granzin1 1 Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) 2 Institute of Structural Biology and Biophysics: Structural Biochemistry (ISB-3) Arrestins play a crucial role in regulation of signal transduction of G protein-coupled receptors (GPCRs) [1]. GPCRs convey a large variety of extracellular stimuli including light, odorants and neurotransmitters to the interior of cells via activation of their cognate G proteins. GPCR signaling is shut off by a conserved two step process, i.e. phosphorylation of the GPCR followed by tight binding of arrestin. Four types of arrestin can be distinguished in vertebrates: rod arrestin (arrestin-1) and cone arrestin (arrestin-4) are restricted to the corresponding compartments of the retina while non-visual β-arrestin-1 (arrestin-2) and β-arrestin-2 (arrestin-3) are widely distributed in various tissues. Arrestins regulate a large number of different GPCRs and serve additional functions beyond desensitization of GPCR signaling [1].

In 1998 we published the first 3-dimensional X-ray structure of arrestin-1 from bovine rod outer segments in the receptor-free form [2]. The overall fold of the basal state of arrestin is conserved across the various arrestins (see above). However, the structure of arrestin in complex with the activated and phosphorylated GPCR remains elusive.

Until now it is unclear how arrestin interacts with rhodopsin. We have used several complementary methods to address local aspects of the arrestin- rhodopsin interaction.

Our light scattering experiments suggested that two rhodopsin molecules bind to the two dome- shaped structural motifs of arrestin (Figure 1) [3]. To investigate the interaction sites of arrestin with rhodopsin various surface regions of recombinant arrestin were sterically blocked by different numbers of fluorophores. By simultaneously modifying both domains with one Alexa 633 moeity the binding capacity was reduced. The presence of two Alexa 633 molecules in each domain prevented binding of rhodopsin to arrestin. This observation indicates that both concave sites participate in binding. These findings are not consistent with the current working model of arrestin binding to rhodopsin (see literature), which requires the accessibility to different regions that

are responsible for the binding and recognition process of P-Rho*. In this case, it is expected that the presence of one dye molecule per arrestin in the concave regions as well as in the area of the helix should be sufficient to exert a clear negative influence on binding efficiency to P-Rho*. This would also be expected when considering earlier investigations which implicate a large number of residues in these regions.

FIG. 1: A possible model of arrestin (residues 6-388, J. Granzin unpublished ) and rhodopsin (Protein Data Bank ID: 1U19) interaction. The docking has been carried out manually without energy minimization and the distance between the receptor monomers is adjusted based on the work of Fotiadis et al. [5].

High resolution liquid state NMR can determine the structure and orientation of peptides in the rhodopsin-bound state (paper submitted, 2009).

FIG. 2a: Crystal structure of uncomplexed arrestin shown as a ribbon representation. Residues 67 to 77 in the unstructured loop connecting β-strands V and VI are colored in yellow, the N- and C-terminal domains of arrestin are depicted in dark blue and green, respectively.

These binding studies indicate that Arr(67-77) competes with arrestin for its binding site on Meta II-rhodopsin. We speculate that the largely α-helical conformation of the Meta II-bound peptide reflects the conformation of the corresponding “finger loop” region in the arrestin-P-Rho* complex (Figure 2a, b). Receptor binding of Arr(67-77) strictly requires rhodopsin activation but is rather insensitive to receptor phosphorylation. These observations support the sequential multi-site binding model of receptor-arrestin interaction proposed by Gurevich and Gurevich [4] and suggest a crucial role of arrerstin loop V-VI in the recognition of rhodopsin activation.

We have started time-resolved fluorescence anisotropy measurements which will give further evidence for the mode of interaction between the two molecules. In addition we will perform time-resolved fluorescence imaging experiments to visualize the interaction of arrestin with activated rhodopsin (in collaboration with U. Alexiev, FU Berlin). Furthermore crystallization of the complex (arrestin-rhodopsin) is in preparation.

FIG. 2b: Arrestin loop V-VI adopts an α-helical structure (boxed) upon binding Meta II. Loop residues 67-77 have been replaced by the NMR structure of Meta II-bound peptide Arr(67-77) followed by energy minimization.

[1] K. Xiao, D.B. McClatchy, A.K. Shukla, Y. Zhao, M. Chen, S.K. Shenoy, J.R. Yates, and R.J. Lefkowitz, Proc. Natl. Acad. Sci. USA 104, 12011-12016 (2007).

[2] J. Granzin, U. Wilden, H.-W. Choe, J. Labahn, B. Krafft, G. Büldt, Nature 391, 918-921 (1998).

[3] D. Skegro, A. Pulvermüller, B. Krafft, J. Granzin, K.P. Hofmann, G. Büldt, R. Schlesinger, Photochem. Photobiol. 83, 385-393 (2007).

[4] V.V. Gurevich and E.V. Gurevich, Trends Pharmacol.Sci. 25, 105-111 (2004).

[5] D. Fotiadis, Y. Liang, S. Filipek, D. A. Saperstein, A. Engel and K. Palczewski, Nature 421, 127-128 (2003).

Signal transduction by sensory rhodopsin II-transducer complex V. Gordeliy1, J. Labahn1, M. Engelhard2, G. Büldt1 1Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) 2Max-Planck-Institute for Molecular Phisiology Microbial rhodopsins, which constitute a family of seven-helix retinal membrane proteins, are distributed throughout the Bacteria, Archaea and Eukaryota. The microbial phototaxis receptor sensory rhodopsin II (NpSRII) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis by modulating the swimming behavior of the bacterium. After excitation by blue-green light NpSRII triggers, by means of a tightly bound transducer protein (NpHtrII), a signal transduction chain. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes. We have obtained X-ray structures of the ground state and photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the transducer in the complex.

Membrane receptors play key roles in different cell functions. Unfortunately, there is no molecular picture of the signal transduction mechanism for any receptor. The same was true for the case of the NpSRII/NpHtrlI complex. Homologs of sensory pathway in which the complex is involved occur in all the three kingdoms of life, most notably in enteric bacteria in which the chemotaxis has been extensively studied. Recent structural and functional studies on the sensory rhodopsinII/transducer complex have yielded new insights into the mechanisms of signal transfer across the membrane. Electron paramagnetic resonance data led to the conclusion that the complex functions as a dimer [1]. However, the atomic resolution structure of the complex as well as molecular events leading to signal transduction were still missing. Our goal was to obtain such information via elucidation of high resolution structure of the complex and its intermediates. The results provided a first atomic model for signal transfer within the membrane part of the receptor. The established mechanism might also be relevant for eubacterial chemoreceptor signaling.

Crystallization of the receptor–transducer complex, a member of the two-component signaling cascade, has been achieved successfully using a shortened transducer comprising the two transmembrane helices (TM1 and TM2). The data showing a signal transfer from receptor to transducer indicate that HtrII-114 forms a

functional complex with its cognate receptor SRII. Thin orange crystals of SRII in complex with HtrII-114 grown in lipidic cubic phase displayed an orthorhombic shape of about 140 μm in size and diffracted to 1.8 Å. The asymmetric unit contains one complex. The expected dimer of the complex is formed by a crystallographic two-fold rotation axis, which is located in the middle of four transmembrane helices: TM1, TM2, TM1’ , TM2’ (where a prime indicates the right-hand complex; Fig. 1). The transmembrane helices F and G of the receptor are in contact with the helices of the transducer [2, 3].

Obviously, the binding of the transducer to helices F and G hardly interferes with the side-chain arrangement of the receptor. A notable exception is found for Tyr 199. The aromatic plane of Tyr 199 has turned in the complex by about 90˚ and is now pointing into the direction of TM2 where its phenolic group forms a hydrogen bond to Nδ(2)-Asn 74 (2.8 Å).

FIG.1: Ribbon diagram of the top view from the cytoplasmic side. α-Helices are in red for the receptor and green for the transducer; β-strands are in blue and coils in grey. The labels of the symmetry related complex are marked by a prime. The crystallographic symmetry axis is located between TM1–TM2 and TM1’–TM2’ .

The F–G loop region fixes the transducer by several contacts as well as by three hydrogen bonds between Thr 189 (SRII), Glu 43 (TM1) and Ser 62 (TM2). A second anchor point is observed in the middle of the membrane where, as

mentioned above, the phenolic hydroxyl of Tyr 199 bridges to Asn 74.

The crystals of the complex were illuminated to freeze-trap the K and M intermediates. In the K state structural changes with respect to the ground state are observed within a sphere of 9 Å diameter around the central water cluster on the extracellular side of the retinal [4]. The isomerization of the retinal bound to Lys 205 gives rise to a displacement of its Cε and Cδ atoms by 1.1 Å. This shift reduces the distance between Lys 205-Cε and the ground state position of water molecule 1 (W1-O) from 3.3 to 2.5 Å. Therefore the pentagonal structure of hydrogen bonds including water molecules W1 to W3 and the oxygen atoms of the aspartic residues Asp 75 and Asp 201 found for the ground state can no longer exist in the K state. W1 is driven away from its original location [2].

FIG. 2: Changes in the structure of helices C and G of ground state (red) and late M state (yellow) including water molecules as red and yellow balls.

The charge separation between the protonated Schiff-base nitrogen and Asp 75-Oδ increases from 4.2 Å to 4.9 Å in the K state. The hydrogen bond structure (Asp 201-Oδ· ·W2· ·W4· ·Arg 72-Nε) is retained, although W2 and W4 shift towards the retinal by about 1.5 Å in the K state. The distance from Asp 201-Oδ to the Schiff-base nitrogen is reduced from 4.9 Å to 2.7 Å as a result of the movement of the Schiff base.

During the transition from K state to M1 state the proton of the Schiff base translocates to Asp 75. In the reaction path from the ground state through K to late M the temperature factor of Asp 75-Oδ2 varies from 18 Å2 (BG) through 31 Å2 (BK) to 22 Å2 (BM) and the distance between Asp 75-Oδ2 and the Schiff-base nitrogen relaxes from 4.9 Å in the K state to 4.3 Å in late M, when proton transfer has occurred.

In late M state a further hydrogen bond connecting helices C and G in the K state through water molecules W2’ and W4’ has vanished. Consequently these helices now have more freedom to move. Difference densities clearly show changes in the position of the main chain that are not present in the K state (for example Arg 72) (Fig 2).

FIG. 3: Schematic picture of helical displacements viewed from the cytoplasmic surface. The ground-state complex is shown in red and the late M-state complex in yellow

Thus, light-activated signal transfer from receptor to transducer originates in the isomerization of retinal, which induces local changes in the hydrogen-bonding network in a region near the retinal (K state). During the transition to the signaling state (late M) these disturbances increase so that in late M fewer hydrogen bonds connect helices C and G. These alterations in the hydrogen-bond network change the pKa values of the Schiff base and Asp 75, thereby enabling proton transfer, which produces a redistribution of charges. Both causes give rise to changes in the tertiary structure of the receptor in late M. Signal transfer to the transducer takes place within the interface of receptor helices F and G and transducer helix TM2. The main effect on TM2 is a clockwise rotation of about 15˚ and a tilt of the helix, with the hinge at Ser 62, that amounts to 0.9 Å at the cytoplasmic side (Leu 77).

New information is of significance not only for bacterial phototaxis and chemotaxis [5], but also for other dimeric receptors, and for the first time might lead finally to a general model of transmembrane signal transduction [6].

[1] Hoff W.D., Jung K.-H. and Spudich J.L. (1997) Annu. Rev.Biophys. Biomol. Struct. 26,223-258

[2] Gordeliy V. I., Labahn J., Engelhard M., Büldt G. et al. Nature 419, 484–-487 (2002).

[3] Royant A., Nollert P., Edman K., Neutze R., Landau E.M., Pebay-Peyroula E. and Navarro J (2001) Proc. Natl. Acad. Sci.USA 98,10131-10136.

[4] Moukhametzyanov R., Klare J., Efremov R., Engelhard M., Büldt G., Gordeliy V. et al. Nature 440, 115-119 (2006)

[5] Spudich J. (2002) Nature Str. Biology 9, 797-799.

[6] Newman R. (2001) Cur. Opinion in Chem. Biol. 5, 723-724.

Vapour diffusion for in-mesophase high throughput crystallization J. Kubicek1, G. Büldt2, F. Schäfer1, J. Labahn2 1Qiagen Research&Development, Hilden 2Institute of structural Biology and Biophysics: Molecular Biophysics (ISB-2) The presented method to crystallize membrane proteins combines the advantages of the meso-phase crystallization method and the classical vapour diffusion crystallization method. It allows fast screening of crystallization conditions employing automated liquid handlers suited for the 96 well crystallization format. Experiments with bacteriorhodopsin proof that by this approach crystals of high quality can be obtained.

For less than hundred membrane proteins their 3-dimensional structure at atomic resolution had been determined though one-third of the proteins inside a cell belong to this class. This state of affairs is especially bothersome as the natural entry points to a cell are membrane proteins and their assemblies: The lacking knowledge of membrane protein structures translates directly into lacking knowledge of biomedical targets and mechanisms.

The small number of known membrane protein structures can be directly traced back to problems in obtaining membrane protein crystals for structural investigations. The methods developed for crystallization of soluble protein are inefficient for membrane proteins. Landau and Rosenbusch [1] used lipidic mesophases to accommodate the specific needs of membrane proteins in a way compatible with crystallization: The lipidic component monoolein self-organizes with water into mesophases [2] (Fig.1). The cubic phase Pn3m consists of a bi-continuous bilayer that separates two channel systems of aqueous phase. The bilayer is locally 2-dimensional like a cell membrane and therefore allows the incorporation of membrane proteins. But it extends continuously through space and supports therefore diffusion of the protein in three dimensions and crystallization upon dehydration.

FIG. 1: Isotherm from monooolein/water phase diagram. The formation of the optically isotropic cubic phases or birefringent lamellar phase can be observed with a polarization-microscope.

The kinetics of the self-organisation of monoolein can be observed by incubating solid monoolein with water. Within an hour ithe formation of cubic phase is observed (Fig.2).

Dehydration of the mesophase can be achieved by lowering the level of humidity. This can be achieved by adding solid salt [1].

1 min 3 min 7 min 25 min 0 min

FIG.2: Swelling experiment with 0.2 mg monoolein and 350 nl H2O at 22°C. The solid, intransparent monoolein phase transforms within 25 minutes almost completely to the optically isotropic cubic phase, whereas the birefringent lamellar phase disappears.

The in-mesophase crystallization [1] unfortunately requires cumbersome manual work like weighting mg-quantities for every single crystallization experiment. This is detrimental to high-throughput screening procedures using automated liquid handling systems as employed successfully for the crystallization of soluble proteins.

In vapour diffusion type crystallization experiments typically a protein solution and a solution containing a precipitating agent are mixed. A small droplet of this mixture will generally be not in equilibrium with undiluted reservoir solution. Generally the vapour pressure of water above these two phases will be different: Water will transfer via the vapour phase into the reservoir solution until the two condensed phases are in equilibrium, typically when the concentration of the precipitating agent in the droplet is approximately the same as in the reservoir (Fig.3, left: broken arrow). This end point of the experiment is to be refined experimentally to reach a droplet composition sufficiently supersaturated to force nucleation and subsequent crystallization of the protein..

In vapour diffusion experiments with monoolein the dehydration can be experimentally realized by enclosing the wetted monoolein together with a reservoir solution which takes up water from the gas phase that separates the two condensed phases (Fig.3).

FIG. 3: Hanging drop vapour diffusion experiment with monoolein: The incubation of monoolein (dark grey) withan aqueous droplet mixed from membrane protein (green)and reservoir (red) solution leads to uptake (arrows) ofwater and membrane protein and subsequent phasetransformation within hours. Over days the formation ofoptically isotropic cubic phase (light grey, middle) isobserved. The loss of water from the droplet towards thereservoir (blue arrow) enhances the incorporation ofprotein into the cubic phase. Over weeks to month (right)the cubic phase reverts back to lamellar phase due to dehydration by vapour diffusion.

When solid monoolein is brought into contact with an aqueous protein droplet it will incorporate protein and take up water. After the fast take-up of water by monoolein the slower loss of water in the droplet by vapour diffusion will further increase the concentration of protein in the aqueous phase (Fig.3, middle). Therefore an increasing amount of the protein will be incorporated into the mesophase according to the Nernst partition law:

K = Caqueous/Xmesophase C,X: concentration, respectively mol-fraction of protein in the respective phases

Lately (Fig.3, right) the protein containing cubic phase will be dehydrated by the reservoir solution and form lamellar phase (Fig.2: right to left).

Nucleation of protein crystallization is thought to be brought-by by the local transformation of cubic phase to lamellar phase.

Experiments with the light-driven bacterial proton pump bacteriorhodopsin from Halobacterium salinarum show that crystallisation occurs upon transformation of the transparent, isotropic, cubic phase to lamellar phase (Fig.4).

FIG.4: Crystallization experiment with Bacteriorhodopsin in monoolein mesophase employing vapour diffusion.

Crystals of bacteriorhodopsin grown in mesophase using vapour diffusion reach a size of 0.25 mm * 0.1 mm and diffract to 1.14 Å (Fig.5), that is considerably further than the best results obtained so far 1.43 Å [3] with other crystallisation methods.

FIG.5: Diffraction experiment with bacteriorhodopsin crystal from in mesophase vapour diffusion. Measured at ERSF, Grenoble, ID14-1 at a wavelength of 0.8 Å. Inset shows observed resolution limit at 1.14 Å

The main parameters of this crystallisation approach [4] that are to be refined experimentally for each type of precipitating agent and for each protein are:

• The ratio of protein and monoolein mass

• The mixing ratio of volumes in the initial drop

• The total initial drop size

[1] E.M. Landau, J.P. Rosenbusch Proc. Natl. Acad. Sci. USA, 93, 14532 (1996)

[2] H. Qiu, M. Caffrey Biomaterials, 21, 223 (2000)

[3] B. Schobert, J. Cupp-Vickery, V. Hornak, S. Smith J. Lanyi

J.Mol.Biol. 321, 715 (2002)

[4] J. Kubicek, J. Labahn, F. Schäfer, G. Büldt Patent pending (2009)

1.5 Å

In vitro co-translational folding A. Katranidis1,4, M. Geritts2, K. Nierhaus3, T. Choli-Papadopoulou4, R. Schlesinger1, J. Fitter1, G. Büldt1 1Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) 2RiNA GmbH, Berlin 3Max-Planck-Institute for Molecular Genetics, Berlin 4Aristotle University of Thessaloniki, Greece

We demonstrate here by single molecule experiments that a green fluorescence protein (GFP) is produced with a characteristic time of five minutes. The fastest GFP molecules appeared already within one minute. Processes precedent to chromophore formation, such as polypeptide synthesis and protein folding, are fast and last not longer than one minute. In our approach a two color single molecule sensitive fluorescence wide-field microscope is employed in order to visualize surface tethered labeled ribosomes and de novo synthesized GFP molecules in real time. Fluorescence of co-translational folded proteins was observed from mature fluorescent GFP molecules which carry 31 additional amino acids at the C terminus remaining linked to the ribosome. Thus it was possible to co-localize fluorescence from labeled ribosomes and from GFP molecules.

Numerous studies showed that protein folding and maturation can differ substantially between de novo synthesized proteins and in vitro refolded proteins [1]. In classical folding studies formerly folded proteins need to be transferred into an unfolded state before the (re-)folding process can be studied. It has been demonstrated in several cases that protein folding takes place already during the elongation of the nascent chain (co-translational folding). Significant differences have been observed between both types of folding with respect to folding rates, to the appearance of folding intermediates, and to the yields. Therefore, one major goal is to understand how polypeptide chain elongation and folding are coupled. In particular single-molecule studies can yield valuable information about these rather asynchronous processes.

In this study we observed green fluorescent proteins (GFP) at a single molecule level after de novo synthesis and folding [2]. Formation of the fluorescent chromophore is a rather slow posttranslational autocatalytic process and the maturation kinetics as well as the folding efficiency between GFP wild type and several mutants differ significantly [3]. We have chosen the GFP Emerald (GFPem) mutant which is characterized by a high folding efficiency and by fast folding and maturation kinetics. GFP synthesis at surface-immobilized fluorescently labeled ribosomes was accomplished by using a fractionated cell-free transcription-translation E. coli system (Figure 1). The sequence of GFPem was elongated by a sequence of 31

amino acids at the C-terminus (spanning the full tunnel length) in order to ensure proper folding of the full length protein outside the tunnel. Suppression of protein release after synthesis keeps the synthesized GFP bound to the ribosome and allows to image GFP fluorescence for extended observation times.

FIG. 1: Schematic view of surface-tethered ribosomes (only the 50 S subunit is shown). The amino-functionalized cover slide is coated with a layer of PEG which is biotinylated at low concentration. By the use of a streptavidin-biotin binding assay fluorescently labeled ribosomes were linked to the surface via biotinylated ribosomal protein L4 (displayed molecules are not on scale). Cell-free synthesized GFPem becomes mature while linked to the ribosome.

For imaging fluorescently labeled ribosomes (with ATTO 655) and emerging GFP molecules we employed a dual color fluorescence wide-field microscope (Figure 2). Our images indicate that approximately 10 – 15% of all visible ribosomes produce a bound mature and fluorescent GFP. In a next series of measurements we monitored the appearance of individual synthesized GFP molecules as a function of time (Figure 3). For this purpose surface-immobilized ribosomes were incubated with a reaction buffer within a closed

imaging chamber and after a dead time of 40 seconds a sequence of images was taken every 15 seconds.

FIG. 2: Scheme of the two-color wide field setup used toimage fluorescently labeled ribosomes and emergingGFP molecules at the same time with single moleculesensitivity.

To our surprise GFPem fluorescence shows up rather fast with a significant fraction within five minutes after initiating polypeptide synthesis. According to the rather limited photostability of GFPem we observe in most cases photo-bleaching after few exposures and in some cases also photo-blinking. The time course of emerging fluorescent GFPem molecules is satisfactorily fitted by a single exponential. The corresponding characteristic time constant for the observed process is 5.3 minutes, which is one of the fastest maturation times for a GFP mutant observed so far. Typical maturation times of other GFP mutants of the S65T type range from 15 to 45 minutes, whereas wild type GFP shows even longer maturation times in the order of 2 hours.

An interpretation of our result has to consider at least three consecutive sub-processes, namely polypeptide synthesis, protein folding, and chromophore formation, as part of the whole biosynthesis. However, in our study GFP molecules become detectable only after chomophore formation. Therefore a characteristic time obtained from the kinetic data is related to the succession of all sub-processes. (i) First, we have to account for the polypeptide elongation with a synthesis rate of about 1-5 amino acid residues per second in cell free systems, while the corresponding in vivo rate is 10-20 residues per second. For our GFP construct with 306 residues (36aa+GFPem+31aa) this would last one to five minutes for a cell free system. (ii) Folding rates of GFP are typically known from refolding studies. The corresponding times range from around 4-5 minutes for concentrated proteins in solution to a few ten seconds in single molecule studies or in chaperonin-mediated refolding. As demonstrated here protein folding seems to be fast in our approach. (iii) Since the characteristic

chromophore formation time requires at least 5-10 minutes for de novo synthesized GFP molecules, we have to assume that the characteristic time constant obtained from our data (5.3 minutes) is related to the chromophore formation. Therefore we conclude that polypeptide synthesis and protein folding together must be faster than one minute.

CCDCamera

Microscopeobjective

Ar+ Laser488 nm

Tube Lens

DichroicMirror

Sample

EmissionFilter

Wedged Mirror

Dye Laser~ 640 nm

Beamsplitter

CCDCamera

Microscopeobjective

Ar+ Laser488 nm

Tube Lens

DichroicMirror

Sample

EmissionFilter

Wedged Mirror

Dye Laser~ 640 nm

Beamsplitter

FIG. 3: As an example integrated peak intensities are shown as a function of time for fluorescent GFP molecules appearing at different times after the initiation of biosynthesis. Fluorescence of individual GFP molecules can only be detected for a few consecutive exposures before photo-bleaching occurs.

High rates of folding and maturation are assumed to play a crucial role to reduce unwanted side reactions, such like misfolding and aggregation, and thereby improve the efficiency of protein biosynthesis in the cell.

Here we demonstrated that co-translational folding can be monitored on single molecule level by employing surface tethered ribosomes. Since the maturation time of the intrinsic GFP chromophore is in the order of minutes this approach does not allow to study faster events which are directly related to protein folding. Therefore, in future we aim to incorporate fluorescent dyes into the elongating nascent chain in order to monitor co-translational folding events by the use of Förster resonance energy transfer (FRET) or by fluorescence anisotropy measurements.

[1] A.H. Fedorov, T.O. Baldwin, J Biol Chem., 1997, 272, 32715-32718.

[2] A. Katranidis, D. Atta, R. Schlesinger, K.H. Nierhaus, T. Choli-Papadopoulou, I. Gregor, M. Gerrits, G. Büldt and J. Fitter, Angewandte Chemie Int. Edit., 2009, 48, 1758-1761

[3] M. Zimmer, Chem. Rev., 2002, 102, 759-781.

Crystal structure of PI3K SH3 domain in complex with an artificial peptide R. Batra-Safferling1, J. Granzin1, S. Hoffmann2, D. Willbold2 1Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) 2Institute of Structural Biology and Biophysics: Structural Biochemistry (ISB-3) Src homology 3 (SH3) domains are small protein modules (~60 residues), found in a number of intracellular signaling proteins, and are mediators of protein-protein interactions [1]. Here we investigated the crystal structures of the SH3 domain from phosphatidyloinositol 3-kinase (PI3K) in the presence of an artificial 12-residue proline-rich peptide PD1R (HSKRPLPPL PSL) (Table1) [2]. The crystal structure of the PI3K SH3-PD1R complex at a resolution of 1.7 Å reveals the type I ligand orientation. The bound peptide has an extended conformation where the central portion forms a left-handed type II polyproline (PPII) helix. The overall structure of SH3 domain shows minimal changes upon ligand binding. In addition, we also attempted crystallization studies with another artificial peptide ligand PD1Y where the anchor residue at position P-3 is a tyrosine. The crystal structure reveals that the ligand is missing but the binding site is occupied by the residues Arg18 and Trp55 from the symmetry related molecule. Considering the above mentioned crystal structures of PI3K SH3 and the published reports, we provide a comparative analysis of protein-ligand interactions that has helped us identify residues which play an important role in defining target specificity.

The role of SH3 domain is to mediate protein-protein interaction via binding to proline-rich motifs in the target proteins enabling the formation of multimeric signaling complexes. SH3 domains recognize unique proline-rich peptides bearing the core sequence PxxP (where P = proline, x = any amino acid), flanked by other residues specific to the domains. Binding of these peptides is not specific and the binding constants vary in milli- to micromolar range. In previous experiments, we identified several artificial peptide ligands from screening a phage displayed peptide library against lymphocyte specific kinase (Lck) SH3 domain [2]. The peptides screened follow a Class I (+xxPxxP) consensus sequence with an average length of 12-residues (+=basic residue; P=conserved residue proline; p=proline preferred) (Figure 1). Our efforts remain to structurally investigate several different SH3 domains in the presence of artificial peptide ligands that bind in μM range. The studies are aimed to first, elucidate the molecular interactions involved in binding mechanism and second, to shed light on the issue of specificity of the ligand binding to SH3 domains.

Recently, we solved the crystal structure of SH3 domain from PI3K kinase in complex with an artificial peptide PD1R at 1.7 Å resolution (Figure 1). The overall β-barrel fold of PI3K SH3 crystal structure is similar to that observed for the SH3 domains. The protein structure consists of two orthogonal β-sheets containing five antiparallel strands (4-11, 28-33, 54-60, 64-70, 74-80), two unique regular helices (34-41, 50-53), two short 310 helices and three loops (Figure 1).

Ligand binding: The bound peptide adopts a left-handed PPII helix conformation, seen for the class I orientation (Figure1). The residues with their side-chains facing the protein are Arg4, Pro7 and Pro10,

FIG. 1: Crystal structure of the PI3K SH3 domain in complex with ligand peptide PD1R. PI3K SH3 domain is shown as ribbon structure where beta strands, loops and alpha helices are colored red, yellow and blue, respectively. Two 310 helices are shown within the loops in dark blue. Residues of the peptide with side chains are shown as stick model with electron density from the final 2Fo-Fc map. The contour level is at 1σ. The amino acid sequence of the peptide PD1R is HSKRPLPPLPSL

Sequence KD value (μM)

PD1R HSKRPLPPLPSL 40

PD1Y HSKYPLPPLPSL 120

TABLE 1. Sequence and KD values of the artificial peptide ligands identified by Tran et al. [2].

where the residue Arg4 is at the anchor position P - 3, and residues Pro7 and Pro10 correspond to the positions P0 and P3, critical for ligand binding. The residues reside on three well-define pockets: The Pro7 sits in the pocket formed from residues Tyr14, Trp55, Pro70, and Tyr73 of the SH3 domain; Pro10 and side chain of residue Leu9 are both in a common pocket, interacting with residues Tyr12 and Tyr73 of the SH3 domain; Arg4 lies sandwiched between the residues Arg18 and Trp55 of the protein, and is surrounded by acidic residues Glu17, Asp21 and Asp68 where it forms salt bridges with Glu17 and Asp21 of the protein.

In a parallel attempt, we tried to co-crystallize PI3K SH3 domain with a modified peptide PD1Y, carrying a tyrosine at anchor position P-3 instead of an arginine (Table 1). Even though the KD values are in μM range, the protein crystallized as free PI3K SH3 domain and not as PI3K SH3-PD1Y complex. However, the ligand binding site is occupied by residues Arg18 and Trp55 of a symmetry related molecule where the side chains of arginine residues are sandwiched between the side chains of two tryptophan residues (Figure 2A). The Trp-Arg-Arg interactions seen are similar to those observed between the anchor residue Arg4 of the peptide PD1R and residues Arg18 and Trp55 of SH3 domain in PI3K SH3-PD1R complex.

It is known from previously solved structures and from the sequence analysis that Trp55 is a highly conserved residue that contacts the ligand and is important in ligand binding. In contrast, structure based alignments show that residue Arg18 is not conserved and a great deal of structural variations are seen at this position upon ligand binding. Such non-conserved positions that participate in ligand binding are predicted to play a role in defining specificity for the ligand. Here, we show that upon substitution of residue arginine at the anchor position by tyrosine in the ligand leads to binding in solution but in the crystal structure, ligand is not present. It is plausible that the Trp-Arg-Arg stack formation (as seen in the complex structure) is entropically a favorable event. The stabilizing role of Trp-Arg-Arg π-π-stacking has been recognized in crystal structures for several receptors. As the residue arginine is absent at the anchor position in the PD1Y ligand, the protein prefers residue Arg18 of the symmetry related protein molecule instead, allowing the crystal packing where the ligand is omitted.

We compared the PI3K SH3-PD1R complex crystal structure with our previously reported NMR

solution structure of hematopoietic cell kinase (Hck) SH3 domain in complex with PD1Y (Figure 2B) [3]. In the later structure, lysine at position P-3 substitutes the function of anchor residue, readjusting the C-terminal residues that cause a kink in the peptide (Figure 2B). Based on our studies, we suggest that the binding mechanism involves two steps – first, binding of the anchor residue and second, binding of the polyprolines.

FIG. 2: A. Interactions mediated by crystal lattice between the PI3K SH3 domain and the symmetry related molecule. Cell boundary is shown in gray line. B. Super-imposition of PI3K SH3 bound PD1R (yellow) with HcK SH3 PD1Y(red). Also shown is the side chain of residue Trp55 of SH3 domains.

In future studies, we would investigate the role of non-conserved residues (like Arg18 of PI3K) which not only play a role in ligand binding, but also in the selection of their respective ligands.

[1] T. Pawson, and G. D. Gish, Cell, 71, 359 (1992).

[2] T. Tran, S. Hoffmann, K. Wiesehan, E. Jonas, C. Luge, A. Aladag and D. Willbold, Biochemistry, 44, 15042 (2005).

[3] H. Schmidt, S. Hoffmann, T. Tran, M. Stoldt, T. Strangler, K. Wiesehan, and D. Willbold, J. Mol. Biol., 365,1517 (2006).

Studies on multi-domain protein folding T. Rosenkranz1, J. Fitter1 1Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) Most of fundamental studies on protein folding have been performed with small globular proteins consisting of a single domain. In vitro many of these proteins are well characterized by a reversible two state folding scheme. However, the majority of proteins in the cell belong to the class of larger multi-domain proteins which often unfold irreversible under in vitro conditions. This makes folding studies difficult or even impossible. A promising approach in this respect is given by single molecule techniques which experienced tremendous methodological advances in recent years. In particular fluorescence based methods can contribute significantly to proceed in understanding fundamental principles of multi-domain protein folding.

In prokaryotic and even more in eukaryotic cells the predominant fraction of the whole proteome belongs to the class of multi-domain proteins. However, because of methodical reasons our existing knowledge about mechanisms and principles of protein folding results mainly from studies on smaller single-domain proteins. In particular competing side reactions such as misfolding and aggregation of non-native states makes folding studies on larger multi- domain proteins often difficult or even impossible under in vitro conditions. Therefore, working at low protein concentrations and employing single molecules studies are well suited to extend our knowledge on multi-domain protein folding. Extremely low protein concentrations (~ nM), as used for example in fluorescence correlation spectroscopy (FCS) studies, can lower or even circumvent aggregation of unfolded states. In principle, this can make refolding studies feasible, which would be hampered by aggregation at higher protein concentrations [1,2] (Fig. 1). In addition, the application of single molecule techniques has an enormous potential for studying the intrinsically heterogeneous process of protein folding, not only for multi-domain proteins. For protein folding studies on single molecule level mainly FCS and energy transfer techniques (FRET: Förster resonance energy transfer and PET: photoinduced energy transfer) have been employed. FCS is an easily applicable technique which needs only one fluorescent dye to be attached to the protein of interest. The diffusion time of the fluorescently labeled protein through the confocal volume of a tightly focused laser beam allows to determine the hydrodynamic radius (Rh) of the protein. Typically,

the native state is compact and exhibits a smaller hydrodynamic radius as compared to the more expanded unfolded state. For α-amylase at least a partial refolding was observed only at extreme low proteins concentrations (~nM) as used in FCS, while at higher protein concentrations (~μM) refolding was hindered due to aggregation (Fig.1B).

0 1 2 30,00

BLA_unfolding BLA_refolding

GndHCl [M]

0,01 0,1 1 10 1000,0

time [ms]

BLA_native BLA_unfol BLA_refol

FIG. 1: (A) Schematic representation of the Bac. Licheniformis α-amylase (BLA) structure. The color code (green, orange, cyan) represents the domain structure. (B) Transitions as observed under unfolding and refolding conditions measured with CD spectroscopy at a protein concentration of about 3 μM. As shown here, proper refolding is not taking place. (C) Autocorrelation curves as measured with FCS for BLA in the native state, in the unfolded stated, and in the refolded state. Here at low protein concentrations (~ 1nM) we observe at least a partial refolding (red line moves back to that for the native state).

Another approach to avoid protein aggregation, in particular of the unfolded state, is to encapsulate single molecules in surface-tethered nano-containers (Fig. 2A). By this kind of encapsulation individual proteins are separated from each other and represent an ideal probe for single molecule studies.

Fig. 2: (A) Scheme of an individual in a polymersomeencapsulated protein labeled with a fluorescent dye. (B) Wide field fluorescence image of surface tetheredpolymerosomes containing Atto655 labeled PGK. (C) A typical time-course of the measured emission intensity asobtained from the integration of an individual spot. Thecorresponding images (see B) were measured every 30 seconds with polymerosomes bound to cover-slides which were built-in a closed imaging chamber suitable forin-situ buffer exchange. The arrows indicate bufferexchange from native to unfolding conditions or viceversa.

Immobilizing biomolecules provides the advantage to observe them individually for extended time periods, unlike in case of freely diffusing molecules

in solution. In order to immobilize individual protein molecules, we encapsulate them in polymeric vesicles made of amphiphilic triblock-copolymers and tether the vesicles to a cover slide surface. A major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein folding studies. The fact that polymeric vesicles possess an extreme stability with respect to various chemical conditions is supported by our observation that harsh unfolding conditions do not perturb the structural integrity of the vesicles. Moreover, polymerosomes prove to be permeable to GdnHCl and thereby ideally suited for unfolding and refolding studies with encapsulated proteins. We demonstrate this with encapsulated phosphoglycerate kinase (PGK), which was fluorescently labeled with Atto655, a dye that exhibits pronounced photoinduced electron transfer (Fig. 2A). In the case of PGK we were able to make use of PET due to the fact that Atto655 attached at a defined position within the protein structure is efficiently quenched by a tryptophan residue located in close proximity to the dye (low emission intensity). Upon unfolding, a structural expansion takes place and the average distance between the dye and the quencher is increased which results in a lower quenching efficiency (high emission intensity, Fig. 2C).

We demonstrated here that proteins encapsulated in polymeric vesicles offer the possibility to study individual proteins for extended time periods. The encapsulation procedure provides a native-like environment for water soluble proteins without detectable hindrance of rotational reorientations. The high structural stability and considerable longevity of the polymerosomes qualifies them to be an ideal tool for single molecule studies. The permeability of triblock-copolymer membranes for GdnHCl and their resistance against structural disintegration at high denaturant concentrations ensure ideal conditions for their use in protein folding studies. At thermodynamic midtransition points (e.g., GdnHCl1/2, T1/2, pH1/2) in particular FRET based approaches provide structural details during folding and unfolding transitions of the proteins. At these conditions the folded and unfolded states have life times in the order of seconds and multiple successive unfolding/ refolding transitions can be observed with FRET for single encapsulated proteins. A major goal for future studies will be the focus on proteins that exhibit unfolding/folding transitions slow enough that trajectories of these transitions can be followed.

[1] K.H. Strucksberg, T. Rosenkranz, J. Fitter, BBA,

2007, 1774(12), 1591-1603

[2] J. Fitter, Cell. Mol. Life Sci., 2009, 66(5), xx-xx

[3] T. Rosenkranz, A. Katranidis, D. Atta, I. Gregor, J. Enderlein, M. Grzelakowski, P. Rigler, W. Meier

and J. Fitter, ChemBioChem, 2009,10,702-709

ISB-3: Structural Biochemistry Director: Prof. Dieter Willbold

The research areas of the Structural Biochemistry group comprise the investigation of precise three-dimensional structures and dynamics of biologically and medically relevant macromolecules in order to fully understand their functions and their related cellular processes. Research is focused on proteins that are involved in diseases like AIDS, SARS and neurodegenerative disorders to study the molecular basis of these diseases and to investigate novel approaches to therapy.

Some of our target proteins are fibril forming or membrane proteins and are therefore not readily amenable to structural investigation. Our research purposes include e.g. the investigation of the structural basis of protein-protein and protein-ligand interactions to explore how viral proteins of HIV and SARS-CoV interact with host cell proteins to use and reprogram the host cell for viral replication and protection against the host immune system.

Furthermore, ultra-sensitive assays potentially suitable for early and non-invasive diagnosis of neurodegenerative disorders like Alzheimer’s disease and prion diseases are developed.

To study these phenomena several molecular biology and biophysical techniques, e.g. liquid and solid state NMR, fluorescence correlation spectroscopy as well as, in cooperation with ISB-2, X-ray crystallography are developed and applied. Moreover, computational biology approaches in the fields of structural biology are performed.

D-peptides for diagnosis and therapy of Alzheimer’s disease S. A. Funke1, T. van Groen2, I. Kadish2, L. Nagel-Steger3, K. Wiesehan1, D. Willbold1,3 1ISB-3: Structural Biochemistry 2Dept. Cell Biology, University of Alabama at Birmingham, AL 35294, USA 3Institute of Physical Biology, Heinrich Heine-Universität Düsseldorf The “amyloid cascade hypothesis” assigns the amyloid-beta-peptide (Aβ) a central role in the pathogenesis of Alzheimer’s disease (AD). We searched for peptides consisting solely of D-enantiomeric amino acids (D-peptides) with strong binding to Aβ(1-42). D-peptides are thought to be protease resistant and less immunogenic than the respective L-enantio-mers. Using mirror image phage display we identified two interesting D-peptides having different properties concerning Aβ cytotoxicity and Aβ aggregation. Peptide D1 is being developed into a probe for in vivo imaging of amyloid plaques in the living brain, wheras peptide D3 has interesting therapeutical properties.

Alzheimer´s disease (AD) is a progressive, neurodegenerative disorder which is characterized by loss of brain functions. More than 20 million people are affected worldwide. The histopatho-logical hallmarks are protein deposits (senile plaques and neurofibrillary tangles) in the brain. One of the typical hallmarks of AD, senile plaques, consists mainly of extracellular Amyloid-β peptide (Aβ) deposits. Aβ is a 4 kDa peptide of 39 to 43 amino acids derived from proteolytic cleavage of the amyloid precursor protein (APP) by two enzymes, β- and γ-secretase. Soluble Aβ polymerizes to form neurotoxic oligomers, which also fold to fibrils, and deposits around neurons. These deposits spread in different areas of the brain and the "amyloid-cascade-hypothesis" assigns those Aβ plaques a central role in the pathogenesis of the disease.

So far, no method for pre-symptomatic diagnosis of AD is available. To improve diagnosis and treatment evaluation, neuroimaging tools, making use of Aβ-binding ligands visualizing amyloid plaques, are developed. A mirror image phage display approach was used in our lab to identify novel and highly specific ligands for aggregated Aβ(1-42).

Mirror image phage display allows the use of phage display to ultimately identify peptides that bind specifically to a given target and consist solely of D-amino acids. D-Amino-acid peptides are known to be less protease sensitive, more resistant to degradation in animals and less or even not at all immunogenic as compared to L-amino-acid peptides. In order to obtain the exact

mirror image of a given protein (L-protein), it is necessary to synthesize a protein with the same amino acid sequence but composed exclusively of D-amino acid residues (D-protein). Such a D-protein can be used as a target for phage display selection like any other target. In the study described herein, we used the D-enantiomer of Aβ as target and a 12-mer randomized peptide library displayed on the surface of M13 bacteriophages to be screened for D-Aβ-binding peptides. For reasons of symmetry, the D-enantiomeric form of the selected 12-mer peptide should interact with the Aβ peptide consisting of L-amino acids.

Here, a phage displayed peptide library with more than 1 x 109 different 12mer peptides was screened for peptides with binding affinity to the mirror image of aggregated Aβ(1-42) [1].

The resulting peptide, D1, binds very specifically to Aβ aggregates with a binding constant in the submicromolar range. Fibrillar deposits derived from other amyloidoses are not stained. As can be seen in Fig. 1, D1 binds to Aβ deposits in brains of AD transgenic mice (APPswe-PSΔ9) in vivo [2]. Although D1 shows interesting properties in reducing Aβ cell toxicity and amyloid formation assays [3], it will mainly be developed as a molecular probe for in vivo imaging of amyloid plaque load in AD patients.

Fig.1: FITC-labelled D1 binds to Aβ plaques in the brains of transgenic APPswe-PSΔ9 mice in vivo. The peptide was directly injected in the brain of the mice. One week after injection, the mice were sacrificed and the brains were investigated by histochemical analysis.

Although there is still controversial discussion if Aβ is the causative agent in AD, inhibition of Aβ production and aggregation are often addressed for therapy development. During a second phage display selection with monomeric or low molecular weight oligomeric D-Aβ(1-42), we obtained peptide D3, which modulates Aβ aggregation and reduces Aβ cytotoxicity in vitro (Fig. 2).

A 1-42 concentration during aggregation (µM)β

00 10 25

** no D310 µM D3100 µM D31 mM D3

no D310 µM D3100 µM D31 mM D3

Fig.2: PC12 cell viability in absence or presence of Aβ and/or D3. Different concentrations of D3 were added to 10 µM Aβ samples or to samples without Aβ as controls. The Aβ/D3mixtures were incubated for 6 days at 37°C and 1:5 diluted into PC12 cell cultures. Cell viability was measured using MTT assay. Percentages of cell viability were derived as follows: The 100 % value was obtained from cells treated neither with the respective peptide nor with Aβ; the value of 0 % was obtained by treatment of the PC12 cells with 0.2 % Triton-X.FITC-labelled

In cooperation with Thomas van Groen (University of Alabama, Birmingham, USA) we carried out investigations on the effects of D3 on Aβ deposits in brains of transgenic mice (APPswe/PSΔ9). These mice develop elevated levels of Aβ at four months of age and at the age of 5 months they show amyloid plaque pathology. An inspection of the brain sections showed a significant reduction of the Aβ-load in the hippocampus and frontal cortex after treatment with D3 (see Fig. 3) [4].

Fig. 3: Influence of D3 on Aβ load in brain tissue sections of transgenic APPswe-PSΔ9 mice. Saline (Control), D1 as a peptide control or D3 were infused into the brains of the mice for four weeks. Animals were transcardially perfused at the end of the infusion period, and coronal sections were cut through the brain, one series of sections was stained with W0-2 (anti human amyloid-β). Representative sections showing the hippocampus and dorsal cortex are shown. Please note the decrease in Aβ staining in the D3 infused brain compared to the control brains.

Detailed analysis of the inflammation (i.e., density of activated astrocytes and microglial cells) around remaining Aβ deposits revealed that the D3 treatment significantly reduced inflammation

processes. The number of activated astrocytes and microglial cells (measured as density of GFAP and CD11b staining) near plaques of equal size was significantly less compared to the control brains (Fig. 4), indicating that D3 changes the inflammatory properties of Aβ.

Figure 4. Influences of D3 on inflammation in brain tissue sections of transgenic APP-PSΔ mice. Saline (Control) or D3 was infused in the brains of the mice for four weeks. Animals were transcardially perfused at the end of the infusion period, and coronal sections were cut through the brain, one series of sections was stained with GFAP or CD11b. Representative sections showing Congo red positive plaques the dorsal cortex are shown. Please note the decrease in inflammation (activated astrocytes and microglia) surrounding Congo red-positive plaques in the D3 infused brain compared to the control brain.

In recent studies, D3 improved the cognitive performance of treated mice in the water maze assay after oral application (unpublished results). We suggest an Aβ (1-42) modulating activity of the peptide as it clearly interacts with early intermediate assemblies of Aβ and promotes fibrillization (unpublished results). Recents studies indicate that soluble Aβ oligomers (also called protofibrills, prefibrillar aggregates and ADDLs) instead of fibrillar forms are the major toxic species in AD. Therefore, agents that interfere with early oligomerization processes might be valuable for therapy of AD. Therefore, D3 is a good starting point for therapy development.

[1] Wiesehan K, Buder K, Linke RP, Patt S, Stoldt M,

Unger E, Schmitt B, Bucci E, Willbold D. ChemBioChem 4, 748-753 (2003).

[2] Van Groen T, Kadish I, Wiesehan K, Funke SA, Willbold D. ChemMedChem 4, 276-282 (2009).

[3] Wiesehan K, Stöhr J, Nagel-Steger K, van Groen T, Riesner D and Willbold D Prot. Eng. Des. Sel. 21, 241-246 (2008).

[4] Van Groen T, Wiesehan K, Funke SA, Kadish I, Nagel-Steger L and Willbold D ChemMedChem 3: 1848-1852 (2008). [This article was evaluated by the Faculty of 1000 Biology as a "must read"]

Human CD4: NMR structure and interactions of C-terminal domain M. Wittlich1,2, B. W. Koenig1,2, S. Hoffmann1,2, D. Willbold1,2 1ISB-3: Structural Biochemistry 2Institute of Physical Biology, Heinrich-Heine-Universität Düsseldorf The type 1 transmembrane glycoprotein CD4 of 58 kDa plays a key role in the adaptive immune response. CD4’s ectodomain serves as main receptor of human immunodeficiency virus (HIV) and binds the HIV-1 envelope glycoprotein gp120. Physical interactions of HIV-1 accessory proteins Nef and Vpu with the cytoplasmic tail of CD4 result in down-regulation of CD4 from the surface of infected cells. Our goal is the detailed characterization of the structure of the membrane-anchored C-terminal domain of CD4 in complex with Vpu and Nef, respectively. Here we present an efficient protocol for recombinant production, purification, isotope labelling, and membrane reconstitution of CD4tmcyt, containing the transmembrane and cytoplasmic domains of CD4. Specific binding of liposome-reconstituted CD4tmcyt with the cytoplasmic domain of Vpu is demonstrated. The NMR structure of CD4tmcyt in membrane mimicking micelles is presented.

Human CD4 consists of an extracellular region of 371 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids (Fig. 1). The CD4 T-lymphocyte coreceptor belongs to the IgG-superfamily and participates in T-cell activation and signal transduction. In addition to these functions, CD4 is the major receptor for HIV infection. The virus is internalized after binding of HIV-1 gp120 to the extracellular domain of CD4. Following infection, two additional viral proteins, Vpu and Nef, interact with CD4, but bind to the cytoplasmic domain of CD4. Vpu-induced degradation of CD4 molecules in the endoplasmatic reticulum requires both proteins to be inserted into the same membrane. The CD4 sequence relevant for this activity is located between amino acids 402-420 [1]. In contrast, Nef acts at the cell surface to mediate the internalization and lysosomal degradation of CD4. Residues 407 to 418 in the cytoplasmic tail of CD4, with special emphasis on the dileucine motif at positions 413 and 414, are necessary and sufficient for down-regulation of CD4 by Nef [2-4].

Recombinant production of transmembrane proteins in E.coli is very challenging. It often results in insoluble protein aggregates, low protein yield, and numerous obstacles during protein refolding and membrane reconstitution. We produced CD4tmcyt as part of a fusion protein containing an N-terminal decahistidine tag and the

soluble protein ubiquitin (Fig. 1) [5,6]. Supplementing all buffers employed for cell lysis, purification, and enzymatic cleavage of the fusion protein with a dedicated mixture of non-ionic surfactants was crucial for increasing solubility of the protein and for the exceptionally high yield of purified CD4tmcyt. We obtained ~6 mg CD4tmcyt per liter of bacterial culture.

FIG. 1: Schematic representation of human CD4 (above) and of the recombinantly produced fusion protein (below). CD4 features a large extracellular domain (EXT), one transmembrane span (TM), and a carboxy-terminalcytoplasmic tail (CYT). The N-terminal tag of the fusion protein consists of yeast ubiquitin preceded by a dipeptide (MG), a decahistdine tag, and the 11 residue linker L1 (SSGHIDDDDKH). Linker L2 contains PreScission and thrombin recognition sequences. Protease cleavage sites are indicated by asterisks. The amino acid sequence of the 70 residue CD4tmcyt protein obtained after PreScission cleavage is shown in the lower part. The authentic CD4 residues 372 to 433 are highlighted (dark orange: TM; light orange: CYT).

CD4tmcyt contains the 60 C-terminal amino acid sequence of wild type human CD4, including five cysteine residues. We also produced the cysteine-free protein CD4mut with substitutions C394S, C397S, C420S, C422S, and C430H at similar yield using the same strategy [6]. We plan to introduce single cysteines into this mutant for attachment of ESR or fluorescence labels.

Both CD4tmcyt and CD4mut were successfully reconstituted into liposomes. Binding of Vpucyt (the C-terminal amino acid residues 39-81 of Vpu) to the CD4 fragments was demonstrated with a liposome centrifugation assay [5,6]. The amount of soluble Vpucyt in the supernatant and in the membrane fraction, respectively, was quantified by reversed phase HPLC (Fig. 2). Our data indicate specific binding of the recombinant CD4 domain to Vpucyt.

FIG. 2: Analysis of CD4mut Vpucyt interaction in POPCmembranes using centrifugation followed by reversedphase chromatography. Dispersions of POPC liposomes(10 mg POPC per sample, 1 mL each) containing varyingamounts of reconstituted CD4mut (0 mg – above; 0.5 mg –middle; 2 mg – below) were incubated with 50 μM Vpucyt. Fully hydrated liposomes were separated from supernatant by centrifugation. The composition ofsupernatant (left column) and pellet fractions (rightcolumn) was analyzed by analytical RPC using a gradientof buffers A and B (dashed line). Position of the VpUcyt(1), CD4mut (2), and POPC (3) peaks are indicated.

We determined the conformation of CD4mut in membrane mimicking dodecylphosphocholine (DPC) micelles using high resolution NMR (Fig. 3) [6]. Two α-helices were identified based on NOE-derived 1H-1H distances and secondary chemical shifts. They extend from M372 to V395 and from M407 to R412, in good agreement with the predicted transmembrane and cytoplasmic helices, respectively. The mutual orientation of the two helices remains undetermined due to lack of long range NOEs. The location of CD4 residues relative to the micelle was studied with paramagnetic probes: Supplementing the buffer with manganese ions (MnCl2) strongly broadens resonances in the cytoplasmic helix and in the interhelical loop, but shows little effect on resonances of the

transmembrane helix. In contrast, incorporation of 16-doxylstearic acid into the hydrophobic core of the micelle affects only resonances in the transmembrane helix.

Our next goal is incorporation of membrane proteins like CD4tmcyt into nanodiscs, a novel phospholipid-based model membrane, followed by NMR structural studies and interaction analysis.

FIG. 3: Schematic representation of the transmembrane and cytoplasmic helices of CD4mut in a detergent micelle. The ribbon diagram reflects the length of the two helices derived from 1H – 1H distances and secondary shift analysis. The position of the two helices with respect to the micelle was deduced from signal broadening caused by paramagnetic probes. The two cylinders surrounding the helices symbolize the space requirement of the amino acid side chains.

[1] M. Y. Chen, F. Maldarelli, M. K. Karczewski, R. L. Willey, and K. Strebel, J.Virol. 67, 3877 (1993)

[2] C. Aiken, J. Konner, N. R. Landau, M. E. Lenburg, and D. Trono, Cell 76, 835 (1994)

[3] S. J. Anderson, M. Lenburg, N. R. Landau, and J. V. Garcia, J. Virol. 68, 3092 (1994)

[4] A. Preusser, L. Briese, A.S. Baur, and D. Willbold, J. Virol. 75, 3960 (2001)

[5] M. Wittlich, K. Wiesehan, B. W. Koenig, and D. Willbold, Protein Expr.Purif. 55, 198 (2007)

[6] M. Wittlich, B. W. Koenig, S. Hoffmann, and D. Willbold, Biochim. Biophys Acta 1768, 2949 (2007)

Mechanism of prion protein conversion and assembly E. Birkmann1,2, J. Stöhr2, T. Kaimann2, G. Panza, D. Willbold1,2, D. Riesner2 1ISB-3: Structural Biochemistry 2Institute of Physical Biology, Heinrich-Heine-University Düsseldorf Prion diseases are a unique group of neurodegenerative diseases because they are know as transmissible. They can occur both spontaneously and genetically caused. The infection is a conversion of host encoded prion protein (PrP) from its cellular isoform PrPC into the pathological and infectious isoform PrPSc [1]. We investigated the conversion process by in vitro studies using recombinant PrP and natural PrPSc. The results determined with our in vitro conversion system and the derived mechanistic models are presented as a brief summary. Furthermore, well characterized intermediates and precursor states during the conversion process as well as kinetic studies of spontaneous and seeded fibrillogenesis are described.

The mechanism of spontaneous prion protein conversion was studied systematically with recombinant (rec) PrP. Our in vitro conversion system is based on the solubilization of PrP in low concentrations (0.2% w/v) of sodium dodecyl sulfate (SDS) under otherwise physiological conditions. The conversion is induced by diluting the SDS. Most extended and systematic studies were carried out with hamster recPrP at neutral pH.

Different conformations could be established. The conformations were characterized with respect to secondary structure as determined by CD spectroscopy and molecular mass as determined by fluorescence correlation spectroscopy and analytical ultracentrifugation. With these methods α-helical monomers, soluble α-helical dimers, soluble but β-sheet rich oligomers of a minimal size of 12–14 PrP molecules, and insoluble amorphous aggregates of β-sheet rich structure were observed. A high activation barrier was found between the α-helical dimers and the β-sheet rich oligomers.

In Fig. 1 the different structures are represented schematically together with their CD-spectra and the EM picture of the polymorphic aggregates. In 0.2% SDS PrP is present in an α-helical and partially random coil conformation denoted with α/R. Addition of 250 mM NaCl shifts PrP into a still soluble state that allows fibril assembly to proceed over several weeks. The structure of the intermediate state PrP* is decisive to switch aggregate formation from the polymorphic

pathway into the fibrillar pathway. Therefore, we called this state fibril precursor state and characterized it by different methods. CD spectroscopy showed a mixture of α-helical and random coil secondary structure. We assume that the α-helical part is identical or close to that which is also present in the fibrillar structure. Studies using analytical ultracentrifugation demonstrated the existence of a monomer-dimer equilibrium.

FIG. 1: Molecule populations involved in the in vitroconversion of recPrP(90-231) induced by lowering SDS concentration. The molecule populations analysed within the in vitroconversion system are summarized. Exemplarily for the methods used to analyse these structures the CD-spectra are presented beneath the different intermediates. Next to the aggregated states the electron micrograph of these structures are shown. The upper part of the figure represents the conversion without added NaCl, the lower part with added NaCl, respectively. (Figure according.[3])

The studies described above indicated a critical role of PrP dimers in the conversion process consistent with research results from other groups. Consequently, we studied the structure of dimers in more detail. We selected conditions which stabilize dimers or drive the dimer-oligomer equilibrium completely to the side of dimers, respectively [4]. recPrP(90-231) in 0.06% SDS 10 mM phosphoric buffer pH 6.8 without additional NaCl is 100% dimeric. To obtain information on the conformation of the dimer, we used the covalent crosslinker EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide), which forms

inter- and intramolecular bonds between directly neighboured amino and carboxyl groups. The bonds were identified by tryptic digestion and subsequent mass spectrometric analysis. Intra- and intermolecular cross-links between N-terminal glycine and three acidic amino acid side chains in the globular part of PrP were identified showing that the N-terminal amino acids (90–124) are not as flexible as known from NMR analysis. When the cross-linked sites were used as structural constraints, molecular modelling calculations yielded a structural model for PrP dimer and its monomeric subunit including the folding of amino acids 90–124 in addition to the known structure of the core domain (Fig. 2). Molecular dynamics simulation with the dimeric structure after release of the crosslink constraints indicated the domain formed by amino acids 90–124 of both momomers to be intrinsically stable. The structural model (Fig. 2) is in agreement with GPI-anchoring of the dimer to the membrane. Demonstrated by arrows the two glycolipid anchors are directed to the lower side where the membrane would be present, whereas the glycosyl groups and the N-termini are directed into the water phase.

FIG. 2: Model of the prion protein dimer. Stereo presentation of the model for the PrP dimer. Thestructure of segment 92–124 (blue) is the result of [4], the structure of segment 125–228 (cyan) is taken from theNMR analysis reported in the literature. Red arrowsrepresent the glycolipid anchor; thick blue arrows,glycosyl groups; and thin blue arrows, N-termini. (Figureaccording [4])

As a model for the infection process fibril formation was also studied when induced by seeds of PrPSc. We used natural PrPSc purified from brain homogenate as seeds. RecPrP was applied as substrate. The fibrillization conditions were identical to those used for spontaneous fibrillization. We monitored and quantitatively described the kinetics of seeded fibril formation. Fibrillization of PrP without seeds takes up to several weeks, whereas seed-enhanced fibrillization was achieved within hours to days (Fig. 3). During the enhanced fibrillization a lag phase is followed by exponential growth and saturation.

FIG. 3: Seeded fibril formation RecPrP (80 ng/μl) seeded with purified PrPSc (diamonds) forms amyloid fibrils readily compared with controls: recPrP + uninfected 1.6 x 10-4 brain equivalents per μl (x); recPrP alone (triangles); purified PrPSc alone (dashes). Fibril formation was monitored by using the ThT fluorescence assay. Electron micrographs: PrPSc

aggregates after NaPTA precipitation, recPrP + PrPSc

after seeding assay. (Figure rearranged according [2])

To compare the in vitro conversion and the seeding efficiency in respect of differences in different species, we adopted the in vitro conversion system to bovine recPrP [5]. We could observe some differences in the structure of the fibril precursor state, but the principle conversion mechanism could be approved as described above with recPrP.

[1] Prusiner SB. (1998) Proc. Natl. Acad. Sci. USA; 95, 13363-13383.

[2] Stöhr J, Weinmann N, Wille H, Kaimann K, Nagel-Steger L, Birkmann, E, Panza G, Prusiner SB, Eigen M & Riesner D (2008) Proc. Natl. Acad. Sci. USA. 105 (7), 2409-2414.

[3] Birkmann E & Riesner D. (2008) Prion, 2 (2), 67-72.

[4] Kaimann T, Metzger S, Kuhlmann K, Brandt B, Birkmann E, Höltje H-D and Riesner D. (2007) J. Mol. Biol. 376, 582-596

[5] Panza G, Stöhr J, Dumpitak C, Papathanassiou D, Weiss J, Riesner D, Willbold D, Birkmann E. (2008) Biochem. Biophys. Res. Commun. 373 (4), 493-497

Solid-state NMR reveals structural differences between fibrils of wild-type and mutant α-synuclein Henrike Heise1, 2, M. Soledad Celej3, Stefan Becker3, Dietmar Riedel3, Avishay Pelah3, Ashutosh Kumar3, Thomas M. Jovin3, and Marc Baldus4 1ISB-3: Structural Biochemistry 2Institute of Physical Biology, Heinrich-Heine-Universität Düsseldorf 3Max-Planck-Institute of Biophysical Chemistry, Göttingen 4Utrecht University, The Netherlands The 140 residue protein α-synuclein is able to form amyloid fibrils and as such, is the main component of protein inclusions involved in Parkinson’s disease. Three point-mutations of α-synuclein have been related to familial early-onset Parkinson’s disease. We have investigated the structure and dynamics of fibrils from full-length α-synuclein and of its disease-related mutant A53T by high-resolution solid-state NMR spectroscopy, Electron Microscopy and Atomic Force Microscopy. In both cases the C-terminus was found to be flexible and unfolded, whereas the main core region is highly rigid and rich in β-sheets. Compared to fibrils from wild-type α-synuclein, the well-ordered β-sheet region is extended. These results demonstrate that a disease-related mutant of α-synuclein differs in both aggregation kinetics and fibril structure.

Parkinson’s disease, the most common neurodegenerative movement disorder, is caused by the loss of dopaminergic neurons in the substantia nigra, which is accompanied by the formation of cytosolic filamentous inclusions called Lewy bodies. The major constituent of these inclusions consists of fibrillar α-synuclein, a 140-residue protein, which in its native conformation is largely unfolded. The central role of α-synuclein aggregation in the etiology of Parkinson’s disease has been further corroborated by the identification of a locus triplication of the gene encoding α-synuclein as well as the three point-mutations A53T, A30P, and E46K as causes of autosomal dominant Parkinson’s disease.

Since their discovery, the three disease-related mutants have been the subject of intense study, as differences in aggregation behavior and morphology with respect to the wild-type form may yield valuable insights into the fundamental processes underlying amyloidogenic diseases. As in the case of wild-type α-synuclein, all three mutants are natively unfolded, and significant acceleration of fibrillization has been observed for the mutants A53T and E46K.

However, the overall morphology of amyloid fibrils in general depends strongly on the fibrillization conditions, including pH, temperature, salt concentration and agitation of the solution. Upon fibrillization of wild-type α-synuclein at physiological temperature and pH, at least two different polymorphic forms, one consisting of straight and the other of irregularly twisted fibrils, have been obtained and characterized [1].

Studies of wild-type α-synuclein by solid-state MAS NMR-spectroscopy have revealed a β-sheet rich core region spanning residues from at least 38 to 94 for the two different polymorphic isoforms, whereas the C-terminus was flexible and unfolded in both cases, and the supramolecular arrangement was found to be a superpleated β-sheet with parallel and in register alignment of β-strands [2] (Figure 2b). In the following, we report studies on fibrils grown from the disease-related A53T mutant of α-synuclein by solid-state NMR Spectroscopy [3].

FIG. 1: 2D 13C/13C correlation spectra of A53T α-synuclein fibrils recorded at effective sample temperatures of ~ 0 °C (red) and -20 °C (black). Homonuclear mixing was achieved by proton-driven spin diffusion for 20 ms.

In Figure 1, two 13C/13C correlation spectra of A53T fibrils, recorded above and below the freezing point, are displayed. While the cross-correlation signals in the high-temperature spectrum are narrower and better resolved, the low-temperature spectrum displays some additional sets of signals, especially in the region typical for non β-strand secondary structures: In hydrated amyloid fibrils, different regions of a protein may exhibit diverse residual mobility, and solid-state NMR spectra based on dipolar transfer mechanisms often show only a subset of all resonances in the protein. Freezing the sample reduces the overall mobility such that flexible parts may give rise to inhomogeneously broadened signals, depending on the degree of disorder. Alternatively, flexible regions can selectively be detected by applying adequate mobility filters in the MAS NMR experiments [4].

FIG. 2: a) Backbone angles obtained with TALOS [5]analysis of the chemical shifts. Only torsion angle pairswhere at least 8 hits were found are plotted, together withthe error bars. For comparison, β-strands determinedearlier for both wild-type forms23 are given together withthe β-strands of A53T. b) superpleated β-sheet within-register parallel alignment of β-strands

The results of our investigations are summarized in Figure 2 a. Fibrils consist of a well-ordered rigid core region rich in β-sheets, and a highly flexible and unfolded C-terminus, whereas the N-terminus lacks high mobility as of residue 22. Site-specific resonance assignments for a large part of the amino acids in the core regions were obtained from 2D homonuclear 13C/13C and heteronuclear 15N/13C correlation spectra. Intrinsic dynamics of the protein were probed by spectroscopy at different temperatures as well as by a combined mobility filter experiment specific for mobile regions of immobilized molecules. According to

our results, almost all resonances indicative of a β-sheet conformation are in rigid protein compartments. Upon freezing the sample, resonances with random coil and α-helical secondary chemical shifts appear in the spectrum, indicating that the fibrils contain regions with increased mobility and structural elements other than β-sheet, while the intensities of resonances in β-strands do not increase unexpectedly. For example, the low-temperature spectrum indicates the existence of at least one valine residue with a chemical shift typical for α-helical conformation, which is neither part of the flexible C-terminus nor of the rigid core region, but belongs to a region of intermediate mobility. Similar to our previous results obtained on wild-type α-synuclein, the C-terminus is unfolded and highly dynamic. The striking difference of the A53T compared with wild-type α-synuclein is an increase in the β-strand character of two leucine residues at the edges of the core region, indicating an extension of the rigid, well-ordered β-sheet rich core region towards at least residue L38 and L100. The accelerated aggregation kinetics itself could result in a different pattern of molecular assembly characterized by an extended β-sheet core region. However, in fibrils of both the mutant and wild-type α-synuclein, the C-terminus extending from at least residue 107 is flexible, whereas the N-terminus extending from residue 22 is rigid. In contrast, in a recent study, obtained on two design mutants A56P and A30P/A56P/A76P, where proline residues were inserted into β-strands in order to retard fibrillization and shift the equilibrium towards early monomers, the N-terminus was found to be flexible as well [6].

These results provide important insights into the effect of changed aggregation properties on the final fibril morphology of one disease-related mutant of α-synuclein. Further studies of this nature may help to delineate the relationship between the etiology of Parkinson’s disease and protein misfolding.

[1] H. Heise, W. Hoyer, S. Becker, O.C. Andronesi, D. Riedel, and M. Baldus, Proc. Natl. Acad. Sci. USA, 102, 15871-15876 (2005).

[2] H. Heise, ChemBioChem, 9, 179-189 (2008).

[3] H. Heise, M.S. Celej, S. Becker, D. Riedel, A. Pelah, A. Kumar, T.M. Jovin, and M. Baldus, J. Mol. Biol. 380, 444-450 (2008).

[4] O.C. Andronesi, S. Becker, K. Seidel, H. Heise, H. S. Young, and M. Baldus, J. Am. Chem. Soc. 127, 12965- 2974 (2005).

[5] Cornilescu, G., Delaglio, F., and Bax, A., J. Biomol. NMR 13, 289-302 (1999).

[6] D.P. Karpinar, M.B.G. Balija, S. Eimer, S. Kügler, B.H. Falkenburger, G. Taschenberger, F. Opazo, H. Heise, A. Kumar, D. Riedel, L. Fichtner, A. Voigt, S. Becker, G.H. Braus, A. Herzig, M. Baldus, H. Jäckle, J.B. Schulz, C. Griesinger, M. Zweckstetter. Pre-fibrillar alpha-synuclein variants with impaired beta-structure increase toxicity in Parkinson`s disease models, submitted (2009).

Surface-FIDA: Single particle detection as diagnostic tool E. Birkmann1, 2, S. A. Funke1, O. Bannach2, F. Henke1, L. Wang1, D. Riesner2, D. Willbold1,2,

1ISB-3: Structural Biochemistry 2Institute of Physical Biology, Heinrich-Heine-University Düsseldorf Prion diseases, Alzheimer’s disease, and Parkinson’s disease are neurodegenerative diseases that are characterized by the formation of protein aggregates during progress of the disease. It is still not known whether these aggregates are causative for or symptoms of these diseases. Many studies show that aggregates or even oligomers of the according proteins are neurotoxic and thus may lead to neurodegeneration. To understand disease-associated or causative mechanisms in respect to protein aggregation, an ultrasensitive tool to quantify these disease-related aggregates is required. In this project we introduce a new diagnostic tool to count and specify even single protein aggregates.

Prions are the causing agent of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt–Jakob disease (CJD) in man, bovine spongiform encephalopathy (BSE) in cattle and Scrapie in sheep. These diseases are characterised by an abnormally folded form of the host-encoded prion protein (PrP). The cellular, i.e. non-pathological isoform of PrP (PrPC) is present in most tissues and is most abundant in the central nervous system. Following infection, PrPC undergoes a conformational change during a post-translational process, leading to altered physicochemical properties such as aggregation, insolubility and β-sheet rich secondary structure. This pathological isoform is designated PrPSc.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder which is characterized by memory loss, confusion and a variety of cognitive disabilities. AD is the most prevalent dementia affecting nearly 2 % of the population in the western world, whereas the risk of AD increases dramatically in individuals beyond the age of 70. Neither a causal therapy for recovery nor a reliable early diagnosis, which could improve present therapeutically or preventional approaches, is yet available. Today, the most reliable diagnosis of AD is the post mortem identification of amyloid plaques and neurofibrillary tangles in the respective brain. The major component of the amyloid plaques is the Amyloid-β (1-42) peptide (Aβ).

Within this project we focussed on the detection of PrP and Aβ particles, because the according diseases are of particular interest as prion

diseases are transmissible and AD is the most prevalent dementia.

In both diseases protein aggregates occur during disease progression. Therefore, we established a highly sensitive and specific tool to detect even single protein aggregates, called surface-FIDA [1, 2, 3] (Fig. 1).

FIG. 1: Surface-FIDA A) Scheme of surface-FIDA; B) Scanning scheme;C) Principal of particle counting: Fluorescence peak caused by the labelled aggregate.

The latest development of this test system is based on fluorescence microscopy using fluorescence intensity distribution analysis (FIDA). It is quantifying the number and size of aggregates simultaneously labelled by two different antibodies for dual colour FIDA [1]. Only aggregates and oligomers but not monomeric proteins are detected. To increase the sensitivity, particles were concentrated in the two dimensional space by immobilizing it to capture antibodies on the surface of the slide. Laser beams are scanning the surface systematically so that even single particles are detected [2, 3].

We successfully established surface-FIDA as a diagnostic tool for prion diseases [2]. The infectious agents of prion diseases are composed

primarily of the pathogenic isoform of the prion PrPSc. In contrast to its cellular isoform, the pathogenic isoform PrPSc forms insoluble aggregates. Hitherto accredited prion tests use the proteinase K (PK)-resistance of PrPSc as a marker for the disease. These prion tests offer only a limited sensitivity because of varying portions of disease related aggregated PrP which is not PK-resistant. Therefore prion protein aggregate detection which does not rely on PK-digestion is favourable because it allows detection of both, PK-resistant and PK-sensitive PrPSc, aggregates.

We could successfully verify our novel test system for correct diagnosis of Scrapie infected hamsters as well as BSE-infected cattle in the clinical stages of diseases. Furthermore, we were able to detect PrP aggregates in the cerebrospinal fluid (CSF) of BSE-infected cattle for the first time [1] (Fig. 2). The use of three different antibodies (capture and two detection probes) but with overlapping epitopes delivered the best specificity within the assay [4].

FIG. 2: Detection of PrP aggregates in cerebrospinal fluid(CSF) of BSE-infected cattle by surface-FIDA Surface-FIDA of BSE-infected and healthy cattle CSF. Inthe histograms the mean values of burst number detectedwithin the surface-FIDA measurements are shown.Different capture probe combinations of the antibodiesD18, Saf32 and 12F10 are used. CSF samples from twodifferent BSE-infected cattle (dark grey) and two different healthy controls (light grey) were analysed. The numberof peaks varied independently of the labelling grade of theprobes

We also established the surface-FIDA assay for the detection of Alzheimer’s Aβ aggregates. We showed that the assay is sensitive enough to detect aggregates in the picomolar range. First measurements show a clear distinction between AD diseased people and non-demented controls by analysing CSF [3, 5] (Fig 3).

During the next steps we will adapt the highly sensitive test system for diagnosis of human prion diseases like Creutzfeldt-Jakob disease and other aggregate related diseases, e.g. Parkinson’s disease. Furthermore we will develop surface-FIDA to an imaging assay.

FIG. 3: Detection of Aβ aggregates in CSF of AD patientsA) The sensitivity of the assay was determined by dilution of the aggregates in CSF. For all measurements (three measurements ± standard deviation), burst numberdetected by 2D-FIDA during 0.5 min are shown. Fluorescence labelled antibodies 6E10 and 19H11 were used as detection probes. B) 2D-surface-FIDA was applied to 20 µl crude CSF of AD patients and control patients not affected by AD. Number of bursts (three measurements ± standard deviation) obtained by 2D-FIDA during 0.5 min are shown.

[1] Birkmann E, Henke F, Weinmann N, Dumpitak C, Funke SA, Willbold D, Riesner D. (2007) Counting of single prion particles bound to a capture-antibody surface (surface-FIDA). Vet. Microbiol. 123, 294-304.

[2] Birkmann E, Schäfer O, Weinmann N, Dumpitak C, Beekes M, Jackman R, Thorne L, Riesner D. (2006) Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion. Biol. Chem. 387, 95-102.

[3] Funke SA, Birkmann E, Henke F, Görtz P, Lange-Asschenfeldt C, Riesner D, Willbold D. (2007) Single particle detection of Abeta aggregates associated with Alzheimer's disease. Biochem. Biophys. Res. Commun., 364 (4), 902-907

[4] Birkmann E, Henke F, Funke SA, Bannach O, Riesner D, Willbold D. (2008) A highly sensitive diagnostic assay for aggregate-related diseases e.g. prion diseases and Alzheimer’s disease. Rejuvenation Res. 11(2), 359-363

[5] Funke SA, Birkmann E, Henke F, Görtz P, Lange-Asschenfeldt C, Riesner D & Willbold D (2008) An ultra-sensitive assay for diagnosis of Alzheimer’s disease. Rejuvenation Res. 11(2), 315-318

NMR structure of the M.Loti K1 channelcyclic nucleotide binding domainS. Schünke1,3

, M. Stoldt1,3, K. Novak2, U. B. Kaupp2,4

, D. Willbold1,3

1 ISB-3: Structural Biochemistry2 ISB-1: Cellular Biophysics3 Institute of Physical Biology, Heinrich-Heine-University, Düsseldorf4 Center for Advanced European Studies and Research (Caesar), Bonn

Cyclic nucleotide-sensitive ion channels, knownas HCN and CNG channels, play crucial rolesin neuronal excitability and signal transductionof sensory cells. HCN and CNG channels areactivated by binding of cyclic nucleotides totheir intracellular cyclic nucleotide-binding do-main (CNBD). However, the mechanism by whichthe binding of cyclic nucleotides opens thesechannels, is not well understood. We reportthe solution structure of the isolated CNBD ofa cyclic nucleotide-sensitive K+ channel fromMesorhizobium loti. The protein consists of awide antiparallel β-roll topped by a helical bundlecomprising five α-helices and a short 310-helix.In contrast to the dimeric arrangement (“dimer-of-dimers”) in the crystal structure, the solutionstructure clearly shows a monomeric fold. Themonomeric structure of the CNBD supports thehypothesis that the CNBDs transmit the bindingsignal to the channel pore independently of eachother.

Ion channels activated by cyclic nucleotides play keyroles in neuronal excitability and signaling of visualand olfactory neurons. They belong to two subfam-ilies: Cyclic nucleotide-gated (CNG) channels, andhyperpolarization-activated and cyclic nucleotide-gated (HCN) channels. Both channel types sharea carboxy-terminal cyclic nucleotide-binding domain(CNBD). HCN channels are activated by hyperpolar-ization and their activity is modulated by cyclic nu-cleotides. In contrast, CNG channels are voltageindependent and require cyclic nucleotides to open.Binding of cyclic nucleotides promotes the openingof the channel. Probably, a conformational change inthe CNBD is propagated to the pore.

Recently, a prokaryotic cyclic nucleotide-sensitiveK+ -channel, designated MloK1, has been identifiedin Mesorhizobium loti. MloK1 harbors six transmem-brane domains (S1-S6), a “GYG” signature sequencefor K+ selectivity, and a conserved CNBD is con-nected via a short C-linker to S6 (Fig. 1). The longerC-linker (∼80 residues) of mammalian CNG chan-nels is important for relaying the binding signal to thechannel gate. Crystal structures of mammalian HCNchannel CNBDs revealed that neighbouring C-linkerscontribute virtually all contacts between subunits inthe tetrameric protein. The crystal structure of the

isolated CNBD of MloK1 suggested that subunits areorganized as dimers. The dimer interface formed bythe short linker has been proposed to be involved inchannel gating [1].

FIG. 1: Subunit topology and assembly of the full-lengthMloK1 cyclic nucleotide-gated K+ channel. MloK1 consistsof four subunits. Each subunit encompasses six transmem-brane segments S1-S6 (yellow) and an intracellular CNBD(shown in ribbon representation).

However, an electron microscopy study of the com-plete channel reveals a four-fold symmetry of subunitarrangement [2]. The CNBDs appear as independentdomains separated by discrete gaps, suggesting thatCNBDs are not interacting with each other. Further-more, the MloK1 channel and the isolated CNBD bindcAMP with similar affinity in a non-cooperative fash-ion [3].

Here we study the solution structure of the monomericCNBD in complex with cAMP by nuclear magneticresonance (NMR) spectroscopy (Fig. 2) and compareit with the structure in the dimer (Fig. 3).

FIG. 2: Solution structure of the isolated cyclic nucleotide-binding domain. A: Superposition of the backbone tracesand all cAMP atoms of the family of 15 NMR structures withthe lowest CYANA target function. Backbone atoms of theN- and C-terminal ends (residues Q216 to V218 and A351to A355) are not shown and were not used for least-squaresuperposition of the structures. B: Ribbon representationof the CNBD structure with the lowest target function. ThecAMP ligand is shown as a stick model. Secondary struc-ture elements are labelled.

The solution structure is very similar to the structureof a monomer in the dimer crystal [1]. The com-parison of all CNBD backbone coordinates (residuesV218 to G350) between solution and crystal struc-ture results in a r.m.s. displacement value of 0.21 nm.However, the coordinates for the N-terminal residues(V218 to P241) which represent the α1-helix, 310-helix and associated loop regions differ remarkably.The α1-helix region (G221 to A231) in the solutionstructure is a straight helix without bending (Fig. 3).In the crystal structure, however, residues R220 toN226 of α1 are bent and form the dimer interface.The two most important insights of our study [4, 5]are that (1) the CNBD even at the high concentra-tion required for NMR measurements is a monomerand that (2) the solution structure, except for the N-terminal C-linker region, is similar to the monomerstructure in the dimer crystal structure. The muchlonger C-linker region of vertebrate CNG and HCNchannels is involved in intra- and intersubunit con-tacts and contributes virtually all contacts betweenthe subunits in the tetrameric crystal structures ofCNBDs from two different HCN channels. The for-mation of dimers and tetramers from monomericCNBDs requires cAMP, suggesting that rearrange-ment of the C-linker interface represents an impor-tant gating event. Thus, the MloK1 channel seems to

be set apart from its vertebrate cousins by the lackof a full-blown C-linker that coordinates intersubunitcontacts. This conclusion is supported by an EMstudy of the complete MloK1 channel. An importantfeature of the EM structure is that the four CNBDsare separated by discrete gaps and that four isolatedCNBDs could be modelled into the electron densitymap. This structure predicts that binding sites act in-dependently. The C-linker contact observed in thecrystal structure was possibly enforced by the pack-ing of dimers in the crystal and that these contactsseem to be functionally irrelevant.In fact, cAMP and several analogues bind non-cooperatively to the monomeric CNBD and thetetrameric full-length MloK1 with virtually identicalhigh affinity [3]. Together with the presented solu-tion structure, this shows that MloK1 cyclic nucleotidebinding sites are functionally independent of eachother [3, 5].

FIG. 3: Comparison of NMR and crystal structure. A: Com-parison of the solution structure (blue) and the crystal struc-ture of the CNBD (light gray). Part of the second domainin the dimer crystal structure that is involved in the dimerinterface is depicted in green. Alignment of the solutionand crystal structure backbones (residues V218 to G350)yielded an r.m.s. displacement of 0.21 nm. A comparisonof the solution and crystal structure excluding N-terminalresidues (V218 to P241) results in an r.m.s. displacementvalue of 0.12 nm. B: CNBD backbone comparison of themonomeric solution structure (shown in blue) and the back-bone of the crystal structure dimer (shown in red).

[1] G. M. Clayton, W. R. Silverman, L. Heginbotham andJ. H. Morais-Cabral, Cell 118, 615-627 (2004).

[2] P. L. Chiu, M. D. Pagel, J. Evans, H. T. Chou, X. Zeng,B. Gipson, H. Stahlberg, and C. M. Nimigean, Struc-ture 15, 1053-1064 (2007).

[3] A. Cukkemane, B. Gruter, K. Novak, T. Gensch,W. Bonigk, T. Gerharz, U. B. Kaupp, and R. Seifert,EMBO Rep. 8, 749-755 (2007).

[4] S. Schünke, M. Stoldt, K. Novak, U. B. Kaupp, andD. Willbold, Biomol. NMR Assign. 1, 179-181 (2007).

[5] S. Schünke, M. Stoldt, K. Novak, U. B. Kaupp, andD. Willbold, EMBO Rep., Manuscript Accepted (2009).

Structural characterization ofGABARAP-ligand interactionsY. Thielmann1

, O.H. Weiergräber2, J. Mohrlüder1, D. Willbold1

1 ISB-3: Structural Biochemistry2 ISB-2: Molecular Biophysics

The GABAA receptor-associated protein (GABA-RAP) plays an important role in intracellulartrafficking of several proteins. It undergoes aC-terminal lipidation process that enables an-choring in the cytosolic leaflet of cellular mem-branes. While the three-dimensional structure ofGABARAP itself has been determined, structuralinvestigation of complexes with its interactionpartners has just commenced. Our recent workhas established that GABARAP features two hy-drophobic binding sites (hp1 and hp2), whichplay an essential role in complex formation withnative ligands, such as calreticulin and the heavychain of clathrin.

Originally, GABARAP has been identified as a proteininteracting with a cytoplasmic loop of the GABAA re-ceptor γ2 subunit. Colocalization with the GABAA re-ceptor in cultured cortical neurons and the presenceof a tubulin binding site qualified this protein as a po-tential adaptor linking the GABAA receptor to the cy-toskeleton. GABARAP and its paralogues are mem-bers of the ubiquitin superfamily which is character-ized by a so-called β-grasp fold. The latter consistsof a central four-stranded mixed β-sheet with two α-helices located on its concave side. A hallmark ofthe GABARAP family is an N-terminal extension con-taining two additional α-helices, which are attachedto the convex face of the β-sheet. Proteins of theGABARAP family are processed by a ubiquitin-typeconjugation machinery, with the final step involvingcovalent linkage to membrane lipids instead of pro-teins. The number of known GABARAP interactionpartners is rising steadily. Current evidence indicatesthat it might play a major role in subcellular transportof various cytosolic and membrane-associated pro-teins.Phage display screening for GABARAP binding pep-tides indicated a high prevalence of tryptophanresidues in potential ligands. Therefore, free in-dole and several derivatives were chosen as probesto characterize the binding properties of GABARAPby nuclear magnetic resonance (NMR) spectroscopy[1]. The specific behaviour of individual resonancesindicated the presence of two apolar grooves on thesurface of GABARAP which display different affinitiesfor indole derivatives. If this binding site is biologi-cally relevant, GABARAP interaction partners shoulddisplay a surface-exposed conserved tryptophan. In-

FIG. 1: Complex of GABARAP with a high-affinity lig-and (K1). GABARAP is depicted as ribbon model with theβ-grasp domain and the N-terminal extension coloured inshades of blue. The peptide is shown with red and graybackbone; hydrophobic side chains involved in complex for-mation are indicated.

deed, such an invariant tryptophan can be found inthe binding motifs of the GABAA receptor γ2 subunitas well as additional physiological ligands, as dis-cussed below.

Among the GABARAP binding peptides identifiedby phage display screening, the "K1" sequence(DATYTWEHLAWP) was found numerous times inindependent experiments. The interaction of thishigh-affinity ligand with GABARAP was studied us-ing NMR spectroscopy and X-ray crystallography[2]. Although the K1 peptide makes contact withGABARAP in its entire length (Fig. 1), complex for-mation appears to be dominated by hydrophobic in-teractions mediated by two tryptophan residues (W6,W11) and a leucine side chain (L9). It is important tonote that ligand binding to GABARAP induces signif-icant conformational changes in the protein.

Combining pulldown experiments and data mining,the heavy chain of clathrin was identified as a physi-ological GABARAP binding partner. NMR titration ofGABARAP with a 13-mer clathrin peptide revealedchanges which are clearly indicative of a direct in-teraction. The biological significance of the clathrin-GABARAP interaction might relate to the GABAA re-ceptor: endocytosis of the receptor is crucial for con-trol of receptor numbers at the postsynaptic mem-brane of neurons. This process depends on the for-

FIG. 2: Proposed structure of the GABARAP-calreticulincomplex. Globular domain and P domain of calreticulin areshown in light and dark red, respectively, the core bind-ing motif (CRT178-188) in blue. GABARAP is displayed assurface representation, with colours indicating residues af-fected in NMR titrations with CRT178-188 (blue) and the entireP domain (red).

mation of coated pits and is mediated by clathrin. Theinteraction with clathrin heavy chain may thereforerepresent an important aspect of GABARAP functionin GABAA receptor trafficking.

Calreticulin is another GABARAP binding partneridentified by a database search and verified us-ing biochemical techniques [3]. NMR spectra ofGABARAP in the presence of calreticulin showedclear indications of complex formation. Surface plas-mon resonance experiments with calreticulin andGABARAP yielded a dissociation constant of 64 nM;this is the strongest interaction reported thus far foreither protein. In a subsequent study, different cal-reticulin fragments were investigated for their inter-action with GABARAP. These included the completeproline-rich (P) domain (aa 177-288) and a shortsegment thereof (aa 178-188) containing the puta-tive GABARAP binding motif [4]. These data sug-gest that the peptide contains the core binding mo-tif, but the adjacent domains of calreticulin accountfor additional contacts. The three-dimensional struc-ture of the GABARAP-CRT178−188 complex could bedetermined by X-ray crystallography. The ligand at-tains an elongated conformation in close contact toGABARAP, interacting with both hp1 and hp2. Astructure model of full-length calreticulin reveals thatits proposed GABARAP binding site is located at theN-terminal junction between the globular domain andthe P domain (Fig. 2).

The minimal machinery required to maintain mem-brane fusion in vivo includes NSF, a hexameric ATP-ase belonging to the AAA (ATPases associated withvarious cellular activities) class. GABARAP has beenidentified as an interaction partner of NSF, whichmodulates the subcellular localization of the ATPase.Whereas crystal structures have been determined forsingle domains of the NSF protomer, a structure ofthe full-length polypeptide is still lacking. Therefore,a homology model of the entire NSF complex wasgenerated, using the related ATPase p97/VCP as atemplate [5]. Docking experiments with GABARAP

FIG. 3: Model of hexameric NSF with docked GABARAPmolecules. NSF subunits are shown in blue and light grey,GABARAP in orange. See text for details.

resulted in the model shown in Fig. 3. The proposedNSF-GABARAP interface is composed of an apolarcore, which is flanked by polar contacts. An interest-ing aspect of the complex model is the proximity ofGABARAP to the nucleotide binding site in the ma-jor ATPase domain of NSF, suggesting a regulatoryimpact on ATP binding and/or hydrolysis. Due to itsC-terminal lipidation, binding of GABARAP to NSF inthis configuration could serve to anchor the complexto membranes.

These investigations have focused on the characteri-zation of GABARAP interactions with artificial as wellas physiological ligands, applying a wide repertoireof methods from molecular and cellular biology, bio-chemistry and structural biology. Together, our datahave provided novel insight into structural founda-tions and dynamic properties of protein-protein inter-actions of GABARAP and its homologues, which ul-timately govern the biological functions of this novelclass of adaptor molecules.

[1] Y. Thielmann, J. Mohrlüder, B. W. Koenig, T. Stangler,R. Hartmann, K. Becker, H.-D. Höltje and D. Willbold,ChemBioChem 9, 1767–1775 (2008).

[2] O. H. Weiergräber, T. Stangler, Y. Thielmann, J.Mohrlüder, K. Wiesehan and D. Willbold, J. Mol. Biol.381, 1320–1331 (2008).

[3] J. Mohrlüder, T. Stangler, Y. Hoffmann, K. Wiesehan,A. Mataruga and D. Willbold, FEBS J. 274, 5543–5555 (2007).

[4] Y. Thielmann, O. H. Weiergräber, J. Mohrlüder and D.Willbold, FEBS J. 276, 1140–1152 (2009).

[5] Y. Thielmann, O. H. Weiergräber and D. Willbold, Pro-teins (in revision).

IBN-2: Bioelectronics Director: Prof. Andreas Offenhäusser

The interfacing of man-made electronics with bio-systems like DNA, redox proteins, enzymes and cells not only allows us to learn about molecular processes in biology, but also paves the way to using it in derived sensory devices. Some of these have already had a profound impact on clinical diagnostics. Technological approaches also can be inspired by biological systems potentially leading to new cognitive and sensory approaches to information processing. Our research aims to develop bioelectronic devices that combine biological systems-from single biomolecules to living cells and organisms-with electronics.

Within the broad field of bioelectronics, we have identified two domains where our expertise in nanotechnology can add value for improving the efficiency and sustainability of current environmental research and health care.

Sensing and imaging Electronic devices that detect trace amounts of biochemicals in the environment or in bodily fluids will allow far earlier detection than current technologies and will therefore facilitate appropriate reactions. Magnetic sensors have evolved due to the ever-increasing need for improved sensitivity. Ultra-sensitive superconducting quantum interference devices (SQUIDs) have a great potential for biomedical sensing. Microwave to terahertz sensing techniques, based on collectively vibrational modes of complex molecules at terahertz frequencies, relaxation of the dielectric function

in the micro- and millimetre wave range, and ionic conductivity at radio frequencies, have great potential for applications in biology, medicine, airline, and public security.

Bioelectronic devices and biomedical applications The use of biomolecules as the building blocks of higher-level functional devices will lead to applications ranging from the integration of biomaterials with electronics in recognition to sensing devices, such as biosensors. Bioelectronics research also exploits the use of biomolecules to perform electronic functions that semiconductor devices currently perform, thereby offering the potential to increase integration in combination with additional functionalities at the nanometer level. Living cells and tissues exhibit an extraordinary range of functions including highly selective biochemical sensing (even in chemically noisy environments), protein synthesis, and information processing. Functional interfaces between neurons and micro-/nanodevices will have the potential to enhance in-vitro applications ranging from basic neuroscience research and disease modelling to drug screening and biosensors. Future in-vivo applications of bioelectronic devices include, for example, stimulating and recording deep-brain activity, managing pain, and restoring damaged nervous pathways.

A Model for the Cell-Sensor Interface and Comparison with Experiment M. Pabst1,2, G. Wrobel1,2, F. Sommerhage1,2, S. Ingebrandt1,2, A. Offenhäusser1,2 1IBN-2: Bioelectronics 2JARA-FIT

Electrogenic cells are able to generate electrical signals which can be measured by various invasive electrophysiological methods such as patch-clamp or sharp microelectrode recordings. Growing cells on the surfaces of e. g. metal microeclectrodes or field-effect transistors allows the recording of an extracellular component of these signals. For an understanding of such extracellular signals it is mandatory to get detailed topographical as well as electrical information about the cell-sensor interface. In a first approximation, this interface can be described by a flat disk between cell membrane and sensor surface. For a correct description of the signals, the electrodiffusion of ions in this interface is modeled by using the stationary Poisson-Nernst-Planck equations. We solve the equations analytically, and derive expressions for the potential, the ionic charge densities, and the seal resistance. The results provide a method for determining the distance h between sensor surface and cell membrane. For human embryonic kidney cells, we receive h ≈ 70 nm. Comparison with transmissions electron microscopy studies shows reasonable agreement.

FIG. 1: Schematic drawing of the cell-sensor interface between a HEK293 and a field-effect transistor (not to scale). The cell is located on the top of a FET gate. The cell is approximated by a half-sphere with a radius a = 15 µm. The cleft height is h < 100 nm [1,2]. The gate area of the FET is about 50 µm2. Smaller letter-size corresponds to smaller a concentration of K+.

The electrical activity of biological cells in vitro can be recorded by measuring changes in the local extracellular voltage with planar metal electrodes or integrated planar field effect transistors (FETs).

These changes in local extracellular voltage result from the flux of ions across the membrane of the cell, mainly sodium (Na+), potassium (K+), and calcium ions (Ca2+). The distribution and strength of the potential near the sensor depends on the geometry of the cleft between attached cell and sensor surface and the specific electrical behavior of the cell membrane.

For a quantitative understanding of the extracellular signals it is necessary to describe the experimental situation. A schematic picture of a typical setup is shown in Fig. 1. Biological cells (here: human embryonic kidney (HEK293) cells expressing a voltage gated K+ channel) are cultured on top of the gate of a FET [1]. Outside the cell and inside the cleft, the region between cell and sensor surface, there is extracellular electrolyte solution. By electrical excitation, the ion channels in the cell’s membrane open and ions can flow from the inside to the outside. While in the upper part of the cell (free membrane) these ions just enter the surrounding electrolyte bath directly, it is different at the attached membrane. Here, the ions have to pass the cleft before entering the bath. The flux of ions into the cleft causes an increase of charge. Due to the connected electric field other charged ions will move: ions with similar charge leave the cleft and move into the surrounding bath while ions of opposite charge are attracted and enter the cleft. The change of charge inside the cleft influences the source drain current under the gate and is sensed by the FET. It can be directly correlated to the membrane current into the cleft.

FIG. 2: Exemplary electrical coupling of a HEK293 cells transfected with K+ channels to a FET gate. A) Voltage-clamp stimulation pulse, B) transmembrane K+ current measured by patch-clamp, and C) corresponding ΔVFET recording.

Fig. 2 shows the comparison of whole-cell membrane currents (IM) and extracellular signal shapes (VFET) recorded with a p-channel FET for HEK293 cells transfected with a K+ channel: (A) Rectangular stimulation pulses from the holding voltage led to (B) whole-cell current IM and (C) corresponding FET signals VFET for activation of the K+ channel. The FET signal shape resembles a combination of different signal components, each with distinct kinetics depending on the respective cell type: (i) capacitive transients, caused by the capacitive coupling of the stimulation pulse, (ii) an increase of VFET to a steady-state amplitude, (iii) a partially instantaneously decline of VFET at the end the pulse, and (iv) a slow relaxation of VFET, that is absent in IM.

For the description of the FET signal shapes the ion flux in the cleft has to be understood. In our approach we apply the Poisson-Nernst-Planck (PNP) equation system, a differential equation system consisting out of the continuity equation, the Nernst-Planck equation and the Poisson equation [2]. The system describes the potential and charge densities inside the cleft. By adapting the PNP system to our experimental situation, the system is reduced to an ordinary inhomogeneous Bessel differential equation. This equation can be solved analytically in the stationary case corresponding to the steady state pulse situation after 100 ms in Fig. 2. As a result we receive for the potential ψ(r)

⎟⎟⎠

⎞⎜⎜⎝

⎛−+⎟⎟

⎞⎜⎜⎝

⎛−∝ 1

)/()/(41)(

LaILrI

Here r is is the radial position inside the cleft, a the radius, LD the Debye-length and I0 the Bessel function. While LD/a is of the order 10-5, the second term can practically neglected giving a parabolic potential, which corresponds to a constant total charge density inside the cleft. From the potential the charge densities can be derived showing a similar parabolic form like the potential. With the above results an expression for the measured voltage signal ΔVFET of the FET in the steady state case can be derived (Fig. 2):

( ) JMJFET IRV δ+=Δ 1

with the ‘cleft resistance’ RJ and the ion current IJM of the cell into the cleft. The correction term δ takes into account the surface properties of the metal gate of the FET underneath the cell. It turns out that δ is of the order of 1, which means half of the signal comes from the gate properties and the other half from the membrane current into the cleft. RJ is given by

BJ hDne

TkqR 208π

with q, kB, T, e0, nBtot, DK, h being a correction factor

close to 1 due to the experimental situation, the Boltzmann constant, temperature, elementary charge, ion density of the surrounding electrolyte

solution, diffusion constant of K+ ions and the height h of the cleft (Fig.1). By correlating ΔVFET as a function of the total cell current IM in steady state case for many cells, the last two formulae allow the calculation of an average height h. From Fig. 3 we receive for RJ = 0.57 MΩ and in consequence 70 nm for the height h.

FIG. 3: Comparison of the steady-state p-channel FET signals (ΔVFET) in dependence on the current through the attached membrane (IJM) for 10 cells.

To compare our theoretical result for the height h, experiments have been made to measure the height directly [3]. Transmission electron microscopy (TEM) was applied to analyze the cell-sensor interface. The studies were complemented by imaging ellipsometry. As a result, it was found for HEK293 cells, that the contact geometry between cell membrane and substrate was dependent on the protein coating used for the substrate. In presence of polylysine, the measured average height h was in the range 35 – 40 nm, which is about half of the theoretical value. Considering the experimental uncertainties for growing cells on substrate, this is a reasonable agreement.

[1] G. Wrobel, R. Seifert, S. Ingebrandt, J. Enderlein, H. Ecken, A. Baumann, U. B. Kaupp, A. Offenhäusser, Cell-transistor coupling: Investigation of potassium currents recorded with p- and n-channel FETs, Biophys. J. 89, 3628-3638 (2005)

[2] M. Pabst, G. Wrobel, S. Ingebrandt, F. Sommerhage, A. Offenhäusser, Solution of the Poisson-Nerst-Planck equations in the cell-substrate interface, Eur. Phys. J. E. – Soft Mat. 24, 1-8 (2007)

[3] G. Wrobel, M. Höller, S. Ingebrandt, S. Dieluweit, F. Sommerhage, H. P. Bochem, A. Offenhäusser, Transmission electron microscopy study of the cell-sensor interface, J. Roy. Soc. Interf. 5 (2008).

Nanostructured Interfaces for Bioelectronic Systems B. Wolfrum1,2, Y. Mourzina1,2, F. Sommerhage1,2, B. Hofmann1,2, D. Brüggemann1,2, A. Offenhäusser1,2 1IBN-2: Bioelectronics 2JARA-FIT

The properties of a bioelectronic interface (interface between an individual cell and a chip transducer) are of primary importance for bioelectronic systems. Nanopatterned surfaces promise to enhance the interface quality regarding to electronic signal transduction, cell adhesion, and selectivity. Electrode materials with increased surface area have the best electrode properties due to a large effective area, favouring a smaller electrode resistance, a larger capacitance, and further decreasing of the feature size. This reduces the noise level and increases the current injection capability. Here, we present a concept of a large-scale patterned gold nanopillar array interface on semiconductor substrates with the aim of functional coupling to electrogenic cells. Nanostructuring is based on the combination of an imprint approach with a nanotemplate-assisted electrodeposition. This new and versatile method can be adopted for different interface materials of electronic circuits and their coupling to both cellular and molecular sensing components.

FIG. 1: Process sketch for the fabrication of the large-scale patterned arrays of Au-nanopillars.

Interfaces with nanoscopic sizes play a vital role in electrochemistry, catalysis, energy storage/conversion devices, cell adhesion studies, bioelectronics, and information technology. Intensive research is focused on patterned nanopillar (NPA) array interfaces of diverse materials. Thereby, the specific and precise positioning of nanofeatures on a large scale is especially challenging.

Fabrication methods of NPAs can be divided into direct lithographic methods utilizing e-beam or focused ion beam (FIB) writing, nanoparticle growth or deposition by self organization, and template-assisted methods. While the direct lithographic methods offer the most versatility they are usually limited to small area patterning due to speed limitations caused by the sequential processing. Methods based on self organization can be applied on a larger scale but often lack the versatility obtained by the direct methods. Furthermore, most fabrication methods based on self organization without template assistance are limited to specific materials.

We therefore combined an imprint approach with a nanotemplate-assisted electrodeposition to create large-scale micro- and nanometer sized arrays of gold nanopillars at predefined positions on a semiconductor substrate. Imprint techniques are often preferred over other direct patterning techniques, because lithography has to be used only for the fabrication of the stamps.

FIG. 2: SEM images of silicon stamps for imprinting aluminum layers (a-d). Top and side view of dotted “FZJ” (a,b) and line patterns (c,d) directly after RIE (a), at different stages of KOH etching (b,c), and after removal of the silicon oxide mask (d). The line width at the stamp tip is 130 nm.

The process for the fabrication of patterned nanopillar arrays is shown in Fig. 1. The Si stamps for the indentation of the aluminum surface (a) are created by lithography combined with anisotropic wet chemical etching of silicon [1]. The stamp patterns have different geometries, Fig. 2, and cover an area of 4x4 mm² on an 11x11 mm² chip, the smallest initial feature size of the stamp being 1 µm. The pattern of the stamp is transferred via imprinting onto a Ti/Au/Al covered substrate (b). Here, we show that an imprint approach on thin aluminum films can be used to define nano- and mircopatterned regions of pores on a substrate, which selectively act as a template for the electrodeposition of nanopillars.

FIG. 3: FZJ-silicon stamp pattern (a) and the reproduction of the stamp pattern as Au nanopillar arrays (b). The inset shows an image enlargement of a single dot feature of (b). Images (c-f) show top and side views of Au nanopillar fences created by using line stamps to indent the aluminum film. The lines extend up to the stamp size of 4 mm.

Patterned aluminum films are subsequently anodized in 0.3 M oxalic acid at 3°C and 40 V to obtain nanoporous alumina membrane (AM) template with an approximate interpore distance of 80 nm (c) [2]. The anodization is stopped after the nanochannels originating from the indented surface reach the underlying gold substrate. The barrier layer, which possibly remains at the bottom of the pores originat ing from the predefined regions, is dissolved in 5% phosphoric acid. AM acts as a template material for the metal deposition, in which gold is exclusively formed inside the nanochannels of the porous alumina. The template material can be selectively etched in alkaline solution to expose the gold nanopillars arrays (d).

In Fig. 3 it is shown how a large-area stamp pattern (Fig. 3a) exhibiting dots of different interspaces can be reproduced with pads of gold nanopillar arrays (Fig. 3b). Each nanopillar array is composed of approximately 60 Au-pillars with a width and height of ~35 and ~300 nm, respectively. The interspace distance and width of

the Au-nanopillars depend on anodization conditions of the aluminum film and the subsequent pore enlargement in phosphoric acid. For example, we fabricated nanopillar arrays with a diameter down to 20 nm and a pillar to pillar distance of ~50 nm by anodizing the aluminum film at 22 V in sulfuric acid (3%). Similar arrays could be used for surface enhanced Raman scattering [3] and cell adhesion studies [4]. Figure 3(c-f) shows images of patterned Au-nanopillar line arrays. The minimum interspace distance of the patterned nanopillar lines is determined by the resolution of the optical lithography used for the stamp fabrication. Smaller interspace distances might be achieved by performing multiple imprints with the same stamp using a precise positioning system.

FIG. 4: SEM image of a rat neuronal cell growing on a gold nanopillar surface (a) and a microelectrode active area modified with gold nanopillar pattern (b).

We found that the nanostructured surfaces presented here were biocompatible with cell lines expressing ion channels as well as primary neuronal cells from the rat cortex, Fig. 4a. We further showed that the nanotemplate-assisted electrodeposition can be combined with a prepatterned electrode array as a precursor for NP growth to create a new type of a 3-D nanostructured interface for coupling with electrogenic cells, Fig. 4b. We propose that this approach can be extended to different interface materials of elec tronic circuits and their coupling to both cellular and molecular sensing components.

Acknowledgements: A. Steffen, J. Müller, H.-P. Bochem, M. Nonn, M. Prömpers, Dr. M. Lepsa, D. Schwaab, Dr. D. Mayer, Dr. S. Ingebrandt, A. Fox, S. Schaal, B. Hermanns.

[1] B. Wolfrum, Y. Mourzina, D. Mayer, D. D. Schwaab, A. Offenhäusser, Small 2 (2006) 1256.

[2] B. Wolfrum, Y. Mourzina, F. Sommerhage, A. Offenhäusser, Nano Letters 6 (2006) 453.

[3] G. Sauer et al., J. Appl. Phys. 2005, 97, 024308.

[4] N. Walter, C. Selhuber, H. Kessler, J. P. Spatz, Nano Lett. 6 (2006) 398.

Functional Networks of Rat Neurons on in-situ Patterned KDI S. Meffert1,2, K. Adamiak3, R. Helpenstein1,2, B. Hofmann1,2, A. Offenhäusser1,2 1IBN-2: Bioelectronics 2JARA-FIT 3DWI, RWTH Aachen University

This study aimed to increase the lifetime and stability of in vitro network of neurons. We tested KDI, a 12 amino acid peptide of γ laminin peptide, using in situ-microcontact printing to create a chemically attached peptide pattern on silicon oxide substrate. By atomic force microscopy (AFM) a pattern thickness of about 2.9 nm which corresponds to a peptide monolayer could be measured. Rat cortical neurons were seeded on the KDI pattern and tested on their viability and functionality. Synaptically connected neurons were confirmed by double patch-clamp recording and immunostaining experiments. The expression of the pre- and postsynaptic proteins vesicular GABA transporter (VGAT) and gephrin could also be evidenced. Our data indicated that a monolayer of in situ-printed KDI peptide is sufficient for neurons adhesion and also for formation of functional and stabilized networks. By future application of these patterns as growth substrate on sensor arrays the extracellular recording of single neurons should be further improved.

There is an increasing need in the field of neuroelectronic devices to immobilize functional proteins on the device surfaces for coupling extracellular signals from neuronal networks and long-term recording. Immobilization on surfaces can be easily accomplished by direct adsorption. However this method results partially thick and inhomogenous protein layers, denaturation of the protein, as well as unstable attachment affecting neurons and network function. Covalent attachment of functional proteins or peptides are one solution and are important when a coated substrate is subjected to a flowing solution or exposed for a long period of time in solution. In this study we aimed to construct a 1) chemically stable, 2) reproducible, 3) thin as possible growth pattern enabling long time stability and functionality of neuronal networks on silicon oxide substrates. Thereby, we used the in situ-microcontact printing (µCP) which was recently established to print proteins/peptides without inducing their denaturation [1]. In situ µCP was

combined with a protocol for covalently attaching peptides to construct a chemically stable peptide pattern of KDI, a 12 AS consisting synthetic peptide deriving from neurite outgrowth-promoting domain of γ laminin. KDI (cystein-terminated KDI peptide, 5 µM) was printed on aminosilanized (APTES, 3-aminopropyl-trietoxy silane) and cross-linker (sulfo-γ-maleimidobutyryloxysuccimide ester) attached glass substrate. To investigate the fidelity of the pattern, carboxyfluorescein labelled-KDI was used for one set of experiments. Printed KDI patterns were first analyzed by phase contrast and fluorescence microscopy. Fluorescence images confirmed the transfer of carboxy-fluorescein labelled KDI on glass substrate and illustrated that the pattern was almost without any interruptions and a high degree of uniformity.

FIG. 1: AFM analysis was performed using Nanoscope IV Multimode Instrument with a Nanoscope IV controller and a 15 µm scanner in tapping mode. The KDI in-situ imprint shows a smooth topography of the printed pattern (A) and a section analysis with a small roughness of about 1nm (A, right). Detailed analysis (B) revealed a layer thickness of 2.868 nm (B, right).

In contrast, no KDI pattern was observable by phase-contrast microscopy which indicates a reduced thickness of this peptide pattern in comparison to the standardly printed protein mix PECM (polylysine/ extracellular matrix gel) [2, 3]. For further characterization of the peptide imprints and measuring the layer thickness atomic force microscopy (AFM) was carried out using a Nanoscope IV Multimode Instrument with a Nanoscope iV controller and a 15 µm scanner. Samples were scanned in tapping mode with resonance frequencies between 260 and 300 kHz. The KDI-imprint showed a smooth topography of the pattern indicating a homogenous peptide coupling to the surface. Further AFM analysis of one section confirmed the slight layer roughness and revealed a thickness of 2.9 nm (Fig.1) which was in the range of the theoretical calculation of a peptide monolayer [4-5]. The KDI pattern was subsequently tested in cell culture on neurons functionality and network stability.

FIG. 2: Phase contrast image of rat cortical neuronsgrown on KDI pattern DIV 7, forming geometricallydefined networks (A). Patterned neurons were fixed andimmunostained with antibodies against the pre- and postsynaptic proteins VGAT (red) and gephyrin (green).Laser scanning images of neurons grown on KDIimmunostained against VGAT and gephrin showing highexpression of both synaptic proteins DIV 7 (B) and DIV 12(C).

Cortical neurons from embryonic (E18) rats were prepared in a chemically controlled medium and seeded (16.000 cells/ cm2) [2] at the KDI patterned glass substrate. The substrates were optically controlled due to adhesion and outgrowth of the cells on the pattern. Figure 2 illustrates the behaviour of the neurons after 7 and 12 days in vitro (DIV7). The aligning of the cell bodies onto the 12µm-nodes of the grid pattern as well as the

guidance of the processes along the lines were achieved with high compliance. The neurons developed a mature morphology and differentiated to network forming neurons. Apart from the morphological evidence the neuronal identity and functionality was confirmed by immunostainings and electrophysiological experiments. Single and double patch clamp experiments elicted spiking neurons and synaptically connected neurons grown on the KDI pattern. The cortical neurons were stained with antibodies against the pre- and postsynaptic proteins, vesicular GABA transporter (VGAT) and gephrin. The expression of both proteins could be identified at DIV 7 and DIV 12. Finally, the network formed on KDI pattern were stable without detaching from the substrate up to DIV 22.

In summary, we were able to construct a chemically stable, homogenous, 2.9 nm-thin KDI and likely monomolecular peptide layer. The peptide layer was tested to be suitable as substrate for adhesion and outgrowing of neurons confined to the pattern geometry. This peptide pattern could further be proved to be suitable for long-time culturing of neurons and due to the stability of networks in cell culture. With these data, we propose that KDI patterned substrate may be useful for extracellular recordings to improve the signal coupling by reducing the cell-device distance.

We thank D. Mayer and S. Gilles for AFM measurements. Financial support came from the Sony Deutschland GmbH.

[1] D. Schwaab, Dissertation at the RWTH Aachen (2007).

[2] A. Vogt, L. Lauer, W. Knoll, A. Offenhäusser, Biotechnol. Prog. 19, 1562 (2003)

[3] S. Böcker-Meffert, T. Decker, S. Schäfer, A. Offenhäusser, PB-10, 5th Int. Symp. Biomimetic Materials Processing (BMMP-5), 2005

[4] M. Scholl, C. Sprössler, M. Denyer, M. Krause, K. Nakajima, A. Maelicke, W. Knoll, A. Offenhäusser, J. Neurosci. Methods 104, 65 (2000)

[5] K. Adamiak, B. Hofmann, S. Böcker-Meffert, A. Offenhäusser, 7th Göttingen Meeting oft he German Neurosciences Society, Germany, 738-5B (2007).

Generation of protein gradients by nanoscale patterning D. Schwaab1,2, P. Zentis1,2, S. Völker1,2, S. Meffert1,2, D. Mayer1,2, A. Offenhäusser1,2 1 IBN-2: Bioelectronics 2 JARA-FIT

Combining high resolution lithography with microcontact-printing by means of hard plastomers it was possible to adjust the size of elements of a protein pattern and simultaneously the distance between them with sub 100 nm resolution. Rat neurons adhered onto these sub 10nm protein layers, apparently integrate over a large number of pattern elements and recognise the pattern as quasi homogeneous. However, the neurites showed pre-dominantly an aligned outgrowth corresponding to the underlying pattern. Thus, the technique proposed in this work maybe path the way to systemically study the influence of nanometer sized purely biochemical gradients on guiding neurons. These discontinuous gradients will further enable to evaluate the role of ECM/ligand spacing and in parallel the effect of defined gradient parameters on axon guiding.

In developing nervous system the path of axons to reach their targets and establish neural circuits is directed by soluble and surface-bound biochemical gradients. Ligands of these gradients are detected by axonal growth cones which probe their environment by extending or retracting their filopodia. Although considerable effort has been directed towards characterizing chemotactic molecules and their receptors, the cellular mechanism by which neuronal growth cones sense these gradients remains generally unknown. It is envisioned to fabricate chemical gradients with length scales relevant to the biological involved cellular structures. The fabrication of continuous surface-bound gradients has been realized by a wide variety of methods [1]. Recently, von Phillipsborn et al. [2] used microcontact-printing to produce geometrically defined discrete gradients consisting of protein-covered spots varying in sizes and spacing. To transfer the concept of discrete gradients to the nanoscale dimensions of extracellular matrix fibrils nanostructuring techniques needs to be adapted. In the scope of this work we have developed a procedure to fabricate discrete purely biochemical gradients of extracellular matrix proteins by means of microcontact printing (µCP). This technique directly transfers molecular inks from a polymer stamp to the target surface [3]. Here we show the transfer of ultra thin (10 nm or less) compact protein layers with sub 100 nm lateral resolution and demonstrate

the use of these films to guide neuronal outgrowth. Polyolefine plastomers (POP) were chosen as stamp material with a Youngs modulus of E=80 MPa (Avinity VP, Ticona). The high stiffness prevents the material from disrupting effects like sagging during printing process and facilitates high pattern fidelity. The plastomer stamps were structured by hot embossing by using a mold made of silicon. The molds contained a pattern with lines and spaces with both constant and varying widths ranging from 1 µm to 75 nm, resulting in a line pattern with different pitches. A mixture of FITC-poly-D-lysine extracellular matrix proteins and (FITC-PDL/ECM) was used as ink. The stamps were dried with a nitrogen stream and pressed onto glass or silicon oxide substrate which was prehydrophilized by oxygen plasma. An uniform transfer of proteins on large length scales was verified by optical fluorescence microscopy (Fig. 1a), indicating also a high pattern fidelity. Images recorded at higher magnification by means of scanning electron microscopy (SEM) did not reveal any major pattern defects. Well-defined protein lines were observed as dark stripes in the SEM images (Fig.1b) with inverted contrast compared to fluorescence images. In the SEM images neither hints of disruptive effects like sagging or pattern collapse nor contrast variation over the imaged area were observed.

FIG. 1: Large scale images of the transfer of FITC-PDL/ ECM ink to a SiO2. (A) Fluorescence microscopy image of the FITC dye coupled to PDL (excitation 488nm, emission 518nm) indicates schematically the area of the SEM image, see right. (B) SEM image at a magnification of 5450 with inverted contrast compared to (A), the dark

The homogeneity of the protein coverage was further investigated with nanoscale resolution using Atomic Force Microscopy (AFM). The AFM images revealed that all structural elements of the pattern were transferred from the stamp to the hydrophilized SiO2 surface, even the 75 nm lines. An analysis of the line width and the deviation between designed pattern and printed features for most structures was smaller 10 % in average. The height of printed protein layers varied between 2 nm and 10 nm. To evaluate the response of neuronal cells to the nanoscale discrete protein gradients described above we analyzed the adhesion and outgrowth of primary neurons on these patterns by SEM. The isolated neurons were obtained from 18 day-old rat embryos (E 18) as described by Vogt et al. [4]. The cells were plated onto patterned SiO2 substrates. After 5-7 days in culture the cells were fixed, critical point dried and coated with a thin layer of platin/iridium.

FIG. 2: SEM image of a patterend SiO2 substrate withoutgrown neurites of rat neurons. The neurites grow inalignement to the pattern of FITC-PDL/ ECM. On the leftside of the image the nano pattern design was superimposed on the original SEM image to highlight thatthe filipodia attached also to the smallest elements of thepattern.

Figure 2 shows a SEM image of a SiO2 substrate after printing of FITC-PDL/ ECM and culturing cortical neurons for 7 days. The image was recorded at the transition from a nanopatterned area to an area homogeneously covered with proteins. The cells adhered to both areas however with distinct differences regarding neuronal outgrowth (data not shown). Averaged over the whole pattern the coverage of the surface with proteins was estimated to 45 % which is consistent with the particular pattern design. Normalized to the coverage with proteins, there was no obvious difference in the number of cells adhered on nanopatterned area (64 ± 5) compared to the area of the surface that were homogeneously covered with proteins 124 ± 29.

The cells adhered to the homogeneously coated and patterned areas with no differences due to the fact that even the largest elements of the pattern (1 µm x 5 µm) were much smaller than the adhesive area (120 µm2) of the soma size of cortical neurons. Furthermore, the SEM images revealed that the neurons extended neurites > 100 µm which also arborized on both particular areas of the substrate. In contrast, clear differences were observed for the growth of neurites on the patterned area compared to the homogeneously coated one. On the patterned surface, neurites showed predominantly an aligned outgrowth corresponding to the underlying pattern indicating that the structure elements of the gradient were recognized in both main directions of the nanopattern which are perpendicular to each other. On the homogeneously coated surface the neurites formed an extensive meshwork of stochastically criss-crossing neurites. For a better analysis of the effect of nanopatterns on the neurite growth SEM images with a higher magnification were recorded. Imaging parameter were selected such (voltage 0.2 kV, inlens detector) that both protein pattern and neurons with outgrown neurites are displayed simultaneously (Fig.2). However the shallow protein layers (2 – 10 nm) were buried underneath the sputtered iridium layer, which results in a rather poor contrast and small protein features are difficult to reveal at some places. Therefore, the designed pattern was superimposed onto the SEM images aligned to the protein pattern underneath in order to highlight the areas which are covered with proteins. The SEM images further confirmed the alignment of the neurite growth with the pattern, even though a few neurites do not follow the underlying pattern. Growth cones could also be imaged demonstrating that the filipodia adhere at different structure elements including the smallest ones with a width of 75 nm. Thus, the technique proposed in this work may path the way to systemically study the influence of nanometer sized purely biochemical gradients on guiding neuronal cells by directing neurite outgrowth. These discontinuous gradients will further enables to evaluate the role of ECM ligand spacing and in parallel also the effect of defined gradient parameters on axon guiding.

[1] K. Dertinger, X. Jiang, Z. Li, V. Murthy, G. Whitesides, PNAS 2002, 1, 12542.

[2] A. von Philipsborn, S. Lang, J. Loeschinger, A. Bernard, C. David, D. Lehnert, F. Bonhoeffer, M. Bastmeyer, Development 2006, 133, 2487

[3] A. Offenhäusser, S. Böcker-Meffert, T. Decker, R. Helpenstein, P. Gasteier, J. Groll, M. Möller, A. Reska, S. Schäfer, P. Schulte, A. Vogt-Eisele, Soft Matter, 2007, 3, 290

[4] A. Vogt, F. Stefani, A. Best, G. Nelles, A. Yasuda, W. Knoll, A. Offenhäusser, J. Neurosci. Meth. 2004, 134, 191.

IBN-4: Biomechanics Director: Prof. Rudolf Merkel

Our research is directed towards a basic understanding of the physical principles, underlying structure and function of living cells. In this vast field we focus on the mesoscopic length scale from molecules to cells where principles of soft matter physics shape biological processes. On this length scale we study mechanical functions and properties of living cells, molecular aggregates, and molecules.

To achieve our goal, we perform complementary experiments on living cells and biomimetic model systems. By studying living cells we gain insight into the physiological relevance of processes and their likely mechanisms. At present, adhesion, locomotion, force generation, and mechano-sensing of living animal cells are at the center of our interest. Further, experiments on model systems designed to mimic bioadhesion and the cytoskeleton enable us to achieve a quantitative understanding. In order to pursue this double strategy we develop and apply advanced methods for measurements on living cells and biomolecules as well as preparation and analysis techniques for tailored biomimetic systems.

Our research strategy traverses the boundaries between physics, chemistry, and biology. Therefore researchers from all three classical sciences form the institute’s scientific staff. Interdisciplinary cooperation on common projects on an everyday basis is one of the major strengths of the institute.

Deformation of Soft Solid Objects:Resolution Limit in Replica MoldingO.D. Gordan1, Bo N.J. Persson2, C.M. Cesa1,*, D. Mayer3, B. Hoffmann1, S. Dieluweit1,R. Merkel1

1 IBN-4: Biomechanics2 IFF-1: Quantum Theory of Materials3 IBN-2: Bioelectronics* now at Philips Research Laboratories, Care & Health Applications, Eindhoven - The Netherlands

The shape of liquid droplets results from theinterplay between surface tension and externalforces. In a similar way, very soft solids are af-fected by surface tension at scales relevant formicro-technology and biology. The characteristicsmoothing length is proportional to the ratio be-tween the surface tension and Young modulus ofthe material.

This can be understood from the following qualita-tive argument: the elastic energy stored in a solid ob-ject, with linear size D and with uniform strain of or-der unity, scales with the volume of the solid as ED3

where E is the elastic modulus. The surface free en-ergy scales with the surface area as γD2, where γ isthe surface free energy per unit area. Thus one mayexpect a strong shape deformation if D is of orderγ/E [1].

As an example, for very soft rubber E may be of or-der 10 kPa and if γ is of order 10 mJ/m2 we getD ≈ 1 µm. Thus, soft micrometer sized solid ob-jects may undergo shape deformations correspond-ing to a strain of order unity. This qualitative conclu-sion has many practical implications, e.g., for micro-technology and biology. Therefore nano and micro-molding using elastic material will produce replicaswith a characteristic smoothing length scale of γ/E,as can be seen in Figure 1.

The experiments were performed using silicone rub-ber prepared using a two part kit, Sylgard 184,purchased from Dow Corning (Midland, MI). Byadjusting the mixing ratio of the base (vinyl ter-minated polydimethylsiloxane) to the curing agent(methylhydrosiloxane-dimethylsiloxane copolymer) inthe range from 10:1 to 55:1 the elasticity of the re-sulting rubber can be tunned between 2 MPa to 10kPa. The mixtures were poured over silicon oxidemolds patterned by conventional optical lithographictechniques. A glass coverslip was placed over thePDMS layer, and the whole ensemble was curedover night at 60 C. Before the molding processthe PDMS was degassed in a desiccator using amechanical pump. Upon curing the mixture under-goes a cross-linking reaction which will produce asolid elastomer. The elasticity of the resulting rub-ber depends on the cross-link density via the mix-ing ratio of the two constituents. In order to preventPDMS sticking to the mold, the silicon oxide masters

FIG. 1: (a, d) AFM images of SiO2 molds, (b, e) stiff 10:1PDMS (1.6 MPa) and (c, f) soft 50:1 PDMS (17 kPa) repli-cas. The PDMS surfaces were scanned in 1% Triton X-100.

were silanizated in vacuum using 1H, 1H’, 2H, 2H’-perfluorooctyl-trichlorosilane (Sigma, St. Louis, MO).Two types of mold patterns were used: the first oneconsisted of a square lattice with 2.5 µm squares andlattice constant of 3.5 µm (Fig. 1a) and the secondone had long trenches of 2, 5, 10, 20, 50, 70 µmwidth, with a 100 µm spacing in between (Fig. 1d).Here it will be demonstrate how the competition be-tween surface free energy and elastic (bulk) energywill modify the surface profile of a solid. Usually,stamps for micro-contact printing, micro-fluidic chan-nels and micro-textured surfaces are produced usingPDMS mixtures of 10:1 (base:to cross-linker) [2] giv-ing accurate replicas at this length scale (microme-ter). In order to illustrate the strong elasticity effect,PDMS rubber with Young moduli of 11, 17, 20, 48,93, 144 kPa and 1.6 MPa was molded from mixturesof 55:1, 50:1, 40:1, 30:1, 35:1 and 10:1, respectively.The comparison between 10:1 and 50:1 can be seenin Figure 1. The shape change due to the surfacetension and elasticity can be calculated starting froma sinusoidal surface profile with amplitude h0 andwavelength λ:

u0 = h0cos

We will assume that h0 << λ. In this case the “re-

laxed" surface profile u1 would still be sinusoidal, witha reduced amplitude h1, but the same wavelength.Thus, the surface displacement in normal direction is

u = u0 − u1 = (h0 − h1)cos

corresponding to the local pressure distribution [3]

p =πE∗(h0 − h1)

where E∗ = E/(1 − ν2), with E the Young modulusand ν the Poisson ratio. Thus, the elastic energy canbe written as

Eel =1

∫d2x u(x)p(x) = A0

πE∗

4λ(h0 − h1)2,

where A0 is the nominal area of the surface. Thesurface energy is:

Es = γ

∫d2x

(du1(x)

≈ A0γπ2

Minimizing the total energy Eel + Es with respect toh1 gives

h1 =h0

1 + 4πγ/λE∗≈ h0

(1− 4πγ

λE∗

). (1)

For surface profiles characterized by some width λand some height h0 we expect the scaling

∆h = h0 − h1 ∼γ

In general a surface with a variation in one directioncan be mathematically described in the Fourier for-malism as:

h0(x) =

∫dqh0(q)eiqx (3)

with q = 2π/λ. Then the relaxed surface profile is

h0(x) =

h0(q)eiqx

1 + 2qγ/E∗(4)

Starting with a surface profile like the one in Figure 1efor h0(x), the surface profile from Figure 1f can becalculated using Equation (4). The calculated pro-files for the 2-, 5-, 10-, and 20- µm -wide lines com-pared with the experimentally measured ones arepresented in Figure 2. As the AFM scan was donein Triton X-100 a surface tension of 8.5 mN/m and astiffness of 11 kPa were considered in the calculation.

Equation (2) indicates that the expected change inheight is proportional to mold depth. This is indeedvisible in Figure 3 which shows the normalized heightchange for the 2 and 5 µm lines produced in differentmolds. Moreover, ∆h/h0 measured at the center ofthe line can be predicted well within the error barsof the AFM measurement knowing only the elasticity,Poisson ratio and the surface tension of the material.

FIG. 2: Measured (solid lines) and calculated (dashedlines) height profiles of lines molded in 11 kPa PDMS using2-, 5-, 10-, and 20- µm -wide lines molds of 531 nm depth.Theory curves were calculated using an interface tension of8.5 mN/m.

FIG. 3: Normalized height change of the PDMS replicasproduced in lines molds (1d) with depths of 531, 299, 184,and 93 nm. Error bars denote the standard deviation ofthe values measured at the different mold depths. Experi-mental results for line widths of 2 µm (triangles) and 5 µm(squares) are shown along with the theoretical results, withan interface tension of 8.5 mN/m.

Even for stiff PDMS (≈ 2MPa) molding fidelity willbecome problematic at smaller length scales, equa-tion (2) giving a good analytical expression to the ruleof thumb ”the stiffer the better”. Equation (4) can beused for exact surface topography calculation of theresulting replica molding samples when the elasticityand the surface tension of the material are known. Aninverse approach would give a direct measure of thesurface tension for soft solids in the case of knownYoung modulus and expected surface profile.

[1] O.D. Gordan, B.N.J. Persson, C.M. Cesa, D. Mayer,B. Hoffmann, S. Dieluweit, R. Merkel - Langmuir 24,6636 (2008)

[2] Y. Xia, G. Whitesides, Annu. ReV. Mater. Sci. 28, 153(1998)

[3] H. Westergaard, Trans. Am. Soc. Mech. Eng., J. Appl.Mech. 6, 49 (1939)

Presenilin 1 Affects Focal Adhesions and Cell Forces via c-Src Regulation D. Waschbüsch1, S. Born1, V. Niediek1, N. Kirchgeßner1, I.Y. Tamboli2, J. Walter2, R. Merkel1, B. Hoffmann1 1IBN-4: Biomechanics 2Department of Neurology, University of Bonn Presenilin 1 (PS) is a critical component of the γ-secretase complex that cleaves trans-membrane proteins. This process plays an essential role in signal transduction and vital functions such as cell adhesion. Here, we found for PS1-/- cells an altered morphology with significantly reduced sizes of focal adhesion sites (FAs) compared to wild-type. Furthermore, cell forces were reduced by 50%. PS1 deficiency was also associated with decreased tyrosine phosphorylation levels of FA specific proteins, which was caused by a transcriptional down-regulation of c-Src kinase. The direct regulatory connection between PS1 and c-Src is ephrinB2, a receptor mediating cell-cell adhesion. EphrinB2 becomes cleaved by PS1 with a subsequent translocation of the ephrinB2 intracellular domain (ICD) into the nucleus, acting as coactivator for c-Src transcription. Therefore, we conclude that γ-secretase is vital for controlling cell adhesion by transcriptional regulation of c-Src via ephrinB2 cleavage.

PS1 is an aspartyl protease forming the active components of the γ-secretase complex, which cleaves transmembrane proteins within their transmembrane domains leading to the release of the two cleavage products from the membrane (1). The freed products can play important roles in signalling pathways by translocating into the nucleus and acting as transcriptional coactivators (2). Presenilin affects many mechanisms and one of these is the ephrinB/Eph receptor mediated cell-cell adhesion. Since ephrins additionally bind to cellular sarcoma protein kinase (c-Src), a protein vitally involved in focal adhesion formation, it is speculated that PS might be an important regulator for switching cell function from a sessile to a dynamic, moving phenotype.

Here, we identified a strong reduction in FA size upon PS1 deficiency associated with a reduction in cell force formation by over 50%. Both effects went along with a decreased phosphorylation level of FA associated proteins and were caused by a PS1 dependent downregulation of c-Src on the level of transcription via ephrinB2 cleavage.

Cell analyses were performed using wild-type mouse embryonic fibroblasts as well as PS1-/- mutant strains. Cell biological procedures were performed according to standard protocols. Cells were either analyzed using phase contrast or transfected with GFP-fusion proteins and analyzed by fluorescence microscopy. Alternatively, cells were fixed and proteins were stained by immunolabelling. Northern blot, western blot as well as qRT-PCR experiments were performed according to standard protocols. Elastomeric substrates were prepared and calibrated (Young’s modulus = 13 kPa, Poisson’s number = 0.5) as described earlier (3).

To characterize the exact function of PS in cell-matrix adhesion processes wild-type (WT) and presenilin 1 knock out mouse embryonic fibroblasts were cultured and used for immunofluorescence analyses against actin and vinculin as marker of focal adhesion sites (Fig. 1). WT cells were characterized by well visible FAs. These sites with an average size of 0.9 µm² were mainly located at the cortex of cells and had an elongated shape. To every FA thick F-actin bundles, called stress fibers, were connected. Very different results were found for PS1-/- cells. Here, the number of FAs was reduced by 40%. The average size of FAs was diminished by 30 to 40% and reached 0.65 µm² on average. Their spatial distribution changed from a cortical to a disperse localization. Prominent actin stress fibers were absent.

For a quantitative measurement of cell forces WT as well as the PS1-/- mutant strain were seeded on micropatterned, soft PDMS-substrates. Displace-ments of the regular micropattern were visualized by RICM and the generating forces were calculated from these data. For single FAs of WT cells, analyses revealed forces in the range of 13 nN (Fig 1B) with a generalized first moment of about 8.5 pNm (σ=4.7 pNm; n=86). PS1-/- cells instead applied forces in the range of just 7 nN per focal adhesion with a generalized first moment of 3.0 pNm (σ=3.1pNm; n=36).

Since earlier experiments indicated an influence of PS on ephrinB1 putatively affecting c-Src activity, we analyzed the phospho-tyrosine levels of FAs in WT and PS1-/-.

Fig. 1 (A) WT as well as PS1-/- cells were stained for actin and vinculin in immunolabelling experiments. (B) Cell types as in (A) were grown on elastomeric substrates and substrate deformations (yellow) were determined. Under-lying forces applied at every FA (red) as well as the generalized first moment (blue) were calculated as described in Cesa et al. (3).

These analyses were performed in fixed cells using a phospho-tyrosine specific antibody and revealed high levels of phosphoryla-tion in FAs of WT cells while phosphorylation of PS1-/- FAs was almost absent (data not shown).

Since phosphorylation in FAs is mainly performed by activated c-Src kinase we analyzed the activation status of this kinase at tyr418 as well as its expression level in WT and PS1-/- cells. As given in Figure 2A, c-Src phosphorylated at tyr418 was decreased by 90% in the absence of PS1 compared to WT. As such result might have been caused by either regulation of autophosphorylation or regulation of expression, northern as well as western analyses for c-Src were performed. Protein levels of c-Src were reduced by 60% in PS1-/- cells (Fig. 2A). Northern analyses identified an almost identical reduction of c-Src transcripts. Here, levels of c-Src mRNA were reduced by 50% in PS1-/- cells (Fig. 2B). Reduced levels of c-Src protein were therefore likely caused by transcriptional downregulation rather than increased protein degradation.

EphrinB2 cytoplasmatic domain is translocated in a PS1 dependent manner into the nucleus. To complete the signal transduction pathway from PS1 function to c-Src activity, we checked γ-secretase targets for a putative influence on c-Src. Since two of them, ephrinB1 and ephrinB2, were also known binding partners of c-Src, we analyzed ephrinB2 in more detail. The experiments revealed c-Src regulation mainly at the transcriptional level. We therefore tested ephrinB2 intracellular domain (EB2-ICD) for transcriptional co-activator function. An EB2ICD-GFP construct was expressed in WT as well as PS1-/- cells and its localization was compared to GFP only.

Fig. 2 Crude protein (A) as well as total RNA (B) were isolated from WT and PS1-/- and analyzed for indicated proteins/mRNAs. α-tubulin and 28S rRNA, respectively, were used as internal standards

Life cell imaging revealed intense nuclear localization of EB2ICD-GFP (Fig. 3A). FAs of transfected PS1-/- cells were also restored in tyrosine phosphorylation intensity and size (1.0 µm², σ=0.4 µm2, n=100 FAs) (Fig. 3A). Furthermore, qRT-PCR experiments revealed a strong increase of c-Src mRNA levels in these cells compared to untransfected PS1-/- cells. A similar increase was observed for c-Src in western analyses (not shown).

Although toxic at high concentrations or enhanced incubation times, a full length EB2-GFP construct, expressed in WT and PS1-/- cells, identified the dependency of EB2-ICD translocation on PS1 function. While nuclear localization could be observed for EB2-GFP in WT cells no such signal was present in PS1-/- (Fig. 3B). These data proof EB2 to be cleaved by γ-secretase and that an EB2 cleavage product is transduced into the nucleus were it functions as transcriptional coactivator for c-Src.

Fig. 3 (A) PS1-/- cells were transfected with an EB2ICD-GFP construct and analyzed in fixed cells for GFP (green) as well as tyrosine phosphorylation of FAs. (B) WT and PS1-/- cells were transfected with full length EB2-GFP and analyzed for nuclear translocation of an EB2-GFP fragment. Note that translocation was only detected in the presence of functional PS1.

[1] B. De Strooper, W. Annaert, P. Cupers, P. Saftig, K. Craessaerts et al. (1999) Nature 398, 518-522

[2] A. Georgakopoulos, P. Marambaud, S. Efthimiopoulos, J Shioi, W. Cui et al. (1999) Mol Cell 4, 893-902

[3] C. M. Ceşa, N. Kirchgeßner, D. Mayer, U. S. Schwarz, B. Hoffmann, R. Merkel (2007) Rev. Sci. Instr. 78, 034301

Heart Muscle Cells Suspended Between Elastic Micropillars A. Kajzar1,2, N. Hersch1, C.M. Cesa1, N. Kirchgeßner1, B. Hoffmann1, R. Merkel1 1Institute of Bio- and Nanosystems 4: Biomechanics 2 Marian Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, 30-059, Krakow, Poland In living animals cells are parts of tissues where they experience a soft environment of polymers and neighbouring cells. However, classical cell culture employs hard and flat substrates such as polystyrene plates for cell attachment. These highly artificial culture conditions lead to cell adaptation which often severely compromises experimental results. Here we developed micropillar arrays from soft rubber as cell culture systems. Heart muscle cells were cultivated within these systems. A comparison of these cells with others that were grown on flat surfaces of identical material revealed much closer to nature cell morphology on micropillars. Moreover, spontaneous myocyte beating lead to micropillar bending that was exploited to determine cell forces. Cell force and micropillar stiffness showed a clear correlation indicating amplitude as decisive feed-back signal for the control of cellular contraction. Within micropillar arrays we cultivated rat heart muscle cells (cardiac myocytes). Cells strongly preferred to span between the elastic micropillars over adhesion to the underlying flat substrate. In addition, the architectures of the cytoskeleton and of protein complexes formed for adhesion were strongly dependent on the environment of the cell. On flat parts of the substrates we observed prominent stress fibres and focal adhesion sites. In contrast, cells suspended between micropillars exhibited well organized myofibres and costameric adhesions at the locations of Z-bands as found in heart tissue. These observations argue for close to nature environmental conditions within elastomeric micropillar arrays. Furthermore, these micropillars could be exploited to determine cell forces. To this end we derived closed expressions for the stiffness of elastically founded pillars.

Sylgard 184 (PDMS*) was carefully mixed in a 20:1 ratio (base:cross-linker) and dispensed onto a microstructured SU-8 master. Subsequently, system was overlayed by a cover slide and PDMS* was crosslinked at 60°C. The next day, cover slides with PDMS micropillars on top were peeled off the masters and glued to the bottom of perforated Petri dishes. Before use, micropillars were fluorescently labelled with DiD [1].

Myocytes were isolated from 19-day old Wistar rat fetal pups as described [2]. 30.000 cells were added to a micropillar substrate and analyzed 2 to

3 days after. Cells were either fluorescently labelled with calcein or fixed for immunofluorescence experiments. Analyses were performed on a confocal microscope (Zeiss, LSM510).

FIG. 1: Myocytes stained for α-actinin (red). Left: Myocyte suspended between micropillars. Image was taken at a height of 20 µm above pillar base. Note that all sarcomer units are aligned in parallel forming the classical striated muscle morphology whereas this morphology is severely disturbed on flat substrates (right). Right: Mycocyte grown on flat substrate besides micropillars. Note the identical scales.

FIG. 2: Laser scanning microscopy images of pillars (red) with adhered cells (green). Side views. Pillar diameters, 10 µm.

FIG. 3: Myocytes were grown within labelledmicropillar arrays for two days. Using a confocalmicroscope pillars were imaged at a height of 15µm above the base. A: Pillar positions during cellrelaxation (top), and contraction (bottom). B:Micropillar displacements along (full lines) and perpendicular (broken lines) to cell orientation. C: Forces of many cells during contraction (filledboxes) and in the relaxed state (open boxes). The lines are linear regressions. Full line: contractionforces, slope 1.6 µm; broken line: forces in therelaxed state, slope 0.6 µm.

Myocytes are a highly organized cell type in heart tissues. They are characterized by repetitive units of actin-myosin fibres, called sarcomers. These units fill the whole cell with parallel orientation to each other and are connected to the environment by specific adhesion structures, named costamers [3]. In contrast, neither the high organization of sarcomers nor costameric adhesion structures can be found when myocytes are cultured on flat and stiff substrates as e.g. classical culture dishes. To

probe the impact of substrate geometry on these cells, we developed an array of elastomeric micropillars as cellular environment. Freshly isolated myocytes were seeded on these arrays and analyzed. Size of myocytes adhered to micropillars decreased compared to those adhered to flat substrate, cf. Fig. 1. In addition, cells were characterized by a strongly reduced number of actin stress fibres. Furthermore, cells grown within micropillar arrays avoided adhesion to the elastic base of the micropillars. Instead, myocytes exclusively span between the pillars for pillar distances of 10 to 30 µm (Fig. 2).

In order to characterize the force generation system of myocytes grown between elastic micropillars in more detail, we determined cell forces from micropillar bending, cf. Fig. 3. Despite the fact that pillar bending is a classical application of continuum mechanics, no closed expressions for the stiffness of micropillars emerging from a substrate of identical material were available. Therefore we derived an approximate treatment based on the Boussinesq theory of the deformation of an elastic half space under the influence of mechanical forces. This approach revealed that micropillars emerging from identical material are by about 30% softer compared to micropillars with clamped bases, i.e. the commonly made assumption overestimates cell forces substantially.

Evaluating cell forces we found a clear cut correlation between micropillar stiffness and cell forces. This can be easiest explained by cell contractions being regulated to achieve a specific amplitude, a conclusion which was further supported by the fact that the slopes of the regression lines coincided with the mean cell contractions in relaxed and contracted state.

Taken together our data clearly show that close to nature cell culture can be achieved using elastic micropillars. As a further benefit, these microsystems enable reliable cell force measurements and valuable insight into mechanical regulation of cellular processes. Applications in functional drug studies on the single cell level are foreseen.

[1] Kajzar, A., C.M. Cesa, N. Kirchgeßner, B.

Hoffmann and R. Merkel, Biophys. J. PMID: 17981895 (2007)

[2] Cesa, C.M., N. Kirchgessner, D. Mayer, U.S. Schwarz, B. Hoffmann and R. Merkel, Rev. Sci. Instrum. 78:034301 (2007)

[3] Alberts, B., D. Bray and J. Lewis, Molecular Biology of the Cell, W.H.Freeman & Co Ltd, Oxford (2006)

Diffusion and Membrane Adhesion:Avidin and E-Cadherin Case StudyS. Fenz1, K. Sengupta1, 2, R. Merkel1

1 IBN-4: Biomechanics2 Centre Interdisciplinaire de Nanoscience de Marseille, CINAM/CNRS-UPR3118, France

Cell-cell adhesion is a highly complex process ofvital importance for any multicellular organism.An abundance of proteins and signal cascadescontribute. Nevertheless, in early stages physi-cal forces dominate as the cell needs some timeto initiate an active response. Here, we present abiomimetic model system for cell-cell adhesionmediated by mobile receptor-ligand pairs. Westudy quantitatively the role of the membranefluidity and the diffusivity of membrane-boundreceptors in biomembrane adhesion applyingbleaching techniques. Membrane adhesion ischaracterized with respect to the inter-membranedistance by high precision micro-interferometry.

In recent years, structure and dynamics of the es-sentially fluid cell membrane and its nanoscale in-homogeneities have attracted much interest. Diffu-sion of lipids and proteins in the plane of the mem-brane impacts directly on the local dynamic structureof the membrane. Diffusion thus plays a vital rolein many membrane related functions including adhe-sion, recognition and transport. To quantitatively in-vestigate the impact of individual parameters on thefluidity and structure of the membrane, well-definedmodel systems were developed. The scope of thisproject was to elucidate the underlying physical prin-ciples.

We applied Continuous Photobleaching (CP) andFluorescence Recovery after Photobleaching (FRAP)to study membrane fluidity as a function of spe-cific protein binding to ligands within the membraneand depending on binding of a second membrane.Membrane adhesion was effected either by biotin-neutravidin (an avidin analogue) or the extracellulardomains of the homophilic cell adhesion moleculeE-cadherin (see FIG 1). To determine the result-ing inter-membrane distance with high precision (± 4nm), traditional Reflection Interference Contrast Mi-croscopy (RICM) theory was extended to account forthe reflections from all five relevant interfaces. Forneutravidin the fate of the receptors during mem-brane adhesion was additionally monitored. A solidsupported lipid bilayer (SLB) served as a model forthe first membrane. It was prepared by the Langmuir-Blodgett Langmuir-Schäfer technique and containeda controlled percentage of ligand lipids. We usedbiotinylated lipids to bind neutravidin and NTA lipids

to bind the E-cadherin Fc chimera via its histag (fordetails on the chimera see FIG 3). Giant unilamel-lar vesicles, prepared by electro-swelling, were em-ployed to mimic the second membrane.

FIG. 1: Sketches of the model systems illustrating themembranes and binding molecules involved. Left: biotin-neutravidin, right: E-cadherin-E-cadherin.

We found that in case of strong receptor-ligand in-teraction (biotin-avidin, Gibbs free energy of bonding∆G0 in solution ∼ 35 kBT) binding of soluble recep-tors to the SLB alone led to reduced diffusion of tracerlipids reflecting a decrease in the overall membranefluidity [1]. This effect scaled with the concentrationof receptors (see Table 1). From theoretical consid-erations, the decrease could be attributed partially tointroduction of obstacles [2] and partially to viscouseffects [3]. The obstacles were formed by biotiny-lated lipids pairwise connected to the same neutra-vidin molecule. This formed one large object, whichdiffused more slowly than single lipids or was com-pletely pinned depending on the receptor concentra-tion. Moreover, the viscous protein layer on the distalside of the SLB introduced extra friction.

biotin bare +NAV +NAV+GUV

1% 2.3±0.2 2.1±0.2 1.8±0.25% 2.2±0.2 1.7±0.2 1.5±0.2

TAB. 1: Diffusion constants [µm2/s] of the tracer lipid in theSLB before and after binding of protein or vesicle for variousconcentrations of biotinylated lipids in the SLB. Error barsrepresent single standard deviations.

Specific binding of a GUV membrane led to addi-tional slowing down of tracers (up to 15%) caused byan increase in obstacle density and enhanced fric-tion due to the adhering membrane. Receptors wereaccumulated in the adhesion zone till full coveragecorresponding to 5% biotinylated lipids was achieved(see FIG 2). Bleaching experiments on the receptorsthemselves revealed a fast decrease in receptor mo-bility with increasing receptor concentration. At fullcoverage the receptors were practically immobilized[1].

FIG. 2: (a) RICM micrograph of an adhering vesicle. (b)Reconstructed height [nm] in the adhesion disc. (c) Fluo-rescence micrograph of accumulated neutravidin in the ad-hesion disc. The initial biotin concentration on the SLB was2%. (d) Intensity profile along the white line in c. The scale-bar is valid for a-c: 10 µm.

In case of weak receptor-ligand interaction (E-cadherin-E-cadherin, ∆G0 ∼ 2 kBT) no significantchange in diffusion of tracer lipids was observedupon protein binding and subsequent vesicle binding[1]. At first glance, the lack of retardation after recep-tor binding is surprising, since the Ecad construct isa dimer and its binding should therefore have intro-duced, as in the strong binding case, mobile obsta-cles in the form of coupled NTA lipids. Nevertheless,the size of the weak protein-lipid complex was onlyone third of the strong one. Thus, it had no measur-able effect on the tracer lipid diffusivity. In order tounderstand the lack of retardation after vesicle bind-ing the inter-membrane distance for both cases wasanalyzed. We measured hexp = (7 ± 1) nm for biotin-neutravidin. This height was in very good agreementwith the protein and linker dimensions known fromcrystallography and X-ray reflectivity (hth = 5.6 nm).

The exact E-cadherin binding configuration is still un-der discussion. So far, three different scenarios wereproposed (see FIG 3) corresponding to hth,1 = 52 nm,hth,2 = 44 nm and hth,3 = 36 nm. We measuredhexp = (54 ± 9) nm strongly favoring the scenariowith only the outmost domain involved [1]. As a re-sult, the inter-membrane distance in the E-cadherin-E-cadherin case was significantly larger than in thebiotin-avidin case. Consequently, the adhering mem-brane generated less friction.

FIG. 3: Three different binding configurations of E-cadherin-E-cadherin are under discussion: overlap of theoutmost domain, the three outmost domains or all domains.

In summary, we gained three new insights:

a) The impact of protein binding and protein medi-ated membrane binding on the fluidity of a mem-brane depends strongly on the geometry of theprotein - especially on the number of binding sitesand their distance as well as the closeness of theinteraction.

b) The high precision micro-interferometric mea-surements of the inter-membrane distance al-lowed to shed light on the hitherto unclear bind-ing configuration of the cell adhesion moleculeE-cadherin. We conclude that E-cadherinmolecules residing on a soft, flexible membranesbind predominately with their first domain.

c) The adhesion assays in a cell-free model systemunequivocally show that accumulation of mobilereceptors does not require active interaction of acell.

These results on diffusion, inter-membrane distanceand receptor accumulation should contribute to theunderstanding of equivalent phenomena in cell-celladhesion.

[1] S. F. Fenz, R. Merkel and K. Sengupta, Langmuir25(2), 1074-1085 (2009).

[2] M. J. Saxton, Biophysical Journal 52(6), 989-997(1987).

[3] E. Evans and E. Sackmann, J. Fluid. Mech. 194, 553-561 (1988).

New Role for Filopodia in Adhesion Formation During Cell Migration C. Schäfer, B. Borm, S. Born, C. Möhl, E.M. Eibl, B. Hoffmann IBN-4: Biomechanics Cell migration is a decisive prerequisite for embryogenesis, wound healing and immune defence. The exact interconnection between focal adhesion (FA) formation and correct guidance is the basis for an efficient functioning of these major biological processes. FAs connect the cytoskeleton of the cell to the environmental matrix to provide anchorage and force transmission. We found an essential role for filopodia in the formation of FAs during cell migration. Since now, FAs were thought to be built up in the lamellipodium. Here we show that nearly all FAs depend on stable filopodia. These FAs were formed along the axis of filopodia containing all tested adhesion proteins and were just increased in size when reached by the lamellipodium. Blocking filopodia fully inhibits FA formation. Therefore, filopodia are the key factors for formation and localization of all FAs and thus for accurate cell movement and direction.

Keratinocytes are epithelial cells derived from human skin which effectively migrate after stimulation. While migrating they form a wide lamellipodium with long finger-like structures, so called filopodia, in direction of migration (see FIG. 1 A). The lamellipodium as characteristic feature at the leading edge of motile cells creates the necessary force to pull the front of the cell in direction of migration via actin polymerisation [1] and locates the origins of new focal adhesions [2]. In contrast, filopodia are thin membrane extensions comprising tight bundles of parallel actin filaments with their proximal end embedded

FIG. 1: Correlation between filopodia and focal adhesion sites. Keratinocytes were grown for one day and subsequently motility induced by EGF (epidermal growth factor). Confocal images in phase contrast (A) and in reflection (B) are shown. Note the localization of focal adhesion sites right behind stably adhered filopodia (black arrows in A and B). Direction of keratinocyte movement=white arrow. Scale bar=10 μm.

in the lamellipodial actin network. Functionally, filopodia are described as structures mainly probing the substrate for proper matrix composition but how filopodia integrate the substrate information into lamellipodial processes especially formation of new adhesion sites is still unclear although association of adhesions with filopodia has been described [3].

FIG. 2: Protein content of filopodial extensions. Motility induced keratinocytes were fixed and immunofluorescently labeled using antibodies against focal adhesion site specific proteins paxillin (A), talin (B) and vinculin (C). Localization of GFP–tensin (D), GFP–VASP (E) and GFP–zyxin (F) were analyzed in living cells. Here, keratinocytes were transfected one day before EGF stimulation and subsequently analyzed by confocal microscopy. White arrows indicate fluorescent signals within filopodia in fluorescence and phase contrast. Note that all proteins can be found in the filopodia extensions. Scale bar=10 μm.

Many motile cells such as keratinocytes exhibit a highly dynamic formation and disassembly of adhesion sites. These adhesion sites become prominent at the leading edge of lamellipodia with an elongated shape upon movement. In our experiments we focus on the interrelation between filopodial and lamellipodial adhesion structures. Strikingly, lamellipodial adhesions were always located in direct extension of filopodia (see FIG. 1). Lamellipodial adhesions were always found right behind stable filopodia whereas cell areas with filopodia unable to attach were characterize by the absence of lamellipodial adhesions.

The given data strongly argued for an important function of filopodia in adhesion site formation of moving cells. To analyze if adhesion structures can already be found in filopodia the protein localization of focal adhesion site specific proteins

like paxillin, talin, tensin, the vasodilator-stimulated phosphoprotein (VASP) and vinculin was tested by immunofluorescence as well as in living cells using green fluorescent protein (GFP)-fusion constructs (see FIG. 2). These data clearly show that each of these proteins is localized in filopodia most likely along their complete lengths with signal intensities highly above cytoplasmic background. To our surprise the same localization was also found for zyxin, a marker protein for mature focal adhesions (see FIG. 2 F). These data suggest that stable filopodia form small but fully assembled focal complex like structures termed here filopodial focal complexes (FX).

FIG. 3: Growth of filopodial FXs to focal adhesions. Keratinocytes were transfected with tensin as GFP-fusion protein and motility stimulated after 24 h of growth. Stably adhered filopodia were analyzed over time in phase contrast (top) and fluorescence (bottom). White arrows indicate filopodial FXs at time point 0 changing to focal adhesions over time. Same position is also indicated in phase contrast in the first image of the series. Note the strong increase in fluorescence as soon as the lamellipodium reaches the filopodial adhesion. Time points are given in seconds. Scale bar=3 μm.

We clearly showed that adhesion structures can already be found in filopodia. Therefore we analyzed adhesion formation in filopodia using live cell imaging (see FIG. 3). Here, all tested adhesion proteins were present along stably adhered filopodia. Their localization labeled parts of the filopodia or covered their whole lengths but was always elongated. As soon as the filopodial adhesion proteins were reached by the lamellipodium, the size of the adhesion structure increased along the former orientation of filopodia. During this enlargement process the spatial orientation of the focal adhesion did not change. These data illuminate that form and localization of focal adhesions is determined by filopodial adhesions.

Since our data on moving keratinocytes clearly showed a dependency of focal adhesion sites on filopodial adhesions, we analyzed whether focal adhesions would be formed in the absence of filopodia. For this reason we blocked filopodia formation by neomycin supplementation. In the presence of neomycin all cells (n=338) were significantly reduced in migration (see FIG. 4 A).

Cells transfected with GFP–vinculin identified a full correlation between the neomycin induced migratory block and inhibited focal adhesion formation. Furthermore, FRAP (Fluorescence Recovery After Photobleaching) analyses on these cells revealed a full maturation state of focal adhesions formed before neomycin treatment but still located at the leading edge of polarized cells (see FIG. 4 B). These results were highly comparable to those found in [4] for focal adhesions of sessile, neomycin untreated keratinocytes while nascent adhesions of migrating cells contained a much higher fraction of exchanging vinculin (see FIG. 4 B, red curve).

FIG. 4: Blocking filopodia inhibits motility and adhesion formation: (A) Migration speeds of EGF stimulated keratinocytes with (n=74) and without (n=62) neomycin treatment were analyzed and are indicated as boxplots. Migration speeds are significantly different (p-value<0.001). (B) GFP–vinculin transfected cells were treated with neomycin. Afterwards, focal adhesions at the leading edge were analyzed by FRAP. Mean value and standard deviation for every time point from normalized curves are given (blue, n=13). For better visualization data were overlaid with experiments on focal adhesions at the leading edge of migrating, neomycin untreated cells (red, n=14, [4]). A single exponential model was fitted to the data describing a simple binding-unbinding kinetics.

Taken all these data together we present new insights in formation and function of filopodia in migrating keratinocytes. It becomes clear that filopodia play a fundamental role during adhesion site formation and thus determine the localization and shape of almost every adhesion site.

[1] A. Ponti, M. Machacek, S.L. Gupton, C.M. Waterman Storer, G. Danuser, Two distinct actin networks drive the protrusion of migrating cells, Science 305 (2004) 1782–1786.

[2] R. Zaidel-Bar, C. Ballestrem, Z. Kam, B. Geiger, Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells, J. Cell Sci. 116 (2003) 4605–4613.

[3] M. Nemethova, S. Auinger, J.V. Small, Building the actin cytoskeleton: filopodia contribute to the construction of contractile bundles in the lamella, J. Cell Biol. 180 (2008) 1233–1244

[4] C. Möhl, N. Kirchgeßner, C. Schäfer, K. Küpper, G. Diez, W.H. Goldmann, R. Merkel, B. Hoffmann, Vinculin exchange dynamics regulates adhesion site maturation and adhesion strength, unpublished.

The Mechanism of Adhesion Strength Adaptation in Living Cells C. Möhl1, N. Kirchgeßner1, C. Schäfer1, K. Küpper1, G. Diez2, W.H. Goldmann2, R. Merkel1, B. Hoffmann1 1IBN-4: Biomechanics 2Center for Medical Physics and Biology, Friedrich-Alexander-University of Erlangen-Nuernberg The morphogenesis and architecture of multicellular organisms is determined by the coordinated adhesion of single cells to each other and to the extracellular matrix (ECM). The mechanical connection between the actin cytoskeleton and the ECM is provided by protein clusters at the cell membrane building distinct adhesion spots which are denoted as focal adhesion sites (FAs). Many proteins incorporated in FAs are constantly exchanging within a timescale of seconds. Here we show, that the exchange of the FA-protein vinculin decreases with proceeding age of FAs and is regulated by phosphorylation at a single tyrosine residue of vinculin. This stabilization of vinculin within the FA goes along with an enhanced force transduction ability of the cell. Based on these findings we consider the phosphorylation-regulated incorporation of vinculin as a mechanism to modulate adhesion strength. We therefore propose a new role of vinculin as a tuneable factor for the mechanical stability of FAs.

Migrating cells are excellent model systems to study the controlled assembly and disassembly of FAs, since their movement is based on a constant adhesion turnover with formation of new adhesions at the front and the release of old adhesions at the cell’s rear. During their lifetime, FAs undergo a regulated development from a nascent to a mature state by changing size, shape and protein composition. Nascent FAs are formed at the front of the migrating cell. During their maturation they increase in size remaining relatively stable in respect to the substrate. While the cell-body moves forward they come closer to the trailing edge of the cell where they finally dissolve. Thus, the coordinated formation and release of FAs determines the direction of migration and is thought to play a key role in directed cell movement like chemotaxis [1].

We investigated the phosphorylation and exchange dynamics of the FA protein vinculin in epithelial cells from human skin (keratinocytes) migrating on a flat surface. As an adaptor protein, vinculin connects other FA proteins with actin filaments and is thought to stabilize the adhesion complex [2]. Quantitative immunostaining studies revealed a high vinculin phosphorylation in the nascent FAs at the cell front. With increasing

maturation state, the phosphorylation of FAs was significantly lowered (see FIG. 1).

FIG. 1: Phosphorylation of vinculin in focal adhesions (FAs) of migrating skin cells. A: Pseudocolor image of the ratio between phosphorylated vinculin and total vinculin within FAs of a migrating cell. The cell shape is marked by a white line and the big arrow points to the direction of migration. Nascent FAs at the cell front (arrowheads) are higher phosphorylated than mature FAs located behind. B: Amount of vinculin phosphorylation in FAs in respect to their distance from the leading edge. Asterisks indicate significant differences of vinculin phosphorylation compared to FAs of stationary cells. Error bars indicate standard deviation. Data were collected from 38 cells.

Since phosphorylation events are known as a mechanism to regulate the binding ability of certain proteins, we explored the exchange kinetics of vinculin in respect to the FA maturation state. Therefore, migrating keratinocytes were transfected with a gene for fluorescently labelled vinculin making it possible to visualize this protein in vivo by light microscopy. To measure the vinculin exchange in single FAs, we photobleached vinculin at these sites and measured the fluorescence recovery over time. If there was no protein exchange, the fluorescence wouldn’t recover at all. In contrast, if all bleached proteins were replaced, the fluorescence would recover completely. Hence, with this method called Fluorescence Recovery after Photobleaching (FRAP) the amount of the stably incorporated against the constantly exchanging vinculin could be determined in single FAs (see FIG. 2 A).

FIG.2: Vinculin exchange dynamics examined by Fluorescence Recovery after Photobleaching (FRAP). A: FRAP experiment at a mature FA of a cell transfected with fluorescently labelled vinculin. FAs appear as bright spots. The bleach-field is marked by a red circle. Bleaching occurs at 0s. Since vinculin is constantly exchanging within the FA, the fluorescence recovers over time. Scale bar: 5 µm. B: Mean fluorescence recovery of vinculin over time from several FRAP experiments: nascent FAs at the cell front (red, n=14), mature FAs at the rear (green, n=11), FAs from sessile cells (blue, n=14), nascent FAs with phosphorylation-inhibitedvinculin (grey, n=13). C: Saturation values of the mean recovery curves shown in B. This value indicates the exchanging fraction of vinculin. Asterisks designate a statistically significant difference from frontal (red) or rear (green) adhesions.

Just like the amount of phosphorylation, the fraction of exchanging vinculin decreased with the FA maturation level indicating that vinculin exchange is regulated by phosphorylation. To verify this theory, cells were transfected with a slightly altered vinculin gene, where phosphorylation at a specific tyrosine residue (TYR1065) was inhibited. The exchange of the inhibited vinculin mutant was in nascent FAs significantly lowered compared to the wild type vinculin confirming the direct interplay between the phosphorylation of vinculin and its binding kinetics (see FIG. 2 B and C).

As Vinculin is essential for mechano-coupling, the regulation of vinculin binding could be a mechanism to adapt the adhesion strength of individual FAs. Therefore, the correlation between FA maturation state and force transduction was examined by traction force microscopy of cells migrating on elastic silicone substrates [3].

FIG.3: Force application during cell locomotion: Fluorescent image of vinculin in a migrating cell with FAs appearing as bright spots. Red arrows mark the force vector for each FA. The white arrow points to the direction of migration. Eigenvectors of the generalizedfirst moment tensor are indicated by green arrows Mature FAs at the rear produce strong inwards directed forces. Nascent FAs at the front produce weak rearwards directed forces. Scale bar: 10 µm.

In these experiments, tractions of the migrating cell produced local substrate deformations which could be quantified by tracking the displacement of fluorescent beads embedded in the substrate. Since the elastic properties of the silicone substrate and the location of FAs as sites of force transduction were known, traction forces could be calculated from these deformations. These experiments revealed that major traction forces were transmitted at the cell’s rear where typically mature FAs are located (see FIG. 3).

Taken together, these experiments confirm the direct interplay between the amount of stablely incorporated vinculin into the FA and its ability to transduce force, whereas the vinculin incorporation could be modified by phosphorylation. Thus, we suggest vinculin to be an important stabilization factor for FAs which is directly regulated by phosphorylation through intracellular signalling pathways.

[1] Webb DJ, Parsons JT, Horwitz AF, Nat Cell Biol 4(4):E97-100 (2002)

[2] Ziegler WH, Liddington RC, Critchley DR, Trends Cell Biol 16(9):453-60 (2006)

[3] Cesa CM, Kirchgessner N, Mayer D, Schwarz US, Hoffmann B, Merkel R, Rev Sci Instrum 78(3):034301 (2007)

Education & Dissemination International Helmholtz Research School International Soft Matter Conference 2007 IFF Spring School 2008 Jülich Soft Matter Days

International Helmholtz ResearchSchool of Biophysics and Soft Matter

The International Helmholtz Research School ofBiophysics and Soft Matter (IHRS BioSoft) pro-vides intensive training in biophysics and softmatter. It also offers a comprehensive frameworkof experimental and theoretical techniques thatwill enable PhD students to gain a deeper under-standing of the structure, dynamics, and functionof complex systems.

In recent years, life science research has undergonea fundamental transition. It has become evident thateven the simplest molecular machines display an as-tounding complexity, leaving alone networks of genesand proteins in a living cell. Thus, there is an ur-gent need for a more quantitative, theory-oriented ap-proach. Soft matter research has, in parallel, madegreat progress in understanding the structure of com-plex multi-component macromolecular systems, theirnon-equilibrium behaviour and their response to ex-ternal fields. A particular focus is laid upon unravelingthe physics of biologically relevant systems. Thus,there is an urgent need for an interdisciplinary grad-uate education.

The IHRS BioSoft is located at Forschungszen-trum Jülich, run in cooperation with the universitiesin Cologne and Düsseldorf and caesar Bonn, andfunded by the Helmholtz Association. Its ultimategoal is to advance the integration and exchange be-tween physics, chemistry, and biology in researchand education. Students benefit not only from lec-tures, seminars, and lab courses given by experts inthe field, but also from courses in transferable skills.Furthermore, they experience the environment pro-vided by a large, multidisciplinary research centre.

In addition to groups within the PoF-BioSoft program,also the group ’Physics of Soft Matter’ headed byProf. S. Egelhaaf in Düsseldorf, the group ’Molec-

ular Sensory Systems’ headed by Prof. U. B. Kauppin Bonn, the ICG-3 (Phytosphere) headed by Prof.U. Schurr in Jülich, the group on ’Molecular PhysicalChemistry’ headed by Prof. C. Seidel in Düsseldorf,and the physical chemistry group headed by Prof.Strey in Cologne participate in the IHRS BioSoft.

The research school accepts fellows for three-yearPhD projects and is open to highly qualified and mo-tivated applicants from all countries. The fellow PhDstudents will be based in one of the groups that arepart of the IHRS, but also participate in interdisci-plinary courses. Other students are welcome to joinmost of these courses as long as there are freeplaces. The lectures of the school usually attract anumber of extra participants that choose the topicsselectively according to their needs.

In 2007, two-semester introductory lecture courses,’Introduction to Statistical Physics’ taught by Prof.Dhont and Prof. Gompper and ’Molecules of Life -Introduction to the Chemistry and Biology of Cells’with various lecturers from within the IHRS BioSoft(Prof. Schurr, Prof. Merkel, Prof. Kaupp, Prof. Sei-del, Prof. Richter, Prof. Büldt, Prof. Willbold, Dr.Enderlein, and Prof. Offenhäusser) were offered tothe students. Both courses equip the students fortheir research projects with important basic knowl-edge: from a physics point of view, entropy and statis-tical physics have a large influence on the behaviourof the systems that are usually mesoscopic. The bi-ological lectures covered systems from amino acidsto the structure and dynamics of entire cells as wellas methods such as X-ray crystallography, fluores-cence spectroscopy, electrophysiology, and opticalmicroscopy.

In 2007 and 2008, the students learned about theimportant tool of ’Computer Simulations in Physicsand Biology’ by a two-semester advanced-seminarcourse that covered various, independent talks ondifferent topics: Monte Carlo and Molecular Dynam-ics Simulations, Polyelectrolytes, Solid State NMR,Evolution of Bacterial Genomic Networks, Meso-scopic Hydrodynamics, Colloids, Proteins, Protein-Ligand Binding, Protein Structure Prediction, andMembrane Proteins. Most of the speakers were fromForschungszentrum Jülich and daily work with themethods and systems they presented: G. A. Vliegen-thart, R. G. Winkler, H. Heise, M. Stoldt, M. Lercher,M. Ripoll, G. Naegele, A. Baumgärtner, M. Zacharias

(IU Bremen), J. Granzin, and W. B. Fischer (NYMU,Taiwan).Very recently, students were offered the one-semester introductory lecture course on ’Cell Biology’by Prof. Müller and Prof. Baumann that included labdemonstrations, and the advanced lecture courseson ’Complex Fluids’ by Prof. Strey (Cologne) and’Rheology’ by Prof. Vermant (Leuven).

Complementary laboratory courses provide the stu-dents with practical experience and strengthen theinterdisciplinary approach. Every year, the two-week’Neutron Scattering’ course (organized by T. Brückel,G. Heger, D. Richter, and R. Zorn) is open for theparticipation of IHRS students. The course pro-vides an extensive training by theoretical lectures andpractical exercises. In a course on ’Optical Spec-troscopy’, G. Schuetz (Linz), J. Enderlein (Tübin-gen), J. Humplickova (Prague), M. Sauer (Bielefeld),J. Hofkens (Leuven), T. Gensch, and J. Heberle(Bielefeld) taught several optical techniques, suchas fluorescence techniques, single-molecule spec-troscopy, reaction-induced infrared difference spec-troscopy, and Raman spectroscopy of biomolecules.The laboratory course ’Recording of Cell Activity’ —for example on Ca2+ imaging in living cells — wasoffered jointly by several institutes within the IHRS.In the last semester, a two-week course ’Fluores-cence Spectroscopy’ by Prof. Seidel in Düsseldorfwas open for IHRS fellows, a one-week course ’CryoTransmission Electron Microscopy’ was organizedexclusively for IHRS students by L. Belkoura and M.Baciu in Cologne and a one-day course on ’NMRSpectroscopy’ was offered by B. König in Jülich.The PhD students regularly present their research inthe Student’s Seminar that is chaired by two IHRSBioSoft faculty members; every talk is followed by along discussion. As the research in the participatinggroups, also the research topics of the PhD projectscover a wide range within biophysics and soft mat-ter. Therefore a talk in the Student’s Seminar is verychallenging, because it needs to be prepared suchthat physicists, chemists, and biologists can bene-fit. Apart from questions and feedback about the re-search, the speakers usually receive also commentsregarding the style of the presentation and whetherit was suitable for the different parts of the audience.Topics of talks that were given include:

• Holographically induced nucleation (R. Hanes,Physics of Soft Matter, Düsseldorf)

• Regulation of HCN channels by phosphory-lation (F. Winkhaus, Molecular Sensory Sys-tems, Bonn)

• NMR as a tool to study protein structures (M.Schwarten, Structural Biochemistry)

• Non-genomic action of progesterone in humansperm (N. Goodwin, Molecular Sensory Sys-tems, Bonn)

• Nanostructured gold electrodes for the func-tional coupling with neuronal cells (D. Brügge-mann, Bioelectronics)

• Microinterferometry: a tool to study membranefluctuations (C. Monzel, Biomechanics)

• Swarm behaviour of self-propelled particles (Y.Yang, Theory of Soft Matter and Biophysics)

• Photo-control of cell networks for extracellularrecording systems (V. Maybeck, Bioelectron-ics)

• Self-assembly in a binary H2O-C12E4 system(I. Savic, Physical Chemistry, Cologne)

• Squeezing actin: a TIRF microscopy study (A.Tsigkri, Soft Condensed Matter)

• Microemulsions as delivery systems (SabineSchetzberg, Physical Chemistry, Cologne)

• Polyelectrolyte electrophoresis (S. Frank, The-ory of Soft Matter and Biophysics)

• Molecular Dynamics simulations of polyethy-lene oxide (PEO) and PEO/PMMA blends (M.Brodeck, Neutron Scattering)

• Combined single-molecule force and fluores-cence spectroscopy (S. Grabowski, MolecularPhysical Chemistry, Düsseldorf)

• How is shoot growth affected by low root tem-perature? (R. Poire, Phytosphere)

• HCN channels in the main olfactory bulb (A.Aho, Molecular Sensory Systems, Bonn)

• Morphologic and physiologic aspects of synap-tic transmission in rat barrel cortex (G. Haack,Cellular Neurobiology)

Fellow PhD students already participated in two ofthe three seminars in transferable skills by ImperialCollege London that are organized by the HelmholtzAssociation. The seminars shall cover various as-pects ranging from group work in the beginning ofthe thesis, presentation techniques up to writing ofapplications towards the end of the PhD project.Currently, first students who have started their PhDprojects within the IHRS BioSoft finish their thesis.

International Soft Matter Conference,Aachen 2007G. Gompper 1, J.K.G. Dhont 2, D. Richter 3

1 IFF-2: Theoretical Soft-Matter and Biophysics2 IFF-7: Soft Condensed Matter3 IFF-5: Neutron Scattering

The International Soft Matter Conference tookplace in Aachen on October 1-4, 2007. It broughttogether about 600 scientists from 35 countriesto discuss all aspects of soft matter science.

With the large number of conferences organized ev-ery year on topics like polymer chemistry and poly-mer physics, colloid chemistry and colloid physics,surfactants in solutions, etc., the aim of an Interna-tional Soft Matter Conference clearly had to be tobring together a balanced mixture of scientists work-ing on all kinds of soft materials — such as polymers,colloids, surfactants, membranes, biomaterials andtheir composites [2, 1]. The need for a unified viewof Soft Matter systems is threefold. First, it has beenrecognized over the last decades that colloids, poly-mers and surfactants are by far not as distinct mate-rials as previously assumed. Indeed, there is essen-tially a continuum of molecules and systems, whichfills the triangle of materials illustrated in Fig. 1. Thetwo main axes of this triangle are, roughly speaking,amphiphilicity as abscissa and elongation or flexibil-ity as ordinate. Let us illustrate this by following theleft-hand side of the triangle from colloids to flexiblepolymers. Traditionally, colloids are hard, sphericalparticles. However, there are also rod-like colloids.As the aspect ratio, the ratio of rod length and rod di-ameter, becomes larger, rods typically become moreflexible. An example is the fd-virus shown in Fig. 1.For even larger aspect ratios, the length exceeds thepersistence length; this is the regime of semi-flexiblepolymers, for which DNA is an example of enormousimportance. Finally, in the limit of very small persis-tence lengths, we arrive at the classical, flexible syn-thetic polymers. Second, mixtures of several com-ponents of colloidal, polymeric or amphiphilic char-acter are becoming increasingly important, becausethey open up the possibility to tune and control mate-rial properties. Well-known examples are the deple-tion interaction between colloidal particles induced bypolymers in solution, the intriguing mesophases inmixtures of spherical and rod-like colloids, the tun-ing of membrane properties by anchored polymersand amphiphilic block copolymers, or the modifica-tion of the properties of polymers melts by addi-tion of colloidal particles to form nano-composites.Third, biological and biomimetic systems share manymacromolecules and properties with Soft Matter sys-

FIG. 1: The components of modern Soft Matter systemscan be arranged in a triangle, which shown that there is acontinuum of molecules and materials which fills the spacebetween spherical colloids, flexible polymers, and surfac-tants.

tems. Indeed, the application of physical conceptsand ideas to biological systems has become one ofthe most intense activities in soft condensed matterin recent years.

With more than 600 participants from about 35 coun-tries (see Fig. 2), 8 plenary talks, 42 invited talks, 85contributed talks, and about 400 posters, the amountof information provided during the conference wasmuch too large to be summarized in a few lines here.Instead, we hope that the list of the plenary speakersand the titles of their talks will give a feeling about theexciting atmosphere and the intensity of the discus-sions during the conference:

• M. Cates (University of Edinburgh, UK) LatticeBoltzmann simulations of nonequilibrium com-plex fluids.

• W. Gelbart (University of California, Los Ange-les, USA) Physical aspects of viral infectivity.

• L. Leibler (ESPCI, Paris, France) Supramolec-ular plastics and rubbers.

• G. Maret (Universität Konstanz, Germany)Elasticity, phonons and melting of colloidalcrystals.

FIG. 2: Number of participants from countries with thelargest participation.

• D. Nelson (Harvard University, USA) Neutralmutations and gene surfing in microorganisms.

• D. Roux (Saint-Gobain, Courbevoie, France)Glass technologies: colloids and active sur-faces.

• H. Tanaka (University of Tokyo, Japan) Me-chanical instability in phase separation, frac-ture, and cavitation.

• D. Weitz (Harvard University, USA) Dripping,jetting, drops and wetting: Structuring new softmaterials with microfluidics.

The conference announcement is shown in Fig. 3.All invited speakers were invited to submit an originalpaper related to the subject of their presentation fora special issue of the European Physical Journal E[3]. All contributions were fully reviewed. This issueprovides a good overview about the current statusand topics of Soft Matter research.

Due to the large interest and overwhelming participa-tion in the conference, the program committee cameto the conclusion that it would be a good idea to orga-nize international soft matter conference every threeyears in the future. Participants were therefore askedfor proposals. These proposals were presented in aplenary session. A public vote showed the largestsupport for the proposal from Granada (Spain). It willtherefore the location of the International Soft Mat-ter Conference 2010. We are looking forward to thismeeting of the Soft Matter community.

[1] European Network of Excellence “SoftComp",Newsletter No.5, December 2007.

[2] G. Gompper, J.K.G. Dhont, and D. Richter, Eur. Phys.J. E 26, 1 (2008).

[3] Special Issue Eur. Phys. J. E 26 (2008).

FIG. 3: Announcement of the International Soft MatterConference 2007.

39th IFF Spring School: Soft Matter – From Synthetic to Biological Materials

The 39th international IFF Spring School took place from 3 March until 14 March at the Forschungszentrum Jülich. Leading scientists from research and industry gave 220 students and young scientists from 25 countries and five continents a comprehensive overview of the interdisciplinary research field "Soft Matter" at the interface between physics, chemistry, biology and the life sciences.

Soft matter is ubiquitous in a vast range of technological applications and is of fundamental relevance in such diverse fields as chemical, environmental, and food industry as well as life sciences. Over the past years, soft matter science has been largely extended in its scope from more traditional areas such as colloids and polymers to the study of biological systems, soft nanoscale materials, and the development of novel composites and microfluidic devices.

Soft and biological materials share fundamental structural and dynamical features including a rich variety of morphologies and non-equilibrium phenomena, self-organisation, an unusual friction-dominated flow dynamics, and a high sensitivity to external fields. These properties emerge from the cooperative interplay of many degrees of freedom, with spatio-temporal correlations that can span a huge range from nano- to millimetres and nanoseconds to days. The key requirements for the advancement in the field of these highly complex soft materials are:

• The development of novel experimental techniques to study properties of individual components in processes and the cooperative behavior of many interacting constituents. The synthesis of complex materials, self-organized and biomimetic systems with novel or unusual properties will broaden the spectrum of applications.

• The exploration of advanced theoretical and computer simulation methods that span the large range of time and length scales and allow to cope with an increasing complexity of molecular constituents. Existing methods need to be extended and new approaches are required to describe systems far from equilibrium, e.g., in life sciences and material processing.

• Structural and novel functional properties of soft and biological materials need to be studied invoking self-organization and hierarchical structure formation, entropic particle interactions and fluid-like aspects of biological materials such as vesicles and cells.

• The unusual dynamics of complex fluids requires special approaches to gain insight into diffusion transport properties, rheology and mesoscopic flow behavior, which are influenced by a delicate interplay of hydrodynamic interactions, thermal fluctuations, and external fields.

The IFF Spring School 2008 at the Forschungszentrum Jülich, Germany, addressed advanced experimental techniques and

applications, and theoretical and computer simulation methods on an undergraduate and graduate student level. Introductory lectures provided the basis of important experimental and theoretical tools. More advanced lectures explained practical aspects of various methods and lead the participants from basic methods to the frontiers of current research.

The lectures covered the following topics:

• Scattering Techniques

• Single Molecule Techniques

• Equilibrium- and Non-equilibrium Statistical Physics

• Microfluidics

• Computer Simulations

• Synthesis

• Self-Organisation

• Flow Properties and Rheology

• Biomechanics

• Macromolecules and Colloids

• Membranes and Interfaces

• Biomimetic Systems

• Glasses and Gels

The school offered about 50 hours of lectures plus discussions, as well as the opportunity to participate in practical courses and visits to the participating institutes at the Forschungszentrum Jülich.

The local media coverage included newspapers, radio and television.

Jülich Soft Matter Days 2008J.K.G. Dhont 1, G. Gompper 2, D. Richter 3

1 IFF-7: Soft Condensed Matter2 IFF-2: Theoretical Soft-Matter and Biophysics3 IFF-5: Neutron Scattering

The Jülich Soft Matter Days is a yearly confer-ence, held at the Gustav-Stresemann-Institut inBonn, Germany. The number of participants islimited to about 220, which ensures an infor-mal atmosphere and avoids parallel sessions.The conference attracts scientists from all con-tinents and has a pronounced international char-acter. An important part of the conference aretwo posters sessions with more than 120 contri-butions.

The aim of these meetings is to bring together sci-entists from all soft matter disciplines and from bio-physics. The systems of interest include colloidaldispersions, polymer-solutions, -mixtures and -melts,block copolymers, binary and ternary amphiphilicsystems (microemulsions), membranes, vesicles andbiological macromolecules. While many of these sys-tems have already been investigated for a long pe-riod of time, only recently their common features andmixtures have come into focus. In addition, in recentyears, systems with various types of complex parti-cles have been studied, where the particles exhibitproperties that are both of colloidal, polymeric andpossibly amphiphilic character. For example, Januscolloids share features of colloids and amphiphiles,and fd-viruses are in between rod-like colloids andstiff polymers. These highly complex systems arecharacterized by structural units with typical lengthscales ranging from nanometers to micrometers. Theexperimental and theoretical investigations as well asthe understanding of the properties of these mate-rials poses enormous challenges due to their highcomplexity, the large number of cooperative degreesof freedom, and the large range of relevant length,time and energy scales. We hope that this confer-ence provided a forum to share and discuss the latestadvances for all researchers in this field.

The topics that have been discussed 2008 in the col-loid session range from Archimedian tilings on quasi-crystalline surfaces (C. Bechinger, Stuttgart), multipleglassy states in star-polymer mixtures (Ch.N. Likos,Düsseldorf), synthesis of nanoparticle-microgel com-posites (L.M. Liz-Marzan, Vigo), to buckling of mi-crocapsules (A. Imhof, Utrecht), colloids at interfaces(W.K. Kegel, Utrecht) and dynamics of charged col-loids (G. Nägele, Jülich).

FIG. 1: Announcement of the Jülich Soft Matter Days2008.

In the polymer session, highlights include talks aboutcapillary wrinkling of floating polymer films (T.P. Rus-sell, Amherst), amplification of tension in branchedpolymers (M. Rubinstein, Chapel Hill), the physicaland biological significance of transition in DNA (K.Yoshikawa, Kyoto), and the rheology of branched en-tangled polymer melts (D.J. Read, Leeds).

In the session on physics of the cell, the main top-ics were the role of DNA conformations in gene con-trol (R. Metzler, München), cell shapes and forces onpatterned substrates (U. Schwarz, Karlsruhe), fiberorganization in living cells (F. Nédélec, Heidelberg),

FIG. 2: Discussions during one of the two the postersession.

and bacterial swarming (J. Vermant, Leuven).

The main interests in the session on proteins were onneutron scattering to obtain information on structure(D.I. Svergun, Hamburg) and inter-domain dynam-ics (R. Biehl, Jülich), NMR experiments and simula-tions on self-similar dynamics (G. Kneller, Orléans),and protein aggregation in chinese century eggs (E.Eiser, Cambridge).

Some of the topics of interest in the session onself-assembly were the kinetics of block-copolymermicelles (R. Lund, San Sebastian) and lipid vesi-cles (M. Nakano, Kyoto), rheology of microphase-separated diblock copolymers (T. Ohta, Kyoto), mi-croscopic swimmers at surfaces (J. Elgeti, Jülich),and cytoskeleton dynamics of liposomes (C. Sykes,Paris).

Finally, the session on hydrodynamics included talkson ordering of colloids by flow and sedimentation(M.J. Solomon, Ann Arbor), micro-fluidic crystals (R.Bar-Ziv, Rehovot), and heat and mass transport atinterfaces (J.-L. Barrat, Lyon).

FIG. 3: The participants meet in the central court of the Gustav-Stresemann-Institut in Bonn.

An important part of conference are the poster ses-sions. The posters were grouped according to thesame topics as the oral contributions. The more than120 poster contributions were split into two postersessions, which gave all participants the opportunityfor lively discussions at the posters, an impression ofwhich is given in Fig.2.

For more information about the 2008 conference andthe upcoming 2009 meeting, please visit the confer-ence webpage http://www.fz-juelich.de/iff/jsmd2008.

We hope that the Jülich Soft Matter Days will continueto be successful as an inter-disciplinary meeting onsoft matter and biophysics.

Publications 2007 2008

Institute of Solid State Research Theoretical Soft Matter and Biophysics (IFF-2) 2007 Belitsky, V.; Maric, N.; Schütz, G. M. Phase transition in a cellular automaton model of a highway on-ramp Journal of Physics A - Mathematical and General, 40 (2007), 11221 - 11243 Brzank, A.; Schütz, G. M. Phase transition in the two-component symmetric exclusion process with open boundaries Journal of Statistical Mechanics: Theory and Experiment, 8 (2007), P08028 Cherstvy, A. G. Effect of a Low-Dielectric Interior on DNA Electrostatic Response to Twisting and Bending Journal of Physical Chemistry B, 111 (2007), 12933 - 12937 Eisenriegler, E.; Bringer, A. Polymer depletion profiles around nonspherical colloidal particles Journal of Chemical Physics, 127 (2007), 034904 Götze, I.; Noguchi, H.; Gompper, G. Relevance of angular momentum conservation in mesoscale hydrodynamics simulations Physical Review E, 76 (2007), 046705 Grosskinsky, S.; Schütz, G. M.; Willmann, R. D. Rigorous results on spontaneous symmetry breaking in a one-dimensional driven particle system Journal of Statistical Physics, 128 (2007), 587 - 606 Gwan, J. F.; Baumgaertner, A. Cooperative Transport in a Potassium Ion Channel Journal of Chemical Physics, 127 (2007), 045103 Gwan, J. F.; Baumgaertner, A. Ion Transport in a Nanochannel Journal of Computational and Theoretical Nanoscience : for all Theoretical and Computational Aspects in Science, Engineering, and Biology, 4 (2007), 50 - 56 Harris, R. J.; Schütz, G. M. Fluctuation theorems for stochastic dynamics Journal of Statistical Mechanics : Theory and Experiment, (2007), P07020 Harris, R. J.; Stinchcombe, R. B.* Scaling approach to related disordered stochastic and free-fermion models Physical Review E, 75 (2007), 031104 Kohyama, T.*; Gompper, G. Defect Scars on Flexible Surfaces with Crystalline Order Physical Review Letters, 98 (2007), 198101-1 - 198101-4 Noguchi, H.; Gompper, G. Swinging and Tumbling of Fluid Vesicles in Shear Flow Physical Review Letters, 98 (2007), 128103 Noguchi, H.; Gompper, G. Transport coefficients of dissipative particle dynamics with finite time step Europhysics Letters, 79 (2007), 36002 Noguchi, H.; Kikuchi, N.*; Gompper, G. Particle-based mesoscale hydrodynamic techniques Europhysics Letters, 78 (2007), 10005

Ripoll, M.; Winkler, R. G.; Gompper, G. Hydrodynamic screening of star polymers in shear flow European Physical Journal E, 23 (2007), 349 - 354 Schütz, G. M.; Harris, R.J.* Hydrodynamics of the Zero-range Process in the Condensation Regime Journal of Statistical Physics, 127 (2007) 2, 419 - 430 Winkler, R. G. Diffusion and segmental dynamics of rodlike molecules by fluorescence correlation spectroscopy Journal of Chemical Physics, 127 (2007), 054904 Winkler, R. G.; Cherstvy, A. G. Adsorption of Weakly Charged Polyelectrolytes onto Oppositely Charged Spherical Colloids Journal of Physical Chemistry B, 111 (2007), 8486 - 8493 Yang, Y.; Burkhardt, T. W.; Gompper, G. Free energy and extension of a semiflexible polymer in cylindrical confining geometries Physical Review E, 76 (2007), 011804

2008 Baumgaertner, A. Concepts in Bionanomachines: Translocators Journal of Computational and Theoretical Nanoscience : for all Theoretical and Computational Aspects in Science, Engineering, and Biology, 5 (2008) 9, 1852 - 1890 Cannavacciuolo, L.; Winkler, R. G.; Gompper, G. Mesoscale simulations of polymer dynamics in microchannel flows Europhysics Letters, 83 (2008), 34007-1 - 34007-6 Chatterjee, S.; Barma, M. Shock probes in a one dimensional Katz-Lebowitz-Spohn Model Physical Review E, 77 (2008), 061124-1 - 061124-8 Cherstvy, A. Protein - DNA interactions: Reaching and Recognizing the Targets Journal of Physical Chemistry B, 112 (2008), 4741 - 4750 Cherstvy, A. G. DNA Cholesteric Phases: The Role of DNA Molecular Chirality and DNA-DNA Electrostatic Interactions Journal of Physical Chemistry B, 112 (2008), 12585 - 12595 Finken, R.*; Lamura, A.*; Seifert, U.*; Gompper, G. Two-dimensional fluctuating vesicles in linear shear flow European Physical Journal E, 25 (2008), 309 - 321 Frank, S.; Winkler, R. G. Polyelectrolyte electrophoresis: Field effects and hydrodynamic interactions Europhysics Letters, 83 (2008), 38004 Gompper, G.; Dhont, J. K. G.; Richter, D. Editorial : A unified view of soft matter systems? European Physical Journal E, 26 (2008), 1 - 2 Großkinsky, S.; Schütz, G. M. Discontinuous Condensation Transition and Nonequivalence of Ensembles in a Zero-Range Process Journal of Statistical Physics, 132 (2008), 77 - 108 Grosskinsky, S.; Chleboun, P.; Schütz, G. M. Instability of condensation in the zero-range process with random interaction Physical Review E, 78 (2008) 3, 030101

Harish, R.; Karevski, D.*; Schütz, G. M. Molecular traffic control for a cracking reaction Journal of Catalysis, 253 (2008), 191 – 199 Huang, C.C. Kinetics and dynamics of wormlike micelles under shear Europhysics Letters, 81 (2008), 58002 McWhirter, J.L. Phase behavior of a simple dipolar fluid under shear flow in an electric field Journal of Chemical Physics, 128 (2008), 034502 Noguchi, H.; Gompper, G. Transport coefficients of off-lattice mesoscale-hydrodynamics simulation techniques Physical Review E, 78 (2008), 016706-1 - 016706-12 Popkov, V.; Salerno, M.; Schütz, G. M. Asymmetric simple exclusion process with periodic boundary driving Physical Review E, 78 (2008) 1, 011122 Priezzhev, V.B.; Schütz, G. M. Exact solution of the Bernoulli matching model of sequence alignment Journal of Statistical Mechanics : Theory and Experiment, (2008), P09007 Ripoll, M.; Holmqvist, P.; Winkler, R. G.; Gompper, G.; Dhont, J. K. G.; Lettinga, M. P. Attractive Colloidal Rods in Shear Flow Physical Review Letters, 101 (2008), 168302-1 - 168302-4 Ripoll, M.; Winkler, R. G.; Mussawisade, K.; Gompper, G. Mesoscale hydrodynamics simulations of attractive rod-like colloids in shear flow Journal of Physics: Condensed Matter, 20 (2008), 404209 Schütz, G. M.; Brandaut, M.; Trimper, S. Exact solution of a stochastic susceptible-infectious-recovered model Physical Review E, 78 (2008), 061132 Tao, Y.-G.; Götze, I.O.; Gompper, G. Multiparticle collision dynamics modeling of viscoelastic fluids Journal of Chemical Physics, 128 (2008), 144902-1 - 144902-12 Verberck, B.; Heresanu, V.; Rouziere, S.; Cambedouzou, J.; Launois, P.; Kovats, E.; Pekker, S.; Vliegenthart, G.; Michel, K.H.; Gompper, G. Fullerene-cubane: X-ray Scattering Experiments and Monte Carlo Simulations Fullerenes Nanotubes and Carbon Nanostructures, 16 (2008), 293 - 300 Vliegenthart, G.; Gompper, G. Mechanical properties of icosahedral virus capsids Journal of Computer-Aided Materials Design, 14 (2007), 111 - 119 Yang, Y.; Elgeti, J.; Gompper, G. Cooperation of Sperm in Two Dimensions: Synchronization, Attraction and Aggregation through Hydrodynamic Interactions Physical Review E, 78 (2008), 061903

Institute of Solid State Research Neutron Scattering (IFF)

2007 Allgaier, J.; Willbold, S.; Chang, T. Synthesis of Hydrophobic Poly(alkylene oxide)s and Amphiphilic Poly(alkylene oxide) Block Copolymers Macromolecules, 40 (2007) 3, 518 - 525 Bóta, A.*; Varga, Z.*; Goerigk, G. Biological systems as nanoreactors: Anomalouls small-angle scattering study of the CdS nanoparticle formation in multilamellar vesicles Journal of Physical Chemistry B, 111 (2007) 8, 1911 - 1915 Bóta, A.*; Varga, Z.*; Goerigk, G. Vesicles as reactors of nanoparticles: an anomalous small-angle X-ray scattering study of the domains rich in copper ions Journal of Applied Crystallography, 40 (2007), s259 - s263 Buchenau, U.; Wischnewski, A.; Ohl, M.; Fabiani, E.* Neutron scattering evidence on the nature of the boson peak Journal of Physics: Condensed Matter, 19 (2007), 205106 Buchenau, U.; Zorn, R.; Ohl, M.; Wischnewski, A. Dielectric and thermal relaxation in the energy landscape Philosophical Magazine, 87 (2007) 3/5, 389 - 400 Chang, J.*; Pailhés, S.*; Shi, M.*; Månsson, M.*; Claesson, T.*; Tjernberg, O.*; Voigt, J.; Perez, V.*; Patthey, L.*; Momono, N.*; Oda, M.*; Ido, M.*; Schnyder, A.*; Mudry, C.*; Mesot, J.* When low- and high-energy electronic responses meet in in cuprate superconductors Physical Review B, 75 (2007), 224508-1 - 224508-6 De Luca, E.*; Waigh, T. A.*; Monkenbusch, M.; Kim, J. S.*; Jeon, H. S.* Neutron spin echo study of the dynamics of micellar solutions of randomly sulphonated polystyrene Polymer, 48 (2007), 3930 - 3934 Faulhaber, E.*; Stockert, O.*; Schmalzl, K.; Jeevan, H.S.*; Deppe, M.*; Geibel, C.*; Steglich, F.*; Loewenhaupt, M.* Spatial separation of antiferromagnetism and superconductivity in CeCu2Si2 Journal of Magnetism and Magnetic Materials, 310 (2007), 295 - 297 Feygenson, M.; Kentzinger, E.; Ziegenhagen, N.; Rücker, U.; Goerigk, G.; Wang, Y.*; Brückel, T. Contrast variation by anomalous X-ray scattering applied to investigation of the interface morphology in a giant magnetoresistance Fe/Cr/Fe trilayer Journal of Applied Crystallography, 40 (2007) 3, 532 - 538 Frank, C.; Frielinghaus, H.; Allgaier, J.; Prast, H. Nonionic Surfactants with Linear and Branched Hydrocarbon Tails: Compositional Analysis, Phase Behavior, and Film Properties in Bicontinuous Microemulsions Langmuir, 23 (2007) 12, 6526 - 6535 Frick, B.*; Koza, M.*; Zorn, R. Editorial European Physical Journal Special Topics: ST, 141 (2007) Frielinghaus, H. Small-angle scattering model for multilamellar vesicles Physical Review E, 76 (2007), 051603-1 051603-8 Goerigk, G.; Huber, K.*; Schweins, R.* Probing the extent of the Sr2+ ion condensation to anionic polyacrylate coils: A quantitative anomalous small-angle x-ray scattering study Journal of Chemical Physics, 127 (2007), 154908-1 - 154908-8

Goll, G.*; Stockert, O.*; Prager, M.; Yoshino, T.*; Takabatake, T.* Low energy excitations in CeBiPt Journal of Magnetism and Magnetic Materials, 310 (2007), 1773 Gutiérrez, J.*; Barandiarán, J. M.*; Bermejo, F. J.*; Mondelli, C.*; Romano, P.*; Fouquet, P.*; Monkenbusch, M. Evidence for two disparate spin dynamic regimes within Fe-substituted La0,7 Pb0,3)Mn1-xFex)O3 (0 kleiner/gleich x kleiner/gleich 0,2) colossal magentoresistive manganites: Neutron spin-echo measurements Physical Review B, 76 (2007) 18, 184401 Holderer, O.; Frielinghaus, H.; Monkenbusch, M.; Allgaier, J.; Richter, D.; Farago, B.* Hydrodynamic effects in bicontinuous microemulsions measured by inelastic neutron scattering European Physical Journal E, 22 (2007), 157 - 161 Iatrou, H.*; Frielinghaus, H.; Hanski, S.*; Ferderigos, N.*; Ruokolainen, J.*; Ikkala, O.*; Richter, D.; Mays, J.*; Hadjichristidis, N.* Architecturally induced multiresponsive vesicles from well-defined polypeptides. Formation of gene vehicles Biomacromolecules, 8 (2007) 7, 2173 - 2181 Ibberson, R. M.*; Prager, M. Temperature-dependent crystal structure analysis of methyl iodide by high-resolution neutron powder spectroscopy Zeitschrift für Kristallographie, 222 (2007), 416 Ioffe, A.; Bodnarchuk, V.; Bussmann, K.; Mueller, R. Larmor labeling by time-gradient magnetic fields Physica B: Condensed Matter, 397 (2007), 108 - 111 Kentzinger, E.; Frielinghaus, H.; Rücker, U.; Ioffe, A.; Richter, D.; Brückel, T. Probing lateral magnetic nanostructures by polarized GISANS Physica B: Condensed Matter, 397 (2007), 43 - 46 Kirstein, O.*; Prager, M.; Grimm, H.; Buchsteiner, A.*; Wischnewski, A. Quasielastic neutron scattering experiments including activation energies and mathematical modeling of methyl halide dynamics Journal of Chemical Physics, 127 (2007), 094504 Klose,F.*; Holtkamp,N.*; Richter,D. Die Megawatt-Spallationsneutronenquelle SNS: Neue Chancen für die Erforschung der kondensierten Materie Physik Journal, 1 (2007),23 Laurati, M.; Stellbrink, J.; Lund, R.; Willner, L.; Richter, D.; Zaccarelli, E.* A system with star-like morphology and interactions Physical Review E, 76 (2007), 041503 Lebedev, D.V.*; Monkenbusch, M.; Shalguev, V.I.*; Lantsov, V.A.*; Isaev-Ivanov, V. V. Dynamic properties of RecA protein filaments from E-coli and P-aeruginosa investigated by neutron spin echo Biofizika, 52 (2007) 5, 799 - 803 Li, H. F.; Su, Y.; Perßon, J.; Meuffels, P.; Walter, J. M.; Skowronek, R.; Brückel, T. Neutron-diffraction study of structural transition and magnetic order in orthorhombic and rhombohedral La7/8Sr1/8Mn1-gammaO3+delta Journal of Physics: Condensed Matter, 19 (2007), 176226-1 -176226-12 Li, H.; Su, Y.; Perßon, J.; Meuffels, P.; Walter, J.*; Skowronek, R.*; Brückel, T. Correlation between structural and magnetic properties of La7/8Sr1/8Mn1-gammaO3 delta with controlled nonstoichiometry Journal of Physics: Condensed Matter, 19 (2007), 016003-1 - 016003-12 Lund, R.; Willner, L.; Richter, D.; Iatrou, H.*; Hadjichristidis, N.*; Lindener, P.* Unraveling the equilibrium chain exchange kinetics of polymeric micelles using small-angle neutron scattering - archtectural and topological effects Journal of Applied Crystallography, 40 (2007), s327 - s331 Maccarrone, S.; Frielinghaus, H.; Allgaier, J.; Richter, D.; Lindner, P.* SANS Study of Polymer-Linked Droplets Langmuir, 23 (2007) 19, 9559 - 9562 Mangiapia, G.; Ricciardi, R.*; Auriemma, F.*; de Rosa, C.*; Lo Celso, F.*; Triolo, R.*; Heenan, R. K.*; Radulescu, A.; Tedeschi, A. M.*; D'Errico, G.*; Paduano, L.* Mesoscopic and Microscopic Investigation on Poly(vinyl alcohol) Hydrogels in the Presence of Sodium Decylsulfate Journal of Physical Chemistry B, 111 (2007) 9, 2166 - 2173

Mattern, N.*; Gemming, T.*; Goerigk, G.; Eckert, J.* Phase separation in amorphous Ni-Nb-Y alloys Scripta Materialia, 57 (2007), 29 - 32 Monkenbusch, M.; Richter, D. High resolution neutron spectroscopy - a tool for the investigation of dynamics of polymers and soft matter Comptes Rendus Physique, 8 (2007), 845 - 864 Niedzwiedz, K.; Wischnewski, A.; Monkenbusch, M.; Richter, D.; Genix, A.-C.*; Arbe, A.*; Colmenero, J.*; Strauch, M.*; Straube, E.* Polymer Chain Dynamics in a Random Environment: Heterogeneous Mobilities Physical Review Letters, 98 (2007), 168301 Oberdisse, J.*; Hine, P.*; Pyckhout-Hintzen, W. Structure of interacting aggregates of silicia nanoparticles in a polymer matrix: Small-angle scattering and reverse Monte-Carlo simulations Soft Matter, 3 (2007) 2, 476 - 485 Prager, M.; Desmedt, A.*; Allgaier, J.; Russina, M.*; Jansen, E.*; Natkaniec, I.*; Pawlukojc, A.*; Press, W. Methyl group rotation and whole molecule dynamics in methyl bromide hydrate Phase Transitions, 80 (2007) 6/7, 473 Prager, M.; Pawlukojc, A.*; Wischnewski, A.; Wuttke, J. Inelastic neutron scattering study of methyl groups rotation in some methylxanthines Journal of Chemical Physics, 127 (2007) 214509 Prager, M.; Sawka-Dobrowolska, W.*; Sobczyk, L.*; Pawlukojc, A.*; Grech, E.*; Wischnewski, A.; Zamponi, M. X-ray diffraction and inelastic neutron scattering study of 2,6-dimethylpyrazine chloranilic acid complex Chemical Physics, 332 (2007), 1 - 9 Prager, M.; Wischnewski, A.; Bator, G.*; Grech, E.*; Pawlukojc, A.*; Sobczyk, L.* INS spectroscopic study of the 1:1 tetramethylpyrazine (TMP) squaric acid (H(2)SQ) complex Chemical Physics, 334 (2007), 148 - 153 Pranzas, P. K.*; Dornheim, M.*; Bösenberg, U.*; Fernandez, J. R. A.*; Goerigk, G .; Roth, S. V.*; Gehrke, R.*; Schreyer, A.* Small-angle scattering investigations of magnesium hydride used as a hydrogen storage material Journal of Applied Crystallography, 40 (2007) Suppl. 1, s383 - s387 Radulescu, A.; Schwahn, D.; Stellbrink, J. Hierarchical structures formed by partially crystalline polymers in solution: from fundamentals to applications - a combined conventional, focusing and ultra-small-angle neutron scattering study Journal of Applied Crystallography, 40 (2007), s97 - s100 Richter, D. Polymer dynamics: from synthetic polymers to proteins Journal of Applied Crystallography, 40 (2007), s28 - s33 Rother, G.*; Melnichenko, Y. B.*; Cole, D. R.*; Frielinghaus, H.; Wignall, G. D.* Microstructural characterization of adsorption and depletion regimes of supercritical fluids in nanopores Journal of Physical Chemistry B, 111 (2007) 43, 15736 - 15742 Rubio-Retama, J.*; Tamimi, F. M.*; Heinrich, M.; Lopez-Cabarcos, E.* Synthesis and Characterization of Poly(magnesium acrylate) Microgels Langmuir, 23 (2007) 16, 8538 - 8543 Rzhevsky, A. A.*; Krichevtsov, B. B.*; Su, Y.; Schneider, C. M. Probing the (001) surface of magnetite crystals by second harmonic generation Journal of Physics: Condensed Matter, 19 (2007), 396006-1 - 396006-10 Schmalzl, K. Volume and pressure dependence of ground-state and lattice dynamical properties of BaF2 from density-functional methods Physical Review B, 75 (2007), 014306-1 - 014306-11 Schmalzl, K.; Strauch, D.* Static Pockels constants pij and pijk of CaF2 and BaF2 under strain from ab initio calculations Physical Review B, 76 (2007), 205106-1 - 205106-8

Schönhals, A.; Schick, C.; Frick, B.*; Mayorova, M.; Zorn, R. Molecular Dynamics in Glass-forming Poly(phenyl methyl siloxane) as investigated by Broadband Thermal, Dielectric and Neutron Spectroscopy Journal of Non-Crystalline Solids, 353 (2007), 3853 - 3861 Schönhals, A.*; Schick, Ch.*; Huith, H.*; Frick, B.*; Mayorova, M.; Zorn, R. Molecular Mobility of Poly (phenyl methyl siloxane) Investigated by Thermal, Dielectric and Neutron Spectroscopy Polymeric Materials Science and Engineering, 97 (2007), 948 - 949 Schwahn, D.; Ma, Y.*; Cölfen, H.* Mesocrystal to Single Crystal Transformation of D,L-Alanine Evidenced by Small Angle Neutron Scattering Journal of Physical Chemistry C, 111 (2007) 8, 3224 - 3227 Schweika, W.; Hermann, R.; Prager, M.; Perßon, J.; Keppens, V.* Dumbbell rattling in thermoelectric zinc antimony Physical Review Letters, 99 (2007), 125501-1 - 125501-4 Shi, Q.*; Voss, J.*; Jacobsen, H.S.*; Lefmann, K.*; Zamponi, M.; Vegge, T.* Point defect dynamics in sodium aluminium hydrides- A combined quasielastic neutron scattering and density functional theory study Journal of Alloys and Compounds, 469 (2007), 446 - 447 Sinibaldi, R.*; Ortore, M. G.*; Spinozzi, F.*; Carsughi, F.*; Frielinghaus, H.; Cinelli, S.*; Onori, G.*; Mariani, P.* Preferential hydration of lysozyme in water/glycerol mixtures: A small-angle neutron scattering study Journal of Chemical Physics, 126 (2007) 23, 235101-1 - 235101-9 Spinozzi, F.*; Mariani, P.*; Saturni, L.*; Carsughi, F.; Bernstorff, S.*; Cinelli, S.*; Onori, G.* Met-myoglobin Association in Dilute Solution during Pressure-Induced Denaturation: an Analysis at pH 4.5 by High-Pressure Small-Angle X-ray Scattering Journal of Physical Chemistry B, 111 (2007), 3822 - 3830 10.1021/jp063427m Steffens, P.*; Sidis, Y.*; Link, P.*; Schmalzl, K.; Nakatsuji, S.*; Maeno, Y.*; Braden, M.* Field-Induced Paramagnons at the Metamagnetic Transition of Ca1.8Sr0.2RuO4 Physical Review Letters, 99 (2007), 217402-1 217402-4 Stellbrink, J.; Niu, A.; Allgaier, J.; Richter, D.; Koenig, B. W.*; Hartmann, R.*; Coates, G. W.*; Fetters, L. J.* Analysis of Polymeric Methylaluminoxane (MAO) via Small Angle Neutron Scattering Macromolecules, 40 (2007), 4972 - 4981 Szekely, N. K.*; Almasy, L.*; Radulescu, A.; Rosta, L.* Small-angle neutron scattering study of aqueous solutions of pentanediol and hexanediol Journal of Applied Crystallography, 40 (2007) Suppl. 1, s307 - s311 Tehei, M.*; Franzetti, B.*; Wood, K.*; Gabel, F.*; Fabiani, E.*; Jasnin, M.*; Zamponi, M.; Oesterhelt, D.*; Zaccai, G.*; Ginzburg, M.*; Ginzburg, B. Z.* Neutron scattering reveals extremely slow cell water in a Dead Sea organism Proceedings of the National Academy of Sciences of the United States of America, 104 (2007), 766 - 771 Vaccaro, M.*; Accardo, A.*; D'Errico, G.*; Schillén, K.*; Radulescu, A.; Tesauro, D.*; Morelli, G.*; Paduano, L.* Peptides and Gd Complexes Containing Colloidal Assemblies as Tumor-Specific Contrast Agents in MRI: Physicochemical Characterization Biophysical Journal, 93 (2007), 1736 - 1746 Vainio, U.*; Pirkkalainen, K.*; Kisko, K.*; Goerigk, G.; Kotelnikova, N. E.*; Serimaa, R.* Copper and copper oxide nanoparticles in a cellulose support studied using anomalous small-angle x-ray scattering European Physical Journal D, 42 (2007), 93 - 101 Varga, Z.*; Bóta, A.*; Goerigk, G. Localization of dihalogenated phenols in vesicle systems determined by contrast variation X-ray scattering Journal of Applied Crystallography, 40 (2007) , s205 - s208 Voigt, J.; Persson, J.; Kim, J. W.*; Bihlmayer, G.; Brückel, Th. Strong coupling between the spin polarization of Mn and Tb in multiferroic TbMnO3 determined by x-ray resonance exchange scattering Physical Review B, 76 (2007), 104431-1 - 104431-5

Voss, J.*; Jacobson, H.S.*; Zamponi, M.; Lefmann, K.*; Vegge, T.*; Shi, Q.* Hydrogen dynamics in Na3AIH6: A combined density functional theory and quasielastic neutron scattering study Journal of Physical Chemistry B, 111 (2007), 3886 Walter, W.*; Borlein, M.*; Eyßelein, F.*; Gehring, M.*; Kozielewski, T.; Kramer, A.*; Monkenbusch, M.; Ohl, M.; Paul, A.; Schrauth, B.*; Tiemann, Ch. Design of a pair of superconducting solenoids for a neutron spin-echo spectrometer at the SNS IEEE Transactions on Applied Superconductivity, 17 (2007) 2, 1209 - 1212 Zhang, F.*; Skoda, M. W. A.*; Jacobs, R. M. J.*; Zorn, S.*; Martin, R. A.*; Martin, C. M.*; Clark, G. F.*; Goerigk, G.; Schreiber, F.* Gold Nanoparticles Decorated with Oligo(ethylene glycol) Thiols: Protein Resistance and Colloidal Stability Journal of Physical Chemistry B, 111 (2007) 49, 12229 - 12237 Zorn, R. Multiple scattering correction of neutron scattering elastic scans Nuclear Instruments and Methods in Physics Research Section A, 572 (2007), 874 – 881

2008 Arbe, A.*; Genix, A.-C.*; Colmenero, J.*; Richter, D. Anomalous Relaxation od Self Assembled Alkyl-Nanodomains in higher order Poly(n-Alkyl Metacrylates) Soft Matter, 4 (2008) 9, 1792 - 1795 Auriemma, F.*; De Rosa, C.*; Ricciardi, R.*; Lo Celso, F.*; Triolo, R.*; Pipich, V. Time-Resolving Analysis of Cryotropic Gelation of Water/Poly(vinyl alcohol) Solutions via Small-Angle Neutron Scattering Journal of Physical Chemistry B, 112 (2008) 3, 816 - 823 Biehl, R.; Hoffmann, B.; Monkenbusch, M.; Falus, P.*; Préost, S.*; Merkel, R.; Richter, D. Direct Observation of Correlated Interdomain Motion in Alcohol Dehydrogenase Physical Review Letters, 101 (2008), 138102-1 - 138102-4 Birczynski, A.*; Ylinen, E.E.*; Punkkinen, M.*; Prager, M.; Szymocha, A.M.*; Lalowicz, Z.T.* Deuteron NMR relaxation, spectra, and evidence for the order-disorder phase transition in (ND4)2PtCl6 Journal of Chemical Physics, 128 (2008), 184510 Bodnarchuk,V. I.*; Kraan,W. H.*; Rekveldt,M. T.*; Ioffe,A. Neutron spin turners with a rotating magnetic field: first experiments Measurement Science and Technology, 19 (2008), 034012 Bosak, A.*; Schmalzl, K.; Krisch, M.*; van Beek, W.*; Kolobanov, V.* Lattice dynamics of beryllium oxide: Inelastic x-ray scattering and ab initio calculations Physical Review B, 77 (2008), 224303-1 - 224303-7 Bose, P.*; Mittal, R.; Choudhury, N.*; Chaplot, S. L.* Inelastic neutron scattering and lattice dynamics of ZrO2, Y2O3 and ThSiO4 Pramana : Journal of Physics, 71 (2008) 5, 1141 - 1146 Bóta, A.*; Varga, Z.*; Goerigk, G. Structural description of the nickel part of a raney-type catalyst by using anomalous small-angle x-ray scattering Journal of Physical Chemistry C, 112 (2008) 12, 4427 - 4429 Brückel, T.; Kentzinger, E.; Mattauch, S.; Paul, A.; Rücker, U.; Voigt, J. A deeper look into magnetic nanostructures using advanced scattering methods Pramana : Journal of Physics, 71 (2008) 5, 895 Buchenau, U.; Schober, H.R. An atomic mechanism for the boson peak in metallic glasses Philosophical Magazine, 88 (2008) 33/35, 3885 – 3900

Burrows, H.D.*; Knaapila, M.*; Monkman, A.P.*; Tapia, M.J.*; Fonseca, S.M.*; Ramos, M.L.*; Pyckhout-Hintzen, W.; Pradhan, S.*; Scherf, U.* Structural Sudies on Cationic Poly9,9- bis [N,N,N-trimethylammonium) Alkyl] Fluorence-Co-1,4-Phenyleneiodides in Aqueous Solutions in the Presence of the Noninioic Surfactant Pentaethyleneglycol Monododecyl Ether (C12E5) Journal of Physics: Condensed Matter, 20 (2008), 104210 Chatterji, T.; Henry, P. F.*; Mittal, R.; Chaplot, S. L.* Negative thermal expansion of ReO3: Neutron diffraction experiments and dynamical lattice calculations Physical Review B, 78 (2008), 134105-1 - 134105-6 Chatterji, T.; Schneider, G. J.; Galera, R. M.* Low-Energy Nuclear Spin Excitations in NdMg3 and NdCo2 Physical Review B, 78 (2008), 012411-1 - 012411-4 Chen, J.*; Jung, P.; Hoffelner, W.*; Ullmaier, H. Dislocation loops and bubbles in oxide dispersion strengthened ferritic steel after helium implantation under stress Acta Materialia, 56 (2008), 250 - 258 Chen, J.*; Jung, P.; Pouchon, M. A.*; Rebac, T.*; Hoffelner, W.* Irradiation creep and precipitation in a ferritic ODS steel under helium implantation Journal of Nuclear Materials, 373 (2008), 22 - 27 Conrad, H.; Brückel, T.; Schäfer, W.*; Voigt, J. POWTEX - the high-intensity time-of-flight diffractometer at FRM II for structure analysis of polycrystalline materials Journal of Applied Crystallography, 41 (2008), 836 - 845 Delcea, M.*; Krastev, R.*; Gutberlet, T.; Pum, D.*; Sleytr, U. B.*; Toca-Herrera, J.L.* Thermal stability, mechanical properties and water content of bacterial protein layers recrystallized on polyelectrolyte multilayers Soft Matter, 4 (2008), 1414 - 1421 Disch, S.; Cheetham, A. K.*; Ruschewitz, U.* Formation of unsaturated C3 hydrocarbons by the protolysis of magnesium sesquicarbide with ammonium halides Inorganic Chemistry, 47 (2008), 969 - 973 Ehlers, G.*; Mamontov, E.*; Zamponi, M.; Faraone, A.*; Qiu, Y.*; Cornelius, A. L.*; Booth, C.H.*; Kam, K.C.*; LeToquin, R.*; Cheetham, A. K.*; Gardner, J. S.* Frustrated spin correlations in diluted spin ice Ho_2-x La_x Ti_2 O_7 Journal of Physics: Condensed Matter, 20 (2008), 235206 Foster, L.J.*; Schwahn, D.; Pipich, V.; Holden, P.J.*; Richter, D. SANS characterization of polyhydroxyalkanoates and their PEgylated hybrids in solution Biomacromolecules, 9 (2008), 314 - 320 Frank, C.*; Frielinghaus, H.; Allgaier, J.; Richter, D. Hydrophilic Alcohol Ethoxylates as Efficiency booster for Microemulsions Langmuir, 24 (2008), 6036 Genix, A.C.*; Arbe, A.*; Arres-Igor, S.*; Colmenero, J.*; Richter, D.; Frick, B.*; Deen, P.* Neutron scattering and dielectric investigation of a dilute blend of poly(ethyleneoxide) in polyethersulfone Journal of Chemical Physics, 128 (2008), 184901 Goel, P.*; Mittal, R.; Chaplot, S. L.*; Tyagi, A. K.* Lattice dynamics of strontium tungstate Pramana : Journal of Physics, 71 (2008) 5, 1135 – 1139 Goldman, A. I.*; Argyriou, D. N.*; Ouladdiaf, B.*; Chatterji, T.; Kreyssig, A.*; Nandi, S.*; Ni, N.*; Bud'ko, S. L.*; Canfield, P. C.*; McQueeney, R. J.* Lattice and magnetic instabilities in CaFe2As2: A single crystal neutron diffraction study Physical Review B, 78 (2008), 100506(R) Gossuin, Y.*; Hocq, A.*; Vuong, Q. L.*; Disch, S.; Hermann, R. P.; Gillis, P.* Physico-chemical and NMR relaxometric characterization of gadolinium hydroxide and dysprosium oxide nanoparticles Nanotechnology, 19 (2008), 475102-1 - 475102-8 Gray, D. L.*; Long, G. J.*; Grandjean, F.*; Hermann, R. P.; Ibers, J. A.* Syntheses, Structure, and a Mössbauer and Magnetic Study of Ba4Fe2I5S4 Inorganic Chemistry, 47 (2008) 1, 94 – 100

Gupta, M.*; Gupta, A.*; Stahn, J.*; Gutberlet, T. Ordering and self-diffusion in FePt alloy film New Journal of Physics, 10 (2008), 053031 Holderer, O.; Monkenbusch, M; Borchert, G.*; Breunig, C.*; Zeitelhack, K.* Layout and performance of the polarizing guide system for the J-NSE spectrometer at the FRM II Nuclear Instruments and Methods in Physics Research Section A, 586 (2008) 1, 90 - 94 Holderer, O.; Monkenbusch, M.; Schätzler, R.; Kleines, H.; Westerhausen, W.; Richter, D. The JCNS neutron spin-echo spectrometer J-NSE at the FRM II Measurement Science and Technology, 19 (2008), 034022 Hu, R.*; Thomas, K. J.*; Lee, Y.*; Vogt, T.*; Choi, E. S.*; Mitrovic, V. F.*; Hermann, R. P.; Grandjean, F.*; Canfield, P. C.*; Kim, J. W.*; Goldman, A. I.*; Petrovic, C.* Colossal positive magnetoresistance in a doped nearly magnetic semiconductor Physical Review B, 77 (2008), 085212-1 - 085212-5 Hüller, A.*; Prager, M.; Press, W.*; Seydel, T. Phase III of solid methane: The orientational potential and rotational tunneling Journal of Chemical Physics, 128 (2008), 034503 Ioffe, A. A new neutron spin-echo technique with time-gradient magnetic fields Nuclear Instruments and Methods in Physics Research Section A, 586 (2008) 1, 31 - 35 Ioffe, A.; Bodnarchuk, V.*; Bussmann, R.; Müller, R.*; Georgii, R.* A new neutron spin-echo spectrometer with time-gradient magnetic fields: First experimental test Nuclear Instruments and Methods in Physics Research Section A, 586 (2008) 1, 36 - 40 Kapnistos, M.*; Lang, M.*; Vlassopoulos, D.*; Pyckhout-Hintzen, W.; Richter, D.; Cho, D.*; Chang, T.*; Rubinstein, M.* Unexpected power-law stress relaxation of entangled ring polymers Nature Materials, 7 (2008), 997 - 1002 Karatas, Y.*; Pyckhout-Hintzen, W.; Zorn, R.; Richter, D.; Wiemhöfer, H.-D.* SANS Investigation and Conductivity of Pure and Salt-Containing Poly(bismethoxy-phosphazene) Macromolecules, 41 (2008), 2212 - 2218 Kentzinger, E.; Rücker, U.; Toperverg, B.; Ott, F.*; Brückel, T. Depth-resolved investigation of the lateral magnetic correlations in a gradient nanocrystalline multilayer Physical Review B, 77 (2008), 104435-1 - 104435-19 Kimber, S. A. J.*; Argyriou, D. N.*; Yokaichiya, F.*; Habicht, K.*; Gerischer, S.*; Hansen, T.*; Chatterji, T.; Klingeler, R.*; Hess, C.*; Behr, G.*; Kondrat, A.*; Büchner, B.* Magnetic ordering and negative thermal expansion in PrFeAsO Physical Review B, 78 (2008), 140503-1 - 140503-4(R) Kolasinska, M.*; Krastev, R.*; Gutberlet, T.; Warszynski, P.* Swelling and Water Uptake of PAH/PSS Polyelectrolyte Multilayers Progress in Colloid and Polymer Science, 134 (2008), 30 - 38 Lebedev, D.V.*; Filatov, M.V.*; Kuklin, A. I.*; Islamov, A. K.*; Stellbrink, J.; Pantina, R.A.*; Denisov, Y.Y.*; Toperverg, B. P.*; Isaev-Ivanov, V. V.* Structural hierarchy of chromatin in chicken erythrocyte nuclei based on small-angle neutron scattering: Fractal nature of the large-scale chromatin organization Crystallography Reports, 53 (2008), 110 - 115 Li, Y.-L.*; Maurel, M.-C.*; Ebel, C.*; Vergne, J.*; Pipich, V.; Zaccai, G.* Self-association of adenine-dependent hairpin ribozymes European Biophysics Journal : with Biophysics Letters, 37 (2008) 2, 173 - 182 Lott, D.*; Fenske, J.*; Schreyer, A.*; Mani, P.*; Mankey, G. J.*; Klose, F.*; Schmidt, W.; Schmalzl, K.; Tartakovskaya, E. V.* Antiferromagnetism in a Fe50Pt40Rh10 thin film investigated using neutron diffraction Physical Review B, 78 (2008), 174413-1 - 174413-10 Lonetti, B.; Kohlbrecher, J.*; Willner, L.; Dhont, J. K. G.; Lettinga, M. P. Dynamic response of block copolymer wormlike micelles to shear flow Journal of Physics: Condensed Matter, 20 (2008), 404207

Manoshin, S.*; Ioffe, A. New modules for the VITESS software package: Time-gradient magnetic fields and neutron refractive lenses Nuclear Instruments and Methods in Physics Research Section A, 586 (2008) 1, 81 - 85 Mamontov, E.*; Zamponi, M.; Hammons, S.*; Keener, W.S.*; Hagen, M.*; Herwig, K.W.* BASIS: A new backscattering spectrometer at the SNS Neutron News, 19 (2008), 22 - 24 Mazumdar, C.*; Rotter, M.*; Frontzed, M.*; Michor, H.*; Doerr, M.*; Kreiyssig, A.*; Koza, M.*; Hiess, A.*; Voigt, J.; Behr, G.*; Gupta, L.C.*; Prager, M.; Loewenhaupt, M.* Crystalline Electric Field Effects in PrNi2B2C: Inelastic Neutron Scattering Physical Review B, 78 (2008), 144422 McGuire, M. A.*; Christianson, A. D.*; Sefat, A. S.*; Sales, B. C.*; Lumsden, M. D.*; Jin, R.*; Payzant, E. A.*; Mandrus, D.*; Luan, Y.*; Keppens, V.*; Varadarajan, V.*; Brill, J. W.*; Hermann, R. P.; Sougrati, M. T.*; Grandjean, F.*; Long, G.* Phase transitions in LaFeAsO: Structural, magnetic, elastic, and transport properties, heat capacity and Mössbauer spectra Physical Review B, 78 (2008), 094517-1 - 094517-10 Mishra, S. K.*; Mittal, R.; Choudhury, N.*; Chaplot, S. L.*; Pandey, D.* Inelastic neutron scattering and lattice dynamics of NaNbO3 and Sr0.70Ca0.30TiO3 Pramana : Journal of Physics, 71 (2008) 5, 1141 - 1146 Proceedings of the International Symposium on Neutron Scattering (15. - 18.01.2008); Pramana - Journal of Physics 71, (2008) 5, 1129 - 1134; ISSN 0304-4289; 2008 Indian; Academy of Sciences, Bangalore Mittal, R. Negative thermal expansion in framework compounds Pramana : Journal of Physics, 71 (2008) 4, 829 - 835 Mittal, R.; Su, Y.; Rols, S.*; Tegel, M.*; Chaplot, S. L.*; Schober, H.*; Chatterji, T.; Johrendt, D.*; Brueckel, Th. Phonon dynamics in Sr[sub 0.6]K[sub 0.4]Fe[sub 2]As[sub 2] and Ca[sub 0.6]Na[sub 0.4]Fe[sub 2]As[sub 2] from neutron scattering and lattice-dynamical calculations Physical Review B, 78 (2008) 22, 224518-1 - 224518-5 Mittal, R.; Su, Y.; Chatterji, T.*; Chaplot, S.L.*; Schober, H.; Rotter, M.*; Johrendt, D.*; Brückel, T. Inelastic neutron scattering and lattice-dynamical calculations of BaFe2As2 Physical Review B, 78 (2008), 104514 Narros, A.*; Arbe, A.*; Alvarez, F.*; Colmenero, J.*; Richter, D. Atomic motions in the alpha-beta merging regime of 1-4 polybutadiene: A molecular dynamics simulation study Journal of Chemical Physics, 128 (2008), 224905 Niedzwiedz, K.; Wischnewski, A.; Pyckhout-Hintzen, W.; Allgaier, J.; Richter, D.; Faraone, A.* Chain Dynamics and Viscoelastic Properties of Poly(ethylene-oxide) Macromolecules, 41 (2008), 4866 Oh, I.-H.*; Mattauch, S.; Heger, G.*; Lee, C.E.* Neutron Diffraction Study of a Rb0.5Tl0.5H2PO4 Single Crystal Journal of the Physical Society of Japan, 77 (2008) 9, 094602-1 - 094602-4 Papagiannopoulos, A.*; Fernyhough, C.M.*; Waigh, T. A.*; Radulescu, A. Scattering Study of the Structure of Polystyrene Sulfonate Comb Polyelectrolytes in Solution Macromolecular Chemistry and Physics, 209 (2008), 2475 - 2486 Pipich, V.; Willner, L.; Schwahn, D. The A-B Diblock copolymer as non-ordering external field in a three component A/B/A-B polymer blend (Part of the "Karl Freed Festschrift") Journal of Physical Chemistry B, 112 (2008) 50, 16170 - 16181 Pipich, V.; Balz, M.*; Wolf, S.E.*; Tremel, W.*; Schwahn, D. Nucleation and Growth of CaCO3 Mediated by the Egg-White Protein Ovalbumin: A Time-Resolved in-situ Study Using Small-Angle Neutron Scattering Journal of the American Chemical Society, 130 (2008) 21, 6879 - 6892 Prager, M.; Desmedt, A.*; Unruh, T.*; Allgaier, J. Dynamics and adsorption sites of guest molecules in methyl chloride hydrate Journal of Physics: Condensed Matter, 20 (2008), 125219

Prager, M.; Grimm, H.; Natkaniec, I.*; Nowak, D.*; Unruh, T.* The dimensionality of ammonium reorientation in (NH4)2S2O8: the view from neutron spectroscopy Journal of Physics: Condensed Matter, 20 (2008), 125218 Radulescu, A.; Fetter, L.J.*; Richter, D. Polymer driven wax crystal control using partially crystalline polymeric materials Advances in Polymer Science, 210 (2008), 1 - 100 Radulescu, A.; Ioffe, A. Neutron guide system for small-angle neutron scattering instruments of the Jülich Centre for Neutron Science at the FRM-II Nuclear Instruments and Methods in Physics Research Section A, 586 (2008) 1, 55 - 58 Ramzi, A.*; Rijcken, C.*; Veldhuis, T.*; Schwahn, D.; Hennink, W.*; van Nostrun, C.* Core-Shell Structure of Degradable, Thermosensitive Polymeric Micelles studfied by Small-Angle Neutron Scattering Journal of Physical Chemistry B, 112 (2008) 3, 784 - 792 Schmalzl, K.; Strauch, D.*; Hiess, A.*; Berger, H.* Temperature dependent phonon dispersion in 2H-NbSe2 investigated using inelastic neutron scattering Journal of Physics: Condensed Matter, 20 (2008), 104240-1 - 104240-3 Schmidt, H.*; Gupta, M.*; Gutberlet, T.; Stahn, J.*; Bruns, M.* How to measure atomic diffusion processes in the sub-nanometer range Acta Materialia, 56 (2008), 464 - 470 Schneider, G. J.; Kerscher, M.; Göritz, D.* Messung der Volumenänderung von Elastomeren bei hohen Dehnraten = Measurement of Volume Changes of Elestomeres at High Strain Rates Kautschuk Gummi Kunststoffe, 6 (2008) , 317 - 321 Schober, H.*; Farhi, E.*; Mezei, F.*; Allenspach, P.*; Andersen, K.*; Bentley, P.M.*; Christiansen, P.*; Cubitt, B.*; Heenan, R. K.*; Kulda, J.*; Langan, P.*; Lefmann, K.*; Lieutenant, K.*; Monkenbusch, M.; Willendrup, P.*; Saroun, J.*; Tindesmans, P.*; Zsigmond, G.* Tailored instrumentation for long-pulse neutron spallation sources Nuclear Instruments and Methods in Physics Research Section A, 589 (2008) 1, 34 – 46 Sinko, K.*; Huesing, N.*; Goerigk, G.; Peterlik, H.* Nanostructure of gel-derived aluminosilicate materials Langmuir, 24 (2008) 3, 949 - 956 Sougrati, M. T.*; Hermann, R. P.; Grandjean, F.*; Long, G. J.*; Brück, E.*; Tegus, O.*; Trung, N. T.*; Buschow, K. H. J.* A structural, magnetic, and Mössbauer spectral study of the magnetocaloric Mn1.1Fe0.9P1-xGex compounds Journal of Physics: Condensed Matter, 20 (2008), 475206-1 - 475206-9 Stellbrink, J.; Lonetti, B.; Rother, G.*; Willner, L.; Richter, D. Shear induced structures of soft colloids: Rheo-SANS experiments on kinetically frozen PEP-PEO diblock copolymer micelles Journal of Physics: Condensed Matter, 20 (2008), 404206 Stelzer, H.; Weissbacher, C.; Soltner, H.; Janssen, F.; Butzek, M.; Kozielewski, T.; Lindenau, B.; Monkenbusch, M.; Ohl, M. Investigation of the temperature rise due to eddy currents in large chopper disks operated at polarized neutron beamlines Nuclear Instruments and Methods in Physics Research Section A, 594 (2008) 2, 228 - 231 Strempfer, J.*; Hupfeld, D.; Voigt, J.; Bihlmayer, G.; Goldman, A. I.*; Brückel, Th. Resonant magnetic x-ray scattering from terbium Journal of Physics: Condensed Matter, 20 (2008), 445208-1 - 445208-7 Theis-Bröhl, K.*; Westphalen, A.*; Zabel, H.*; Rücker, U.; McCord, J.*; Höink, V.*; Schmalhorst, J.*; Reiss, G.*; Weis, T.*; Engel, D.*; Ehresmann, A.*; Toperverg, B. P.* Hyper-domains in exchange bias micro-stripe pattern New Journal of Physics, 10 (2008), 093021-1 - 093021-21 Teixeira, S.C.M.*; Zaccai, G.*; Ankner, J.*; Bellissent-Funel, M.C.*; Bewley, R.*; Blakeley, M.P.*; Callow, P.*; Coates, L.*; Dahint, R.*; Dalgliesh, R.*; Dencher, N. A.*; Forsyth, V. T.*; Fragneto, G.*; Frick, G.*; Gilles, R.*; Gutberlet, T.; Haertlein, M.*; Hauß, T.*; Häußler, W.*; Heller, W.T.*; Herwig, K.*; Holderer, O.; Juranyi, F.*; Kampmann, R.*; Knott, R.*; Krueger, S.*; Langan, P.*; Lechner, R. E.*; Lynn, G.*; Majkrzak, C.*; May, R. P.*; Meilleur, F.*; Mo, Y.*; Mortensen, K.*; Myles, D.A.A.*; Natali, F.*; Neylon, C.*; Niimura, N.*; Ollivier, J.*; Ostermann, A.*; Peters, J.*; Pieper, J.*; Rühm, A.*;

Schwahn, D.; Shibata, K.*; Soper, A.K.*; Strässle, Th.*; Suzuki, J.*; Tanaka, I.*; Tehei, M.*; Timmins, P.*; Torikai, N.*; Unruh, T.*; Urban, V.*; Vavrin, R.*; Weiss, K.* New Sources and Instrumentation for Neutrons in Biology Chemical Physics, 345 (2008), 133 - 155 Varga, Z.*; Bóta, A.*; Goerigk, G. Unbinding Transition in Lipid Multibilayers Induced by Copper(II) Ions Journal of Physical Chemistry B, 112 (2008) 29, 8430 – 8433 Xu, X. S.*; Angst, M.; Brinzari, T. V.*; Hermann, R. P.; Musfeldt, J. L.*; Christianson, A. D.*; Mandrus, D.*; Sales, B. C.*; McGill, S.*; Kim, J.-W.*; Islam, Z.* Charge order, dynamics, and magneto-structural transition in multiferroic LuFe2O4 Physical Review Letters, 101 (2008), 227602-1 227602-4 Yan, H.*; Frielinghaus, H.; Nykanen, A.*; Ruokolainen, J.*; Saiani, A.*; Miller, A.F.* Thermoreversible lysozyme hydrogels: properties and an insight into the gelation pathway Soft Matter, 4 (2008) 6, 1313 Zamponi, M.; Wischnewski, A.; Monkenbusch, M.; Willner, L.; Richter, D.; Falus, P.*; Farago, B.*; Guenza, M.G.* Cooperative dynamics in homopolymer melts: a comparision of theoretical predictions with neutron spin echo experiments Journal of Physical Chemistry B, 112 (2008), 16220 - 16229 Zelenak, V.*; Badanicova, M.*; Halamova, D.*; Cejka, J.*; Zukal, A.*; Murafa, N.*; Goerigk, G. Amine-modified ordered mesoporous silica: Effect of pore size on carbon dioxide capture Chemical Engineering Journal, 144 (2008) 2, 336 - 342 Zorn, R.; Mayorova, M.; Richter, D.; Frick, B.* Inelastic neutron scattering study of a glass-forming liquid in soft confinement Soft Matter, 4 (2008), 522 – 533

Institute of Solid State Research Soft Matter (IFF-7)

2007 P. Ballesta, M. P. Lettinga, and S. Manneville Superposition rheology of shear-banding wormlike micelles J. Rheology (2007) Vol .51 (5), 1047-1072 Blochowicz, T*; Gögelein, C.; Spehr, T.*; Müller, M.*; Stühn, B.* Polymer-induced transient networks in water-in-oil microemusions studied by small-angle x-ray and dynamic light scattering Physical Review E, 76 (2007) 4, 041505 Dhont, J. K. G.; Wiegand, S.; Duhr, S.*; Braun, D.* Thermodiffusion of Charged Colloids: Single-Particle Diffusion Langmuir, 23 (2007), 1674 - 1683 Fan, T.-H.*; Dhont, J. K. G.; Tuinier, R. Motion of a sphere through a polymer solution Physical Review E, 75 (2007), 011803 Fan, T.-H.*; Xie, B.*; Tuinier, R. Asymptotic analysis of tracer diffusivity in nonadsorbing polmyer solutions Physical Review E, 76 (2007), 051405 Fleer, G. J.*; Skvortsov, A. M.*; Tuinier, R. A Simple Relation for the Concentration Dependence of Osmotic Pressure and Depletion Thickness in Polymer Solutions Macromolecular Theory and Simulations, 16 (2007), 531 - 540 Fleer, G. J.*; Tuinier, R. Analytical phase diagram for colloid-polymer mixtures Physical Review E, 76 (2007), 041802 Fleer, G. J.; Tuinier, R. The critical endpoint in phase diagrams of attractive hard spheres Physica A, 379 (2007), 52 - 58 Gapinski, J.; Patkowski, A.; Banchio, A. J.; Holmqvist, P.; Meier, G.; Lettinga, M. P.; Nägele, G. Collective diffusion in charge-stabilized suspensions: Concentration and salt effects Journal of Chemical Physics, 126 (2007), 104905 Holmqvist, P.; Dhont, J. K. G.; Lang, P. R. Colloidal dynamics near a wall studied by evanescent wave light scattering: Experimental and theoretical improvements and methodological limitations Journal of Chemical Physics, 126 (2007), 044707 Holmqvist, P.; Kleshchanok, D.; Lang, P. R. Unexpected slow near wall dynamics of spherical colloids in a suspension of rods Langmuir, 23 (2007), 12010 - 12015 Jungblut, S.*; Tuinier, R.; Binder, K.*; Schilling, T.* Depletion induced isotropic-isotropic phase separation in suspensions of rod-like colloids Journal of Chemical Physics, 127 (2007), 244909 K. Kang, S. Sprunt Light-Controlled Polymerization Kinetics for the Photostabilization of Cholesteric Fingerprint Rolls Mol.Cryst.Liq.Cryst., (2007) Vol. 466, pp. 23-38 Kang, K.; Wilk, A.*; Patkowski, A.*; Dhont, J. K. G. Diffusion of spheres in isotropic and nematic networks of rods: Electrostatic interactions and hydrodynamic screening Journal of Chemical Physics, 126 (2007), 214501

Kita, R.*; Polyakov, P.; Wiegand, S. Ludwig-Soret Effect of Poly(N-isopropylacrylamide): Temperature Dependence Study in Monohydric Alcohols Macromolecules, 40 (2007) 5, 1638 - 1642 Kleshchanok, D.; Lang, P. R. Steric Repulsion by Adsorbed Polymer Layers Studied with Total Internal Reflection Microscopy Langmuir, 23 (2007), 4332 -4339 Kohlbrecher, J.*; Bollhalder, A.*; Vavrin, R.*; Meier, G. A high pressure cell for small angle neutron scattering up to 500 MPa in combination with light scattering to investigate liquid samples Review of Scientific Instruments, 78 (2007), 125101 Lang, P. R. Depletion interaction mediated by polydisperse rods Journal of Chemical Physics, 127 (2007), 124906 Lettinga, M. P.; Grelet, E.* Self-diffusion of rodlike viruses through smectic layers Physical Review Letters, 99 (2007), 197802 McPhie, M. G.; Nägele, G. Long-time self-diffusion of charged colloidal particles: Electrokinetic and hydrodynamic interaction effects Journal of Chemical Physics, 127 (2007), 034906 Patkowski, A.*; Gapinski, J.*; Meier, G.; Kriegs, H. Isotropic Brillouin spectra of liquids having an internal degree of freedom Journal of Chemical Physics, 126 (2007), 014508 Polyakov, P.; Zhang, M.*; Müller-Plathe, F.*; Wiegand, S. Thermal diffusion measurements and simulations of binary mixtures of spherical molecules Journal of Chemical Physics, 127 (2007), 014502 Rediguieri, C.F.*; de Freitas, O.*; Lettinga, M. P.; Tuinier, R. Thermodynamic Incompatibility and Complex Formation in Pectin/Caseinate Mixtures Biomacromolecules, 8 (2007), 3345 - 3354 Rodriguez-Fernández, J.; Perez-Juste, J.; Liz-Marzán, L.M.; Lang, P. R. Dynamic Light Scattering of Short Au Rods with Low Aspect Ratios Journal of Physical Chemistry C, 111 (2007), 5020 - 5025 Tuinier, R.; Taniguchi, T.*; Wensink, H. H.* Phase behavior of a suspension of hard sperocylinders plus ideal polymer chains European Physical Journal E, 23 (2007), 355 - 365 Wiegand, S.; Ning, H.; Kita, R.* Universal concentration dependence of the Soret coefficient in aqueous systems Journal of Non-Equilibrium Thermodynamics, 32 (2007) 3, 193 Wiegand, S.; Ning, H.; Kriegs, H. Thermal diffusion forced Rayleigh scattering setup optimized for aqueous mixtures Journal of Physical Chemistry B, 111 (2007), 14169 Zhang, Z.; Berns, A. E.; Willbold, S.; Buitenhuis, J. Synthesis of poly(ethylene glycol) (PEG)-grafted colloidal silica particles with improved stability in aqueous solvents Journal of Colloid and Interface Science, 310 (2007), 446 - 455 Zhang, Z.; Buitenhuis, J. Synthesis of Uniform Silica Rods, Curved Silica Wires, and Silica Bundles Using Filamentous fd Virus as a Template Small, 3 (2007), 3, 424 - 428

2008 Banchio, A. J.*; McPhie, M. G.; Nägele, G. Hydrodynamic and electrokinetic effects on the dynamics of charged colloids and macromolecules Journal of Physics: Condensed Matter, 20 (2008), 404213-1 - 404213-14

Banchio, A. J.*; Nägele, G. Short-time transport properties in dense suspensions: From neutral to charge-stabilized colloidal spheres Journal of Chemical Physics, 128 (2008), 104903 Blanco, P.*; Polyakov, P.; Bou-Ali, M.M.*; Wiegand, S. Thermal diffusion and molecular diffusion values for some alkane mixtures: a comparison between thermogravitational column and thermal diffusion forced Rayleigh scattering Journal of Physical Chemistry B, 112 (2008), 8340 - 8345 Dhont, J. K. G.; Briels, W. J.* Gradient and vorticity banding Rheologica Acta, 47 (2008), 257 - 281 Dhont, J. K. G.; Briels, W. J.* Single-particle thermal diffusion of charged colloids: Double-layer theory in a temperature gradient European Physical Journal E, 25 (2008), 61 - 76 Fleer, G. J.*; Tuinier, R. Analytical phase diagrams for colloids and non-adsorbing polymer Advances in Colloid and Interface Science, 143 (2008), 1 - 47 Gögelein, C.; Nägele, G.; Tuinier, R.; Gibaud, T.*; Stradner, A.*; Schurtenberger, P.* A simple patchy colloid model for the phase behavior of lysozyme dispersions Journal of Chemical Physics, 129 (2008), 085102 Grelet, E.*; Lettinga, M. P.; Bier, M.*; vanRoij, R.*; van der Schoot, P.* Dynamical and structural insights into the smectic phase of rod-like particles Journal of Physics: Condensed Matter, 20 (2008), 494213 Holmqvist, P.; Kleshchanok, D.; Lang, P. R. Interaction potential and near wall dynamics of spherical colloids in suspensions of rod-like fd-virus European Physical Journal E, 26 (2008), 177 - 182 Kang, K.; Dhont, J. K. G. Double-layer polarization induced transitions in suspensions of colloidal rods EPL : a Letters Journal Exploring the Frontiers of Physics, 84 (2008), 14005 Kang, K.; Lettinga, M. P.; Dhont, J. K. G. Is vorticity-banding due to an elastic instability? Rheologica Acta, 47 (2008), 499 - 508 Kleshchanok, D.; Tuinier, R.; Lang, P. R. Direct measurement of polymer-induced forces Journal of Physics: Condensed Matter, 20 (2008), 073101 Kriegs, H.; Meier, G.; Gapinski, J.*; Patkowski, A.* The effect of intramolecular relaxations on the damping of longitudinal and transverse phonons in polysiloxanes studied by Brillouin spectroscopy Journal of Chemical Physics, 128 (2008), 014507 Lonetti, B.; Kohlbrecher, J.*; Willner, L.; Dhont, J. K. G.; Lettinga, M. P. Dynamic response of block copolymer wormlike micelles to shear flow Journal of Physics: Condensed Matter, 20 (2008), 404207 McPhie, M.; Nägele, G. Nonmonotonic density dependence of the diffusion of DNA fragments in low-salt suspensions Physical Review E, 78 (2008), 060401(R) Meier, G.; Kriegs, H. A high pressure cell for dynamic light scattering up to 2kbars with conservation of plane of polarization Review of Scientific Instruments, 79 (2008), 013102 Meier, G.; Vavrin, R.*; Kohlbrecher, J.*; Buitenhuis, J.; Lettinga, M. P.; Ratajczyk, M.* SANS and dynamic light scattering to investigate the viscosity of toluene under high pressure up to 1800 bar Measurement Science and Technology, 19 (2008), 034017 Ning, H.; Datta, S.*; Sottmann, T.*; Wiegand, S. Soret effect of nonionic surfactants in water studied by different transient grating setups Journal of Physical Chemistry B, 112 (2008), 10927 - 10934

Ning, H.; Dhont, J. K. G.; Wiegand, S. Thermal-Diffusive Behavior of a Dilute Solution of Charged Colloids Langmuir, 24 (2008), 2426 - 2432 Nygard, K.*; Satapathy, D.K.*; Bunk, O.*; Diaz, A.*; Perret, E.*; Buitenhuis, J.; Pfeiffer, F.*; David, C.*; van der Veen, J.F.* Structure of confined fluids by y-ray interferometry using diffraction gratings Optics Express, 16 (2008) 25, 20522 - 20529 Patkowski, A.*; Gapinski, J.*; Fluerasu, A.*; Holmqvist, P.; Meier, G.; Lettinga, M. P.; Nägele, G. Structure and dynamics of colloidal suspensions studied by means of XPCS Acta Physica Polonica A, 114 (2008) 2, 339 - 350 Polyakov, P.; Wiegand, S. Systematic study of the thermal diffusion in associated mixtures Journal of Chemical Physics, 128 (2008), 034505 Polyakov, P.; Müller-Plathe, F.*; Wiegand, S. Reverse nonequilibrium molecular dynamics calculation of the Soret coefficient in liquid heptane/benzene mixtures Journal of Physical Chemistry B, 112 (2008) 47, 14999 - 15004 Ripoll, M.; Holmqvist, P.; Winkler, R. G.; Gompper, G.; Dhont, J. K. G.; Lettinga, M. P. Attractive Colloidal Rods in Shear Flow Physical Review Letters, 101 (2008), 168302-1 - 168302-4 Sprakel, J.*; Spruijt, E.*; Cohen Stuart, M.A.*; Besseling, N. A. M.*; Lettinga, M. P.; van der Gucht, J.* Shear banding and rheochaos in associative polymer networks Soft Matter, 4 (2008) 8, 1696 - 1705 Tuinier, R.; Fan, T.-H.* Scaling of nanoparticle retardation in semi-dilute polymer solutions Soft Matter, 4 (2008), 254 - 257 Tuinier, R.; Smith, A.*; Poon, W. C. K.*; Egelhaaf, S. U.*; Aarts, D.G.A.L.*; Lekkerkerker, H. N. W.*; Fleer, G. J.* Phase diagram for a mixture of colloids and polymers with equal size EPL : a Letters Journal Exploring the Frontiers of Physics, 82 (2008), 68002

Institute for Structural Biology and Biophysics Cellular Biophysics (ISB-1) 2007 Cukkemane, A.; Grüter, B.; Novak, K.; Gensch, T.; Bönigk, W.; Gerharz, T.; Kaupp, U. B.; Seifert, R. Subunits act independently in a cyclic nucleotide-activated K+ channel EMBO Reports, 8 (2007) 8, 749 - 755 Dedecker, P.*; Muls, B.*; Hofkens, J.*; Enderlein, J.; Hotta, J.-I.* Orientational effects in the excitation and de-excitation of single molecules interacting with donut-mode laser beams Optics Express, 15 (2007) 6, 3372 - 3383 Dertinger, T.; Pacheco, V.*; von der Hocht, I.; Hartmann, R.*; Gregor, I.; Enderlein, J. Two focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements ChemPhysChem, 8 (2007) 3, 433 - 443 Eckel, R.*; Walhorn, V.*; Pelargus, C.*; Martini, J.*; Enderlein, J.; Nann, T.*; Anselmetti, D.*; Ros, R.* Fluorescence-emission control of single CdSe nanocrystals using gold-modified AFM tips Small, 3 (2007) 1, 44 - 49 Enderlein, J. Nucleotide specificity versus complex heterogeneity in exonuclease activity measurements Biophysical Journal, 92 (2007) 5, 5156 - 5158 Gensch, T.; Komolov, K. E.*; Senin, I. I.*; Philippov, P. P.*; Koch, K.-W.* Ca2+-dependent conformational changes in the neuronal Ca2+-sensor recoverin probed by the fluorescent dye Alexa647 Proteins - Structure Function and Bioinformatics, 66 (2007), 492 - 499 Gilbert, D.*; Franjic-Würtz, C.*; Funk, K.*; Gensch, T.; Frings, S.*; Möhrlen, F.* Differential maturation of chloride homeostasis in primary afferent neurons of the somatosensory system International Journal of Developmental Neuroscience, 25 (2007), 479 - 489 Gregor, I.; Enderlein, J. Time-resolved methods in biophysics. 3. Fluorescence lifetime correlation spectroscopy Photochemical and Photobiological Sciences, 6 (2007), 13 - 18 Haber-Pohlmeier, S.; Abarca Heidemann, K.; Körschen, H. G.; Kaur Dhiman, H.*; Heberle, J.; Schwalbe, H.*; Klein-Seetharaman, J.; Kaupp, U. B.; Pohlmeier, A. Binding of Ca2+ to glutamic acid-rich polypeptides from the rod outer segment Biophysical Journal, 92 (2007), 3207 - 3214 Kapusta, P.*; Wahl, M.*; Benda, A.*; Hof, M.*; Enderlein, J. Fluorescence lifetime correlation spectroscopy Journal of Fluorescence, 17 (2007) 1, 43 - 48 Mataruga, A.; Kremmer, E.*; Müller, F. Type 3a and type 3b OFF cone bipolar cells provide for the alternative rod pathway in the mouse retina Journal of Comparative Neurology, 502 (2007), 1123 - 1137 Melnikov, S. M.*; Yeow, E. K. L.*; Uji-i, H.*; Cotlet, M.*; Müllen, K.*; De Schryver, F. C.*; Enderlein, J.; Hofkens, J.* Origin of simultaneous donor-acceptor emission in single molecules of peryleneimide-terrylenediimide labeled polyphenylene dendrimers Journal of Physical Chemistry B, 111 (2007) 4, 708 - 719 Mohrlüder, J.; Stangler, T.; Hoffmann, Y.; Wiesehan, K.; Mataruga, A.; Willbold, D. Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library FEBS Journal, 274 (2007) 5543 - 5555 Schröder-Lang, S.*; Schwärzel, M.*; Seifert, R.; Strünker, T.; Kateriya, S.*; Looser, J.*; Watanabe, M.*; Kaupp, U. B.; Hegemann, P.*; Nagel, G.* Fast manipulation of cellular cAMP level by light in vivo Nature Methods, 4 (2007), 39 - 42

Sykora, J.*; Kaiser, K.*; Gregor, I.; Bönigk, W.; Schmalzing, G.*; Enderlein, J. Exploring fluorescence antibunching in solution to determine the stoichiometry of molecular complexes Analytical Chemistry, 79 (2007) 11, 4040 - 4049 von der Hocht, I.; Enderlein, J. Fluorescence correlation spectroscopy in cells: Confinement and excluded volume effects Environmental and Molecular Pathology, 82 (2007) 2, 142-146 Wahl, M.*; Rahn, H.-J.*; Gregor, I.; Erdmann, R.*; Enderlein, J. Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels Review of Scientific Instruments, 78 (2007), 033106 Zettl, H.*; Zettl, U.*; Krausch, G.*; Enderlein, J.; Ballauff, M.* Direct observation of single molecule mobility in semidilute polymer solutions Physical Review E, 75 (2007), 061804

2008 Funk, K.*; Woitecki, A.*; Franjic-Würtz, C.*; Gensch, T.; Möhrlen, F.*; Frings, S.* Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons Molecular Pain, 4 (2008), 32 - 43 Harzheim, D.; Pfeiffer, K.H.; Fabritz, L.*; Kremmer, E.*; Buch, T.*; Waisman, A.*; Kirchhof, P.*; Kaupp, U. B.; Seifert, R. Cardiac pacemaker function of HCN4 channels in mice is confined to embryonic development and requires cyclic AMP EMBO Journal, 27 (2008), 692 - 703 Kaupp, U. B.; Kashikar, N.D.; Weyand, I. Mechanisms of sperm chemotaxis Annual Review of Physiology, 70 (2008), 93 - 117 Knop, G.C.; Seeliger, M.W.*; Thiel, F.; Mataruga, A.; Kaupp, U.B.; Friedburg, C.*; Tanimoto, N.*; Müller, F. Light response in the mouse retina are prolonged upon targeted deletion of the HCN1 channel gene European Journal of Neuroscience, 28 (2008) 2221 - 2230 Mach, T.*; Chimerel, C.*; Fritz, J.*; Fertig, N.*; Winterhalter, M.*; Fütterer, C. Miniaturized planar lipid bilayer: increased stability, low electric noise and fast fluid perfusion Analytical and Bioanalytical Chemistry, 390 (2008), 841 - 846 Whitehead, J.*; Vignjevic, D.*; Fütterer, C.; Beaurepaire, E.*; Robine, S.*; Farge, E.* Mechanical factors activate ß-catenin-dependent oncogene expression in APC1638N/+ mouse colon HFSP Journal, 2 (2008) 286 - 294

Institute for Structural Biology and Biophysics Molecular Biophysics (ISB-2)

2007 Berndt, A.*; Kottke, T.; Breitkreuz, H.*; Dvorsky, R.*; Hennig, S.*; Alexander, M.*; Wolf, E.* A Novel Photoreaction Mechanism for the Circadian Blue Light Photoreceptor Drosophila Cryptochrome Journal of Biological Chemistry, 282 (2007), 13011 – 13021 Gmelin, W.*; Zeth, K.*; Efremov, R.; Heberle, J.*; Tittor, J.*; Oesterhelt, D.* The Crystal Structure of the L1 Intermediate of Halorhodopsin at 1.9 Å Resolution Photochemistry and Photobiology, 83 (2007), 369 - 377 Gorshkova, Yu.E.; Gordeliy, V. I. Investigation of Interaction of Dimethylsulfoxide with Lipid Membranes via Small-Angle Neutron Scattering Crystallography Reports, 52 (2007), 535 - 539 Haber-Pohlmeier, S.; Abarca Heidemann, K.; Körschen, H. G.; Kaur Dhiman, H.*; Heberle, J.; Schwalbe, H.*; Klein-Seetharaman, J.; Kaupp, U. B.; Pohlmeier, A. Binding of Ca2+ to glutamic acid-rich polypeptides from the rod outer segment Biophysical Journal, 92 (2007), 3207 - 3214 Immeln, D.*; Schlesinger, R.; Heberle, J.*; Kottke, T.* Blue Light Induces Radical Formation and Autophosphorylation in the Light-sensitive Domain of Chlamydomonas Cryptochrome Journal of Biological Chemistry, 282 (2007) 30, 21720 - 21728 Majerus,T.*;Kottke,T.*;Laan,W.*;Hellingwerf,K.*; Heberle, J.* Time-resolved FT-IR spectroscopy traces signal relay within the blue-light receptor AppA. Chemphyschem., 12 (2007) 8, 1787-1789 Mennes, N.*; Klare, J. P.*; Chizhov, I.*; Seidel, R.*; Schlesinger, R.; Engelhard, M.* Expression of the halobacterial transducer protein HtrII from Natronomonas pharaonis in Escherichia coli FEBS Letters, 581 (2007) 7, 1487 – 1494 Mironova, O. S.; Budyak, I. L.; Büldt, G.; Schlesinger, R.; Heberle, J. FT-IR difference spectroscopy elucidates crucial interactions of sensory rhodopsin I with the cognate transducer Htrl Biochemistry, 46 (2007) 33, 9399 – 9405 Murugova, T. N.*; Gordeliy, V. I.; Kuklin, A. I*.; Solodovnikova, I.M.*; Yaguzhinsky, L. S.* Study of Three-Dimensionally Ordered Structures of Intact Mitochondria by Small-Angle Neutron Scattering Crystallography Reports, 52 (2007) 3, 521 - 524 Poetsch, A.*; Berzborn, R. J.*; Heberle, J.*; Link, T. A.*; Dencher, N. A.*; Seelert, H.* Biophysics and Bioinformatics Reveal Structural Differences of the Two Peripheral Stalk Subunits in Chloroplast ATP Synthase Journal of Biochemistry, 141 (2007), 411 - 420 Rogachev, A.V.; Cherniy, A.; Ozerin, A. N.; Gordeliy, V. I.; Kuklin, A. I. Spherical sector model for describing the experimental small-angle neutron scattering data for dendrimers Crystallography Reports, 52 (2007), 500 – 504 Skegro, D.*; Pulvermuller, A.*; Krafft, B.*; Granzin, J.; Hofmann, K. P.*; Büldt, G.; Schlesinger, R. N-terminal and C-terminal Domains of Arrestin Both Contribute in Binding to Rhodopsin (dagger) Photochemistry and Photobiology, 83 (2007), 385 - 393 Strucksberg, K.H.; Rosenkranz, T.; Fitter, J. Reversible and irreversible unfolding of multi-domain proteins Biochimica et Biophysica Acta, 1774 (2007), 1501 - 1603

2008 Budyak, I.L.*;Mironova, O.S.*;Yanamala, N.*;Manoharan,V.*;Büldt,G.; Schlesinger,R.; Klein-Seetharaman,J. Flexibility of the Cytoplasmic Domain of the Phototaxis Transducer II from Natronomonas pharaonis Journal of Biophysics, in press Jiang, X.*; Zaitseva, E.*; Schmidt, M.*; Siebert, F.*; Engelhard, M.*; Schlesinger, R.; Ataka, K.*; Vogel, R.*; Heberle, J.* Resolving voltage-dependent structural changes of a membrane photoreceptor by surface-enhanced IR difference spectroscopy Proceedings of the National Academy of Sciences of the United States of America, 105 (2008) 34, 12113 – 12117 Stadler, A.M.; Digel, I.*; Artmann, G.M.*; Embs, J.P.*; Zaccai, G.*; Büldt, G. Hemoglobin Dynamics in Red Blood Cells: Correlation to Body Temperature Biophysical Journal, 95 (2008) , 1-13 Thielmann, Y.; Weiergräber, O. H.; Mohrlüder, J.; Koenig, B. W.; Hartmann, R.; Stangler, T.; Wiesehan, K.; Willbold, D. Characterization of the binding surface of the human protein GABARAP From Computational Biophysics to Systems Biology (CBSB08) : proceedings / ed.: U. H. E. Hansmann, J. H. Meinke, S. Mohanty, W. Nadler, O. Zimmermann. - Jülich, John von Neumann Institut für Computing, 2008. - (NIC Series ; 40). - 978-3-9810843-6-8. - Weiergräber, O. H.; Stangler, T.; Thielmann, Y.; Mohrlüder, J.; Wiesehan, K.; Willbold, D. Ligand binding mode of GABA(A) receptor-associated protein Journal of Molecular Biology, 381 (2008), 1320 - 1331

Institute for Structural Biology and Biophysics Structural Biochemistry (ISB-3) 2007 Birkmann, E.; Henke, F.; Weinmann, N.; Dumpitak, C.; Groshup, M.; Funke, A.; Willbold, D.; Riesner, D. Counting of single prion particles bound to a capture-antibody surface (surface-FIDA) Veterinary Microbiology, 123 (2007) 294-304 Dertinger, T.; Pacheco, V.; von der Hocht, I.; Hartmann, R.; Gregor, I.; Enderlein, J. Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements ChemPhysChem, 8 (2007) 433-443 Elfrink, K.; Nagel-Steger, L.; Riesner, D. Interaction of the Cellular Prion Protein with Raft-like Lipid Membranes. Biological Chemistry, 388 (2007) 79-90. Funke, S. A.; Birkmann, E.; Henke, F.; Görtz, P.; Lange-Asschenfeldt, C.; Riesner, D.; Willbold, D. Single particle detection of amyloid-β aggregates associated with Alzheimer’s disease Biochemical and Biophysical Research Communications, 364 (2007) 902-907. Hänel, K.; Willbold, D. SARS-CoV accessory protein 7a directly interacts with human LFA-1 Biological Chemistry, 388 (2007), 1325-1332. Hoffmann, S.; Jonas, E.; König, S.; Preusser-Kunze, A.; Willbold, D. Nef protein of human immunodeficiency virus type 1 binds its own myristoylated N-terminus Biological Chemistry, 388 (2007) 181-183 Leliveld, S. R.; Korth, C. The use of conformation-specific ligands and assays to dissect the molecular mechanisms of neurodegenerative diseases Journal of Neuroscience Research, 85 (2007) 2285-2297 Mohrlüder, J.; Hoffmann, Y.; Stangler, T.; Hänel, K.; Willbold, D. Identification of clathrin heavy chain as a direct binding partner for the GABA type A receptor associated protein GABARAP Biochemistry, 46 (2007) 14537-14543. Mohrlüder, J.; Stangler, T.; Wiesehan, K.; Hoffmann, Y.; Mataruga, A.; Willbold, D. Identification of calreticulin as ligand of GABARAP by phage display screening of a peptide library FEBS Journal, 274 (2007) 5543-5555 Muhs, A.; Hickmann, D. T.; Pihlgren, M.; Chuard, N.; Giriens, V.; Meerschman, C.; van der Auwera, I.; van Leuven, F.; Sugawara, M.; Weingertner, M. C.; Bechinger, B.; Greferath, R.; Kolonko, N.; Nagel-Steger, L.; Riesner, D.; Brady, R. O.; Pfeifer, A.; Nicolau, C. Liposomal vaccines with conformation-specific amyloid peptide antigens define immune response and efficacy in APP transgenic mice Proceedings of the National Academy of Sciences of the United States of America, 104 (2007) 23, 9810-9815 Nagel-Steger L, Demeler B, Willbold D, Aggregate size and shape distributions in amyloid-β peptide solutions. NIC Workshop 2007, From Computational Biophysics to System Biology 2007. Eds. Ulrich. E. Hansmann, Jan Meinke, Sandipan Mohanty, Olav Zimmermann. NIC Series Volume 36, 235-237 (2007) Scheidt, H. A.; Vogel, A.; Eckhoff, A.; König, B. W.; Huster, D. Solid state NMR characterization of the putative membrane anchor of TWD1 from Arabidopsis thaliana European Biophysics Journal, 36 (2007) 393-404 Schmidt, H.; Hoffmann, S.; Tran, T.; Stoldt, M.; Stangler, T.; Wiesehan, K.; Willbold, D. Solution structure of a Hck SH3 domain ligand complex reveals novel interaction modes Journal of Molecular Biology, 365 (2007) 1517-1532

Schünke, S.; Novak, K.; Stoldt, M.; Kaupp, U. B.; Willbold, D. Resonance assignment of the cyclic nucleotide binding domain from a cyclic nucleotide-gated K+ channel in complex with cAMP Biomolecular NMR Assignments, 1 (2007) 179-181 Spiess, H. W.; Jeschke, G.; König, B. W.; Willbold, D. Magnetische Resonanzspektroskopie Nachrichten aus der Chemie, 55 (2007) 288-293 Stangler, T.; Tran, T.; Hoffmann, S.; Schmidt, H.; Jonas, E.; Willbold, D. Competitive displacement of full-length HIV-1 Nef from Hck SH3 domain by a high affinity artificial peptide Biological Chemistry, 388 (2007) 611-615 Stangler T, Hartmann R, Willbold D, König BW, Modern high resolution NMR for the study of structure, dynamics and interactions of biological macromolecules. in Progress in Physical Chemistry Vol. 1 / Different Aspects of Intermolecular Interaction, Oldenbourg, München (2007) Stellbrink, J.; Niu, A. Z.; Allgaier, J.; Richter, D.; König, B. W.; Hartmann, R.; Coates, G. W.; Fetters, L. J. Analysis of polymeric methylaluminoxane (MAO) via small angle neutron scattering Macromolecules, 40 (2007) 4972-4981 Wiesehan, K.; Funke, S. A.; Fries, M.; Willbold, D. Purification of recombinantly expressed and cytotoxic human amyloid-beta peptide Journal of Chromatography B, 856 (2007) 229-233 Wittlich, M.; König, B. W.; Hoffmann, S.; Willbold, D. Structural characterisation of the transmembrane and cytoplasmic domains of human CD4. Biochimica et Biophysica Acta: Biomembranes, 1768 (2007) 2949-2960 Wittlich, M.; König, B. W.; Willbold, D. Expression, purification, and membrane reconstitution of a CD4 fragment comprising the transmembrane and cytoplasmic domains of the receptor Protein Expression and Purification, 55 (2007) 198-207

2008 Bailly, A.; Sovero, V.; Vincenzetti, V.; Santelia, D.; Bartnik, D.; König, B. W.; Mancuso, S.; Martinoia, E.; Geisler, M. Modulation of P-glycoproteins by auxin transport inhibitors is mediated by interaction with immunophilins. Journal of Biological Chemistry 283 (2008) 21817-21826 Birkmann E.; Riesner D. Prion infection – seeded fibrillization or more? Prion, 2 (2008) 67-72 Birkmann, E.; Henke, F.; Funke, S. A.; Bannach, O.; Riesner, D.; Willbold, D. A highly sensitive diagnostic assay for aggregate-related diseases e.g. prion disease and Alzheimer's disease. Rejuvenation Research, 11 (2008) 359-363 Elfrink, K.; Ollesch J.; Stöhr J.; Willbold D.; Riesner D.; Gerwert K. Structural changes of PrPc upon membrane anchoring. Proceedings of the National Academy of Sciences of the United States of America 105 (2008) 10815-10819 Funke, S. A.; Birkmann, E.; Henke, F.; Görtz, P.; Lange-Asschenfeldt, C.; Riesner, D.; Willbold, D. An ultra sensitive assay for early diagnosis of Alzheimer's disease. Rejuvenation Research, 11 (2008) 315-318 Gardiennet, C.; Loquet, A.; Etzkorn, M.; Heise, H.; Baldus, M.; Böckmann, A. Structural constraints for the Crh protein from solid-state NMR experiments. Journal Biomolecular NMR 40 (2008) 239-250 Hartmann R, Stangler T, Koenig BW, Willbold D, Exploring protein-ligand interactions by solution NMR. in ‘Proteomics Sample Preparation’, J. v. Hagen, Wiley-VCH (2008)

Heise, H. Solid-State NMR Spectroscopy of Amyloid Proteins. ChemBioChem 9 (2008) 179-189 Heise, H.; Celej, M. S.; Becker, S.; Riedel, D.; Pelah, A.; Kumar, A.; Jovin, T. M.; Baldus, M. Solid-state NMR reveals structural differences between fibrils of wild-type and disease-related A53T mutant alpha-synuclein Journal of Molecular Biology, 380 (2008) 444-450 Kaimann, T.; Metzger, S.; Kuhlmann, K.; Brandt, B.; Birkmann, E.; Höltje, H. D.; Riesner, D. Molecular model of an alpha-helical prion protein dimer and its monomeric subunits as derived from chemical cross-linking and molecular modeling calculations. Journal of Molecular Biology, 376 (2008), 582-596 Leliveld, S. R.; Bader, V.; Hendriks, P.; Prikulis, I.; Sajnani, G.; Requena, J.R.; Korth, C. Insolubility of disrupted-in-schizophrenia 1 disrupts oligomer-dependent interactions with nuclear distribution element 1 and is associated with sporadic mental disease. Journal of Neuroscience, 28 (2008) 15, 3839-3845 Leliveld, S. R.; Stitz, L.; Korth, C. Expansion of the octarepeat domain alters the misfolding pathway but not the folding pathway of the prion protein Biochemistry, 47 (2008) 23, 6267-6278 Müller, C. B.; Loman, A.; Pacheco, V.; Koberling, F.; Willbold, D.; Richtering, W.; Enderlein, J. Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy. Europhysics Letters 83 (2008) 46001 Nagel-Steger L, Demeler B, Hochdörfer K, Schrader T, Willbold D, Modulation of aggregate size and shape distributions of amyloid-β peptide solutions by a designed β-sheet breaker. NIC Workshop 2007, From Computational Biophysics to System Biology CBSB08. Eds. Ulrich. E. Hansmann, Jan H. Meinke, Sandipan Mohanty, Walter Nadler, Olav Zimmermann. NIC Series Volume 40, 333-336 (2008) Panza, G.; Stöhr, J.; Birkmann, E.; Riesner, D.; Willbold, D.; Baba, O.; Terashima, T.; Dumpitak, C. Aggregation and amyloid fibril formation of the prion protein is accelerated in presence of glycogen. Rejuvenation Research 11 (2008) 365-369 Panza, G.; Stöhr, J.; Dumpitak, C.; Papathanassiou, D.; Weinmann, N.; Weiß, J.; Riesner, D.; Willbold, D.; Birkmann, E. Spontaneous and BSE-prion-seeded amyloid formation of full length recombinant bovine Prion Protein. Biochemical and Biophysical Research Communications 373 (2008) 493-497 Stöhr, J.; Weinmann, N.; Wille, H.; Kaimann, T.; Nagel-Steger, L.; Birkmann, E.; Panza, G.; Prusiner, S. B.; Eigen, M.; Riesner, D Mechanisms of prion protein assembly into amyloid. Proceedings of the National Academy of Sciences of the United States of America, 105 (2008) 2409-2414 Thielmann, Y.; Mohrlüder, J.; König, B. W.; Stangler, T.; Hartmann, R.; Hoeltje, H. D.; Willbold, D. An indole binding site major determinant of the ligand specificity of the GABA type A receptor associated protein GABARAP. ChemBioChem, 9 (2008) 1767-1775 Thielmann Y, Weiergräber OH, Mohrlüder J, Koenig BW, Hartmann R, Stangler T, Wiesehan K, Willbold D. Characterization of the binding surface of the human protein GABARAP. NIC Workshop 2007, From Computational Biophysics to System Biology CBSB08. Eds. Ulrich. E. Hansmann, Jan H. Meinke, Sandipan Mohanty, Walter Nadler, Olav Zimmermann. NIC Series Volume 40, 401-403 (2008) van Groen, T.; Wiesehan, K.; Funke, S. A.; Kadish, I.; Nagel-Steger, L.; Willbold, D. Reduction of Alzheimer's Disease Amyloid Plaque Load in Transgenic Mice by D3, a D-enantiomeric Peptide Identified by Mirror Image Phage Display. ChemMedChem, 3 (2008) 1848-1852 Weiergräber, O. H.; Stangler, T.; Thielmann, Y.; Mohrlüder, J.; Wiesehan, K.; Willbold, D. Ligand binding mode of GABA(A) receptor-associated protein Journal of Molecular Biology, 381 (2008), 1320-1331 Wiesehan, K.; Stöhr, J.; Nagel-Steger, K.; van Groen, T.; Riesner, D.; Willbold, D. Inhibition of cytotoxicity and amyloid fibil formation by a D-amino acid peptide that specifically binds to Alzheimer’s disease amyloid peptide Protein Engineering Design and Selection, 21 (2008), 241-246

Wittlich, M.; König, B. W.; Willbold, D. Structural consequences of phosphorylation of two serine residues in the cytoplasmic domain of HIV-1 VpU. Journal of Peptide Science, 14 (2008) 804-810

Institute of Bio- and Nanosystems Bioelectronics (IBN-2)

2007 Akram, R.*; Eker, T.*; Bozbey, A.*; Fardmanesh, M.*; Schubert, J.; Banzet, M. Effect of rf Pumping Frequency and rf Input Power on the Flux to Voltage Transfer Function of rf-SQUIDs Applied Superconductivity IEEE Transactions on Applied Superconductivity, 17 (2007) 2, 676 - 679 Barannik, A. A.*; Cherpak, N. T.*; Prokopenko, Yu. V.*; Filipov, Yu. F.*; Shaforost, E.N.; Shipilova, I. A.* Two-layered disk quasi-optical dielectric resonators: electrodynamics and application perspectives for complex permittivity measurements of lossy liquids Measurement Science and Technology, 18 (2007) 7, 2231 - 2238 Belyaev, A. E.*; Naumov, A. V.*; Tarasov, G. G.*; Komarov, A. V.*; Tacano, M.*; Danylyuk, S. V.; Vitusevich, S. A. Nature of low-energy optical emission in doped AlGaN/GaN heterostructures Journal of Applied Physics, 101 (2007), 033709-1 - 033709-5 Cesa, C. M.; Kirchgeßner, N.; Mayer, D.; Schwarz, U. S.*; Hoffmann, B.; Merkel, R. Micropatterned silicon elastomer for high resolution analysis of cell force patterns Review of Scientific Instruments, 78 (2007) 3, 034301 Christiaens, P.*; Abouzar, M. H.; Poghossian, A.; Wagner, T.; Bijnens, N.*; Williams, O. A.*; Daenen, W. H.*; Haenen, K.*; Douheret, O.*; DHaen, J.*; Mekhalif, Z.*; Schöning, M. J.; Wagner, P.* pH sensitivity of nanocrystalline diamond films Physica Status Solidi A, 204 (2007) 9, 2925 - 2930 Danilchenko, B. A.*; Zelensky, S. E.*; Drok, E. A.*; Belyaev, A. E.*; Kochelap, V. A.*; Lüth, H.; Vitusevich, S. A. Enhancement by electric field of high-speed photoconductivity in AlGaN/GaN heterostructures Applied Physics Letters, 90 (2007) 15, 152102-1 - 152102-3 Fu, Z.*; Wu, A.*; Vilarinho, P. M.*; Kingon, A. I.*; Wördenweber, R. Low dielectric loss BaNd2Ti5O14 thick films prepared by an electrophoretic deposition technique Applied Physics Letters, 90 (2007), 052912 Gasteier, P.*; Reska, A.; Schulte, P.; Salber, J.*; Offenhäusser, A.; Moeller, M.*; Groll, J.* Surface Grafting of PEO-Based Star-Shaped Molecules for Bioanalytical and Biomedical Applications Macromolecular Bioscience, 7 (2007) 8, 1010 - 1023 González, M. P.; Hollmann, E.; Wördenweber, R. Quantitative analysis of the guidance of vortices in superconducting films with magnetic dots Journal of Applied Physics, 102 (2007), 063904 He, M.; Klushin, A. M.; Klein, N. Meandering Bicrystal Josephson Junction arrays in a hemispherical Fabry-Perot Resonator Superconductor Science and Technology, 20 (2007), s413 - s418 He, M.; Klushin, A. M.; Yan, S. L.*; Klein, N. Optimization of Bicrystal Josephson Junctions and Arrays in a Fabry-Perot Resonator IEEE Transactions on Applied Superconductivity, 17 (2007) 2, 934 - 937 Hollmann, E.; Wördenweber, R. Analysis of Defects in Epitaxial Oxide Thin Films via X-Ray Diffraction Technology Thin Solid Films, 515 (2007) 7/8, 3530 Ingebrandt, S.; Han, Y.; Nakamura, F.*; Poghossian, A.; Schöning, M. J.; Offenhäusser, A. Label-free detection of single nucleotide polymorphisms utilizing the differential transfer function Biosensors and Bioelectronics, 22 (2007), 2834 - 2840 Jung, A.; Gronewold, T. M. A.*; Tewes, M.*; Quandt, E.*; Berlin, P. Biofunctional structural design of SAW sensor chip surfaces in a microfluidic sensor system Sensors and Actuators B, 124 (2007), 46 - 52

Jung, A.; Wolters, B.; Berlin, P. (Bio)functional surface structural design of substrate materials based on self-assembled monolayers from aminocellulose derivatives and amino(organo)polysiloxanes Thin Solid Films, 515 (2007) 17, 6867 - 6877 Krause, H.-J.; Wolters, N.; Zhang, Y.; Offenhäusser, A.; Miethe, P.*; Meyer, M. H. F.*; Hartmann, M.*; Keusgen, M.* Magnetic particle detection by frequency mixing for immunoassay applications Journal of Magnetism and Magnetic Materials, 311 (2007), 436 - 444 Kuzel, P.*; Kadlec, F.*; Petzelt, J.*; Schubert, J.; Panaitov, G. Highly tunable SrTiO3 thin film structures for applications in the terahertz range Applied Physics Letters, 91 (2007), 232911 Meszaros, G.; Kronholz, S.; Karthäuser, S.; Mayer, D.; Wandlowski, Th. Electrochemical Fabrication and Characterization of Nanocontacts and nm-sized Gaps Applied Physics A, 87 (2007), 569 - 575 Meyburg, S.*; Stockmann, R.; Moers, J.; Offenhäusser, A.; Ingebrandt, S. Advanced CMOS process for floating gate field-effect transistors in bioelectronic applications Sensors and Actuators B, 128 (2007), 208 - 217 Meyer, M. H. F.*; Hartmann, M.*; Krause, H.-J.; Blankenstein, G.*; Müller-Chorus, B.*; Oster, J.*; Miethe, P.*; Keusgen, M. CRP determination based on a novel magnetic biosensor Biosensors and Bioelectronics, 22 (2007), 973 - 979 Meyer, M. H. F.*; Krause, H.-J.; Hartmann, M.*; Miethe, P.*; Oster, J.*; Keusgen, M.* Francisella tularensis detection using a magnetic labels and a magnetic biosensor based on frequency mixing Journal of Magnetism and Magnetic Materials, 311 (2007), 259 - 263 Meyer, M. H. F.*; Stehr, M.*; Bhuju, S.*; Krause, H.-J.; Hartmann, M.*; Miethe, P.*; Singh, M.*; Keusgen, M.* Magnetic biosensor for the detection of Yersinia pestis Journal of Microbiological Methods, 68 (2007), 218 - 224 Offenhäusser, A.; Böcker-Meffert, S.; Decker, T.; Helpenstein, R.; Gasteier, P.*; Groll, J.*; Möller, M.*; Reska, A.; Schäfer, S.; Schulte, P.; Vogt-Eisele, A.* Microcontact printing of proteins for neuronal cell guidance Soft Matter, 3 (2007) 3, 290 - 298 Pabst, M.; Wrobel, G.; Ingebrandt, S.; Sommerhage, F.; Offenhäusser, A. Solution of the Poisson-Nernst-Planck equations in the cell-substrate interface European Physical Journal E, 24 (2007), 1 - 8 Petrychuk, M. V.*; Belyaev, A. E.*; Kurakin, A. M.; Danylyuk, S. V.; Klein, N.; Vitusevich, S. Mechanisms of current formation in resonant tunneling AlN/GaN heterostructures Applied Physics Letters, 91 (2007) 22, 222112 Poghossian, A.; Abouzar, M. H.; Amberger, F.; Mayer, D.; Han, Y.; Ingebrandt, S.; Offenhäusser, A.; Schöning, M. J. Field-effect sensors with charged macromolecules: characterization by capacitance-voltage, constant-capacitance, impedance-spectroscopy and atomic-force microscopy methods Biosensors and Bioelectronics, 22 (2007), 2100 - 2107 Poghossian, A.; Ingebrandt, S.; Abouzar, M. H.; Schöning, M. J. Label-free detection of charged macromelecules by using a field-effect-based sensor platform: Experiments and possible mechanisms of signal generation Applied Physics A, 87 (2007), 517 - 524 Qiu, L.; Zhang, Y.; Krause, H.-J.; Braginski, A. I.; Burghoff, M.*; Trahms, L.* Nuclear magnetic resonance in the earth's magnetic field using a nitrogen-cooled superconducting quantum interference device Applied Physics Letters, 91 (2007), 072505 Qiu, L.; Zhang, Y.; Krause, H.-J.; Braginski, A. I.; Usoskin, A.* High-temperature superconducting quantum interference device with cooled LC resonant circuit for measuring alternating magnetic fields with improved signal-to-noise ratio Review of Scientific Instruments, 78 (2007), 054701 Schöning, M. J.; Abouzar, M. H.; Poghossian, A.; Han, Y.; Offenhäusser, A.; Ingebrandt, S. Markierungsfreie DNA-Detektion mit Silizium-Feldeffekt-Sensoren - Messeffekte oder Artefakte? Technisches Messen, 74 (2007) 9, 466 - 474

Schöning, M. J.; Kloock, J. P. About 20 years of silicon-based thin-film sensors with chalcogenide glass materials for heavy metal analysis: Technological aspects of fabrication and miniaturization (Review) Electroanalysis, 19 (2007) 19/20, 2029 - 2038 Sosso, A.*; Andreone, D.*; Lacquaniti, V.*; Klushin, A. M.; He, M.; Klein, N. Metrological Study of YBCO Josephson Junction Arrays Integrated in a Fabry-Perot Resonator IEEE Transactions on Applied Superconductivity, 17 (2007) 2, 874 - 877 Turek, M.; Ketterer, L.*; Claßen, M.*; Berndt, H. K.*; Elbers, G.*; Krüger, P.*; Keusgen, M.*; Schöning, M. J. Development and electrochemical investigations on an EIS- (electrolyte-insulator-semiconductor) based biosensor for cyanide detection Sensors, 7 (2007), 1415 - 1426 Wagner, T.; Maris, R. J.*; Ackermann, H.-J.*; Otto, R.; Beging, S.; Poghossian, A.; Schöning, M. J. Handheld measurement device for field-effect sensor structures: Practical evaluation and limitations Sensors and Actuators B, 127 (2007) , 217-223 Wagner, T.; Molina, R.; Yoshinobu, T.*; Kloock, J. P.; Biselli, M.*; Canzoneri, M.*; Schnitzler, T.*; Schöning, M. J. Handheld multi-channel LAPS device as a transducer platform for possible biological and chemical multi-sensor applications Electrochimica Acta, 53 (2007), 305 - 311 Wördenweber, R.; Hollmann, E.; Kutzner, R.; Schubert, J. Induced Ferroelectricity in Strained Epitaxial SrTiO3 Films on Various Substrates Journal of Applied Physics, 102 (2007), 044119-1 - 044119-5 Wördenweber, R.; Hollmann, E.; Mahmood, A.; Schubert, J.; Pickartz, G.; Lee, T. K. Ferroelectric properties of compressively strained epitaxial SrTiO3 films on sapphire Journal of the European Ceramic Society, 27 (2007), 2899 - 2902 Wrobel, G.; Höller, M.*; Ingebrandt, S.; Dieluweit, S.; Sommerhage, F.; Bochem, H. P.; Offenhäusser, A. Transmission electron microscopy study of the cell-sensor interface Journal of the Royal Society Interface, 10 (2007), 1094 - 1098 Wrobel, G.; Zhang, Y.; Krause, H.-J.; Wolters, N.; Sommerhage, F.; Offenhäusser, A. Influence of the first amplifier stage in MEA systems on extracellular signal shapes Biosensors and Bioelectronics, 22 (2007), 1092 - 1096 Wu, Y. H.; Panaitov, G.; Zhang, Y.; Klein, N. Design and fabrication of in-plane resonant microcantilevers Microelectronics Journal, 39 (2007), 44 - 48 Yeung, C. K.*; Sommerhage, F.; Wrobel, G.; Offenhäusser, A.; Chan, M.*; Ingebrandt, S. Drug Profiling Using Planar Microelectrode Arrays Analytical and Bioanalytical Chemistry, 387 (2007), 2673 - 2680 Zhang, Y.; Qiu, L.; Krause, H.-J.; Hartwig, S.*; Burghoff, M.*; Trahms, L.* Liquid state nuclear magnetic resonance at low fields using a nitrogen cooled superconducting quantum interference device Applied Physics Letters, 90 (2007), 182503

2008 Abouzar, M. H.; Poghossian, A.; Razavi, A.; Besmehn, A.; Bijnens, N.*; Williams, O. A.*; Haenen, K.*; Wagner, P.*; Schöning, M. J. Penicillin detection with nanocrystalline-diamond field-effect sensor Physica Status Solidi A, 205 (2008) 9, 2141 - 2145 Barannik, A. A.*; Bunyaev, S. A.*; Cherpak, N. T.*; Vitusevich, S. A. Quasioptical Sapphire resonators in the form of a truncated cone Journal of Lightwave Technology, 26 (2008) 17, 3118 - 3123 Beigmohamadi, M.*; Niyamakom, P.*; Farahzadi, A.*; Effertz, C.*; Kremers, S.*; Brüggemann, D.; Wuttig, M.* Structure and morphology of perylene films grown on different substrates Journal of Applied Physics, 104 (2008) 1

Belyaev, A. E.*; Raicheva, V.G.*; Kurakin, A. M.; Klein, N.; Vitusevich, S. A. Investigation of spin-orbit interaction in AlGaN/GaN heterostructures with large electron density Physical Review B, 77 (2008) 3, 035311 Bettaeib, L.*; Kokabi, H.*; Poloujadoff, M.*; Sentz, A.*; Krause, H.-J. Non destructive testing (NDT) with high-Tc rf SQUIDs Journal of Physics : Conference Series, 97 (2008), 012263 Borstlap, D.; Schubert, J.; Zander, W.; Offenhäusser, A.; Ingebrandt, S. High-k dielectric layers for bioelectronic applications IEICE Transactions on Electronics, E91-C (2008) 12, 1894 - 1898 Crisan, A.*; Wördenweber, R.; Hollmann, E.; Kutzner, R.; Button, T. W.*; Abell, J. S.* Thermally-induced self-assembling nanotechnology of gold nano-dots on CeO2-buffered sapphire for superconducting films Journal of Optoelectronics and Advanced Materials, 10 (2008), 1370 - 1373 Gilioli, E.; Baldini, M; Bindi, M.; Bissoli, F.; Calestani, D.; Pattini, F.; Rampino, S.; Rocca, M.; Zannella, S.; Wördenweber, R. Pulsed Electron Deposition (PED) of Single Buffer Layer for 'low-cost' YBCO Coated Conductors Journal of Physics : Conference Series, 97 (2008), 012197 Gordan, O. D.*; Persson, B. N. J.*; Cesa, C. M.; Mayer, D.; Hoffmann, B.; Dieluweit, S.; Merkel, R. On Pattern Transfer in Replica Molding Langmuir, 24 (2008), 6636 - 6639 Klushin, A. M.; He, M.; Levitchev, M. Yu.; Kurin, V. V.; Klein, N. Optimization of the coupling of mm wave power to arrays of high-Tc Josephson junctions Journal of Physics : Conference Series, 97 (2008), 012268 Kurakin, A.; Vitusevich, S.; Danylyuk, S.; Hardtdegen, H.; Klein, N.; Bougrioua, Z.*; Danilchenko, B. A.*; Konakova, R. V.*; Belyaev, A. E.* Mechanism of mobility increase of the two-dimensional electron gas in AlGaN/GaN heterostructures under small dose gamma irradiation Journal of Applied Physics, 103 (2008), 083707 Kuzel, P.*; Kadlec, C.*; Kadlec, F.*; Schubert, J.; Panaitov, G. Field-induced soft mode hardening in SrTi03/DySc03 multilayers Applied Physics Letters, 93 (2008), 052910-1 - 052910-3 Lukashenko, A.*; Wördenweber, R.; Ustinov, A. V.* Imaging of vortex flow in microstructured high-Tc films by laser scanning microscope Physica C, 468 (2008), 552 - 556 Meier, M.; Nauenheim, C.; Gilles, S.; Mayer, D.; Kügeler, C.; Waser, R. Nanoimprint for future non-volatile memory and logic devices Microelectronic Engineering, 85 (2008), 870 - 872 Qiu, L. Q.; Zhang, Y.; Krause, H.-J.; Braginski, A. I. SQUID-detected NMR in Earth's magnetic field Journal of Physics : Conference Series, 97 (2008), 012026 Reska, A.; Gasteier, P.*; Schulte, P.; Möller, M.*; Offenhäusser, A.; Groll, J.* Ultrathin Coatings with Change in Reactivity over Time Enable Functional In Vitro Networks of Insect Neurons Advanced Materials, 20 (2008), 2751 - 2755 Schröper, F.; Brüggemann, D.; Mourzina, Y.; Wolfrum, B.; Offenhäusser, A.; Mayer, D. Analyzing the electroactive surface of gold nanopillars by electrochemical methods for electrode miniaturization Electrochimica Acta, 53 (2008), 6265 - 6273 Sommerhage, F.; Helpenstein, R.; Rauf, A.; Wrobel, G.; Offenhäusser, A.; Ingebrandt, S. Membrane allocation profiling: A method to characterize three-dimensional cell shape and attachment based on surface reconstruction Biomaterials, 29 (2008), 3927 – 3935 Vitusevich, S. A.; Kurakin, A. M.; Klein, N.; Petrychuk, M. V.*; Naumov, A. V.*; Belyaev, A. E.* AlGaN/GaN High Electron Mobility Transistor Structures: Self-Heating Effect and Performance Degradation IEEE Transactions on Device and Materials Reliability, 8 (2008) 3, 543 - 548 Vitusevich, S. A.; Kurakin, A. M.; Konakova, R. V.*; Belyaev, A. E.*; Klein, N. Improvement of interface properties of AlGaN/GaN heterostructures under gamma-radiation Applied Surface Science, 255 (2008) 3, 784 - 786

Institute of Bio- and Nanosystems Biomechanics (IBN-4)

2007 Beltramo, G.; Ibach, H.; Müller, J. E.; Giesen, M. A Novel Approach to Measure the Step Line Tension and the Step Dipole Moment on Vicinal Au(001) Electrodes Surface Science, 601 (2007), 1876 - 1885 Cesa, C. M.; Kirchgeßner, N.; Mayer, D.; Schwarz, U. S.*; Hoffmann, B.; Merkel, R. Micropatterned silicon elastomer for high resolution analysis of cell force patterns Review of Scientific Instruments, 78 (2007) 3, 034301 Hartwig, B.*; Borm, B.; Schneider, H.*; Arin, M. J.*; Kirfel, G.*; Herzog, V.* Laminin-5-deficient human keratinocytes: Defective adhesion results in a saltatory and inefficient mode of migration Experimental Cell Research, 313 (2007) 8, 1575 - 1587 Ikonomov, J.*; Starbova, K.*; Giesen, M. Measurements of Step and Kink Energies and of the Step Edge Stiffness from Island Studies on Pt(111) Physical Review B, 75 (2007), 245411-1 - 24541-8 Ikonomov, J.; Starbova, K.; Giesen, M. Island coalescence and diffusion along kinked steps on Cu(001): Evidence for a large kink Ehrlich-Schwoebel barrier Surface Science, 601 (2007) 5, 1403 - 1408 Limozin, L.*; Sengupta, K. Modulation of Vesicle Adhesion and Spreading Kinetics by Hyaluronan Cushions Biophysical Journal, 93 (2007), 3300 - 3313 Merkel, R.; Kirchgeßner, N.; Cesa, C. M.; Hoffmann, B. Cell Force Microscopy on Elastic Layers of Finite Thickness Biophysical Journal, 93 (2007), 3314 - 3323 Pichardo-Pedrero, E.*; Beltramo, G.; Giesen, M. Electrochemical annealing and its relevance inmetal electroplating: An atomistic view Applied Physics A, 87 (2007), 461 - 467 Pichardo-Pedrero, E.; Giesen, M. Comparative STM Studies on island equilibrium shapes, shape fluctuations and island coalescence on Au(001) electrodes in chloric and sulfuric acid solutions Electrochimica Acta, 52 (2007) 18, 5659 - 5668 Schaap, I.A.T.*; Hoffmann, B.; Carrasco, C.*; Merkel, R.; Schmidt, C. F.* Tau protein binding forms a 1 nm thick layer along protofilaments without affecting the radial elasticity of microtubules Journal of Structural Biology, 158 (2007), 282 - 292 Semmrich, C.*; Storz, T.*; Glaser, J.*; Merkel, R.; Bausch, A. R.*; Kroy, K.* Glass transition and rheological redundancy in F-actin solutions (from the cover) Proceedings of the National Academy of Sciences of the United States of America, 104 (2007), 20199 - 20203 Wrobel, G.; Höller, M.*; Ingebrandt, S.; Dieluweit, S.; Sommerhage, F.; Bochem, H. P.; Offenhäusser, A. Transmission electron microscopy study of the cell-sensor interface Journal of the Royal Society Interface, 10 (2007), 1094 - 1098

2008 Beltramo, G. L.; Ibach, H.; Linke, U.; Giesen, M. Determination of the step dipole moment and the step line tension on Ag(001) electrodes Electrochimica Acta, 53 (2008), 6818 - 6823

Biehl, R.; Hoffmann, B.; Monkenbusch, M.; Falus, P.*; Préost, S.*; Merkel, R.; Richter, D. Direct Observation of Correlated Interdomain Motion in Alcohol Dehydrogenase Physical Review Letters, 101 (2008), 138102-1 - 138102-4 Gordan, O. D.*; Persson, B. N. J.*; Cesa, C. M.; Mayer, D.; Hoffmann, B.; Dieluweit, S.; Merkel, R. On Pattern Transfer in Replica Molding Langmuir, 24 (2008), 6636 - 6639 Kajzar, A.*; Cesa, C. M.*; Kirchgeßner, N.; Hoffmann, B.; Merkel, R. Toward Physiological Conditions for Cell Analyses: Forces of Heart Muscle Cells Suspended between Elastic Micropillars Biophysical Journal, 93 (2008), 1854 - 1866 Scheijen, F. J. E.*; Beltramo, G. L.; Hoeppener, S.*; Housmans, T. H. M.*; Koper, M. T. M.* The electrooxidation of small organic molecules on platinum nanoparticles supported on gold: influence of platinum deposition procedure Journal of Solid State Electrochemistry, 12 (2008), 483 - 495 Smith, A.*; Sengupta, K.; Goennewein, S.*; Seifert, U.*; Sackmann, E.* Force-induced growth of adhesion domains is controlled by receptor mobility Proceedings of the National Academy of Sciences of the United States of America, 105 (2008), 6906 - 6911 Tschersich, K. G.; Fleischhauer, J.P.; Schuler, H.* Design and characterization of a thermal hydrogen atom source Journal of Applied Physics, 104 (2008), 034908

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