Abstracts

of Invited Talks and of Contributed Talks

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FTIR-Microscopy Studies of Fibres Formed by Cyt c and Lipids

Juha-Matti Alakoskela¹, Arimatti Jutila¹, Sani Marttila², Raija Turunen², Sinikka Pyhäjoki², Erik Goormaghtigh³, and Paavo Kinnunen^1,4

¹University of Helsinki, Finland, ²Crime Laboratory, National Bureau of Investigations, Vantaa, Finland, ³Free University of Brussels, Belgium , and ⁴MEMPHYS - Center for Biomembrane Physics, University of Southern Denmark

The formation of amyloids is involved in the pathogenesis of several diseases. By selecting right kind of solvent environment in vitro the process of amyloid formation can apparently be triggered for almost any protein, (Fandrich et al., 2001). In vivo the environment of lipid membranes could play an important role in the formation of amyloid fibres. Increase in membrane cholesterol content appears to enhance amyloid formation (Yip et al., 2001; 2002), and likewise electrostatic interactions with negatively charged lipids are important for membrane association of amyloid-b peptides (Ege and Lee, 2004; Lindström et al., 2002; Terzi et al., 1997; Vargas et al., 2000).

Importantly, recent results suggest a more general role for negatively charged phospholipids in amyloid formation. Knight and Miranker (2004) showed that negatively charged phospholipid vesicles catalyse the amyloid fibre formation of islet amyloidogenic polypeptide. In a contemporary study from our group, it was found that negatively charged lipids induce formation of very large fibres (up to 1-2 mm in length) by several proteins: lysozyme, insulin, glyceraldehyde-3-phosphate dehydrogenase, myoglobin, transthyretin, cytochrome c, histone H1, and R-lactalbumin. The charged lipids are also incorporated into the resulting fibre. Additionally, in Congo Red staining the fibres showed typical light-green birefringence characteristic of amyloid (Zhao et al., 2004). Similar behaviour was later found for endostatin (Zhao et al., 2005). Cytochrome c was selected for further structural studies.


Fibres were formed in a container with magnetic stirring, and then separated and classified under light microscope. A variety of fibres formed. The fibres, in case of cyt c, may be grossly classified as red, colourless, and blue fibres (and those between) based on colour seen under light microscope. FTIR microscopy revealed that red fibres display mostly native like cyt c spectra, while blue fibres displayed FTIR spectra with high peaks at ≈1613 cm^-1 and ≈1631 cm^-1 typical of β sheets and amyloids. Suprisingly, structural differences are evident not only between fibres but also in spectra recorded along single fibres.

These results support the conclusions of the previous study from our group. Yet, the phenomenon remains poorly characterized and understood.

References:
Ege, C. and Lee, K. Y. C., 2004, "Insertion of Alzheimer's Ab40 peptide into lipid monolayers". Biophys. J. 87: 1732-1740.
Fandrich M., Fletcher M. A., and Dobson C. M., 2001, "Amyloid fibrils from muscle myoglobin". Nature 410: 165-166.
Knight J. D. and Miranker A. D., 2004, "Phospholipid catalysis of diabetic amyloid assembly". J. Mol. Biol. 341: 1175-1187.
Lindström, F., Bokvist, M., Sparrman, T., Gröbner, G., 2002, "Association of amyloid-β peptide with membrane surfaces monitored by solid state NMR". Phys. Chem. Chem. Phys. 4: 5524-5530.
Terzi, E., Hölzemann, G., and Seelig, J., 1997, "Interaction of Alzheimer β-amyloid peptide(1-40) with lipid membranes". Biochemistry 36: 14845-14852.
Vargas, J., Alarcon, J. M., and Rojas, E., 2000, "Displacement currents associated with the insertion of Alzheimer disease amyloid β-peptide into planar bilayer membranes". Biophys. J. 79: 934-944.
Yip, C. M., Elton, E. A., Darabie, A. A.; Morrison, M. R., and McLaurin, J., 2001, "Cholesterol, a modulator of membrane-associated Aβ-fibrillogenesis and neurotoxicity". J. Mol. Biol. 311: 723-734.
Yip, C. M., Darabie, A. A., and McLaurin, J., 2002, "Aβ-42-peptide assembly on lipid bilayers. J. Mol. Biol. 318: 97-107.
Zhao, H., Tuominen, E. K. J., and Kinnunen, P. K. J., 2004, "Formation of amyloid fibers triggered by phosphatidylserine-containing membranes". Biochemistry 43: 10302-10307.
Zhao, H., Jutila, A., Nurminen, T., Wickström, S. A., Keski-Oja, J., and Kinnunen, P. K. J., 2005, "Binding of endostatin to phosphatidylserine-containing membranes and formation of amyloid-like fibers". Biochemistry 44: 2857-2863.


Dr. Juha-Matti Alakoskela
Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine/Biochemistry
POB 63, FIN-00014 University of Helsinki, Finland
Email: jmalakos@cc.helsinki.fi
WWW: http://www.biophysics.helsinki.fi


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Bilayer Elasticity, Lipid Curvature and Ion Channel Function

Olaf S. Andersen¹, Roger E. Koeppe II ², and Jens A. Lundbæk¹

¹Weill Medical College of Cornell University and ²University of Arkansas

Changes in the chemical composition of lipid bilayers - due to changes in lipid composition or the adsorption of amphiphiles - alter lipid bilayer material properties and protein function. To explore the relative importance of changes in bilayer elastic moduli (bilayer elasticity) and lipid spontaneous curvature (or lateral pressure profile) for the modulation of protein function, we use gramicidin channels as molecular force transducers to monitor how experimental manipulations of lipid bilayers change the disjoining force the lipid bilayer imposes on bilayer-spanning inclusions. To understand the implications for complex channel function, we examined how changes in bilayer elasticity, as monitored using gramicidin channels, alter the gating of voltage-dependent sodium channels. We show that the changes in sodium channel gating can be "predicted" from the changes in gramicidin channel function - and that changes in bilayer elasticity quantitatively are more important than changes in spontaneous curvature.

References:
J.A. Lundbæk, O.S. Andersen, T. Werge, and C. Nielsen, "Cholesterol-induced protein sorting: an analysis of energetic feasibility", Biophysical J. 84 2080-2089 (2003).
R.L. Goforth, A.-K. Chi, D.V. Greathouse, L.L. Providence, R.E. Koeppe II, and O.S. Andersen, "Hydrophobic coupling of lipid bilayer energetics to channel function", J. Gen. Physiol. 121 477-493 (2003).
T.-C. Hwang, R.E. Koeppe II, and O.S. Andersen, "Genistein can modulate channel function by a phosphorylation-independent mechanism: importance of bilayer mechanics and hydrophobic mismatch", Biochemistry 42 13646-13658 (2003).
J.A. Lundbæk, J.A., P. Birn, A.J. Hansen, R. Søgaard, C. Nielsen, J. Girshman, M.J. Bruno, S.E. Tape, J. Egebjerg, D.V. Greathouse, G.L. Mattice, R.E. Koeppe II, and O.S. Andersen, "Regulation of sodium channel function by bilayer elasticity - the importance of hydrophobic coupling: effects of micelle-forming amphiphiles and cholesterol", J. Gen. Physiol. 123 599-621 (2004).
T.M. Suchyna, S.E. Tape, R.E. Koeppe II, O.S. Andersen, F. Sachs, and P.A. Gottlieb, "Bilayer-dependent inhibition of mechanosensitive channels by neuroactive peptide enantiomers", Nature 430 235-240 (2004).


Olaf Sparre Andersen
Thomas H. Meikle, Jr., Professor of Medical Education
Department of Physiology and Biophysics
Weill Medical College of Cornell University
1300 York Avenue, Rm C-501B
New York, NY 10021-4896, USA
Tel: +1 212 746-6350/6259
Fax: +1 212 746-8690
Email: sparre@med.cornell.edu
WWW: http://www.med.cornell.edu/gradschool/fac/andersenO.html


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Analytical Derivation of Lateral Pressure Profile from Microscopic Model of Lipid Bilayer

Svetlana Baoukina and Sergey I. Mukhin

Moscow State Institute for Steel and Alloys, Moscow, Russia

Flexible string model of hydrocarbon chain is used to derive analytical expression for the lateral pressure profile across the hydrophobic core of the membrane [1]. Pressure profile influences the functioning of the embedded proteins and is difficult to measure experimentally [2,3]. In our model hydrocarbon chain is represented as a flexible string of finite thickness with a given bending rigidity [4]. In the mean-field approximation we substitute entropic repulsion between neighboring chains in lipid membrane with effective potential. The effective potential is determined self-consistently. Arbitrary chain conformation is expanded over eigenfunctions of the self-adjoint operator of chain energy density. Lateral pressure distribution across the bilayer is calculated using path integral technique [5]. We found that the pressure profile is mainly formed by sum of the partial contributions of a few discrete lowest energy "eigenconformations". Dependences on temperature and area-per-lipid of the lateral pressure produced by hydrocarbon chains are found. Theory gives the redistribution of lateral pressure in the hydrophobic core of lipid bilayer caused by temperature change. Chain contribution to the area compressibility modulus and the temperature coefficient of area expansion are calculated. Experimentally observable predictions of the theory include chain orientational order parameter.

References:
[1] S. Baoukina, S.I. Mukhin, Biophys. J. Supplement. 88 (2005).
[2] R.S. Cantor, Biophys. J. 76, 2625 (1999).
[3] D. Marsh, Biochem. Biophys. Acta, 1286, 183 (1996).
[4] D. Nelson, "Defects and Geometry in Condensed Matter Physics", Cambridge University Press, Cambridge (2002).
[5] H. Kleinert, "Path integrals in Quantum Mechanics, Statistics and Polymer Physics", World Scientific, Singapore (1995).


Svetlana Baoukina
Theoretical Physics Department, Physics and Chemistry Faculty
Moscow State Institute for Steel and Alloys
4, Leninsky Pr., MISA, 119049, Moscow, Russia
Email: svt_lana19@yahoo.com



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Regulating the Activity of Membrane Proteins: Can Agonists Act both as Aqueous Ligands and as Bilayer Solutes/Adsorbants?

Robert S. Cantor

Department of Chemistry, Dartmouth College, Hanover, NH, USA, and
MEMPHYS - Center for Biomembrane Physics, University of Southern Denmark

The function of many integral membrane proteins involves conformational equilibria that can be modulated not only by direct binding of aqueous solutes to well-defined protein sites, but also indirectly, mediated by bilayer physical properties that vary with membrane composition. We have explored one such indirect mechanism involving the lateral pressure profile, by which changes in lipid characteristics, as well as the solubilization or adsorption of small solutes can have a significant effect on protein activity.

In more recent work, we examine the consequences of having the same agonist influence protein activity by both mechanisms. The fundamental differences between these two types of mechanisms, both kinetic and thermodynamic, can result in time-dependent protein activity that is quite similar to various characteristics of desensitization and deactivation observed in some membrane proteins. In particular, we speculate on the possible relevance to the behavior of fast neurotransmitter receptors in postsynaptic membranes, and potentially to the molecular mechanism of general anesthesia.


Robert S. Cantor
Professor
Department of Chemistry, Dartmouth College
Hanover, NH 03755, USA
Tel: +1 603 646-2504
Fax: +1 603 646-3946
Email: robert.cantor@dartmouth.edu
WWW: http://www.dartmouth.edu/~rcantor


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Single Molecule Tracking as a Tool to Unravel the Dynamic Membrane Organization
of the μ Opioid Receptor

N. Destainville, F. Daumas, C. Millot, M. Corbani, A. Lopez, D.S. Dean, and L. Salomé

Université Paul Sabatier, Toulouse, France

Monitoring of the movements of membrane proteins (or lipids) by single particle tracking enables to get reliable insights into the complex dynamic organisation of the plasma membrane constituents. We investigated by this technique the diffusional behavior of the human μ opioid receptor (a G protein coupled receptor) at the surface of living cells. The trajectories of the receptors revealed a diffusion mode combining a short term confined diffusion with a long term random walk. A detailed statistical analysis led us to propose a model where the receptors have a diffusion confined to a domain which itself diffuses, the confinement being due to long-range attractive interprotein interactions, such as interactions mediated by the membrane. The existing models of the dynamic organisation of the cell membrane cannot explain our results. Our model of Brownian interacting proteins is consistent with the experimental observations and accounts for the variations found with the domain size of the short term and long term diffusion coefficients. The changes in the dynamic membrane organization of the μ opioid receptors accompagnying the binding of peptide agonist and antagonist will be discussed, as well as the possible role of hydrophobic mismatch.


Dr. Nicolas Destainville
Laboratoire de Physique Théorique, IRSAMC
Université Paul Sabatier
118, route de Narbonne, 31062 Toulouse cedex 4, France
Tel : +33 5 61 55 60 48
Fax : +33 5 61 55 60 65
Email: destain@irsamc.ups-tlse.fr
WWW: http://www.lpt.irsamc.ups-tlse.fr/~destain/


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Dynamics of Lipid Bilayers from Molecular Dynamics Simulations

Olle Edholm

Theoretical Biological Physics, Department of Physics, The Royal Institute of Technology,
Stockholm, Sweden

Molecular dynamics simulations of lipid bilayers can now be performed for times up to the order of several hundred ns for small systems. This means that a large part of the time scales that are important for NMR relaxation can be covered. This includes the dynamics of water at the interface as well as the complex correlated rotations that occur inside the hydrocarbon region of a lipid bilayer. Even the translational diffusion of single lipid molecules can be followed to get reasonable estimates for lateral diffusion coefficients. We also start to see large scale undulatory motions but can not yet say much about their dynamics based on the necessarily quite short simulations that can be performed on these large systems.

One important observation from the molecular dynamics simulations is that angular orientation correlation functions of C-H ( C-D) vectors are strongly non-exponential on all time scale from fractions of ps and up. A stretched exponential or power law decay describes them better. This is yields after Fourier transformation data that are consistent with the experimentally observed frequency dependence of nuclear magnetic relaxation rates. The motion responsible for this decay of the correlation functions occurs on a local spatial scale and can best be described by trans/gauche isomerizations in the hydrocarbon tails. These isomerizations are, however, strongly correlated within chains and between neighboring chains. This gives rise to a wide distribution of relaxation times that results in the stretched decay of the correlation functions.

Reference:
E. Lindahl and O. Edholm, "Molecular Dynamics simulations of NMR relaxation rates and slow dynamics in lipid bilayers",
J. Chem. Phys. 115 (2001) 4938-4950.


Olle Edholm
Professor
Theoretical Biological Physics, Department of Physics
Stockholm Centre for Physics, Astronomy and Biotechnology
The Royal Institute of Technology
SE-10691 Stockholm, Sweden
Tel: +46 8 55 37-8168
Fax: +46 8 55 37-8216
Email: oed@theophys.kth.se
WWW: http://www.theophys.kth.se/~oed/


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Forces Involved in Membrane Tube Formation. Numerical Simulations Can Refine Experimental Findings

Andrea Fera

Institut Marie-Curie, Paris, France

[no abstract available]


Dr. Andrea Fera
Post-doc researcher
Institut Marie-Curie, Paris, France
Postal address:
AMOLF Institute, Kruislaan 407
1098SJ Amsterdam, The Netherlands
Tel: +31 20 608-1391
Fax: +31 20 608-1288 or +31 20 668-4106
Email: andrea.fera@curie.fr or fera@amolf.nl


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Pressure Profile Studies of All-Atom Lipid Bilayer Models

Justin R. Gullingsrud

University of California, San Diego

Lipid bilayer tension is distributed quite inhomogeneously, but until recently the effect of varying lipid composition, tension, and small molecule concentration could be computationally investigated only with coarse-grained models. High-performance computing and efficient parallel molecular dynamics codes have enabled the systematic study of pressure profiles under a wide variety of conditions using all-atom models with explicit solvent. We computed pressure profiles with reproducible features at the single Angstrom length scale. Compared with PE headgroups, lipids with PC headgroups were found to shift the lateral pressure out of the hydrophobic core and into the headgroup region by an amount that is independent of area per lipid. POPE bilayers simulated at areas smaller than the tension-free value exerted dramatically higher lateral pressure in a narrow region at the start of the aliphatic chain. Stretching of POPC bilayers increased tension predominantly in the same region. Finally, we consider the results of pressure profile analysis in the context of two membrane channels: MscL, a tension-gated mechanosensitive channel, and KcsA, whose tertiary stability has been shown to be affected by the incorporation of tetrafluoroethanol.


Dr. Justin R. Gullingsrud
Department of Chemistry/Biochemistry
University of California, San Diego, MC 0365
9500 Gilman Drive, La Jolla, CA 92093-0365, USA
Tel: +1 858 534-2913
Fax: +1 858 534-7697
Email: jgulling@mccammon.ucsd.edu
WWW: http://www.ks.uiuc.edu/~justin/


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From Artifacts and Barostats to Pressure Profiles, Bending Rigidities, and 'Toy Proteins': A Tour of Methodological and Physical Aspects of Lipid Bilayer Simulations by DPD

Ask F. Jakobsen, Ole G. Mouritsen, and Gerhard Besold

MEMPHYS–Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark

We report about the application of Dissipative Particle Dynamics (DPD) simulation to coarse-grained lipid bilayer models. Both pure systems and bilayers with additives (small molecules as well as simplified models of transmembrane proteins) will be considered. Some of the work presented here is still in progress.

Despite the fact that DPD has attracted more and more attention in recent years as an intriguing technique in mesoscopic modeling of soft matter systems, some basic methodological aspects still require special attention and further development. In this context we first report briefly about our findings concerning artifacts which may affect various physical quantities in DPD simulations. Basically they arise when the time increment is chosen too large in the numerical integration of the equations of motion. In DPD these artifacts may easily remain unnoticed due to the softness of the non-bonded interactions. – A patch of unconstrained bilayer will assume a state of minimum (zero) lateral tension. For bilayer simulations it is therefore recommended to choose a statistical ensemble in which the surface tension can be chosen as an independent variable. We present an algorithm which implements a combined thermostat and barostat in DPD so that constant normal pressure and constant surface tension can be established in a very efficient way. - As a last methodological aspect, we report about first results from a linear response method used to obtain bending rigidities in DPD simulations of bilayers (work in progress).

In the last part of this contribution we focus on physical (mechanical) properties of a lipid bilayer. Its lateral organization, pressure profiles as well as bending rigidities, are all affected and partly change dramatically when additives are incorporated into the bilayer. By adding small amphiphilics or small hydrophobic molecules that partition into the bilayer, the changes in area compressibility, bending rigidity, and lateral pressure profiles can be studied in detail, and we will present some of the results here. – A particularly complex aspect of biomembrane organization is the conformational equilibrium of trans-membrane proteins. A shift of this equilibrium by changing the physical properties of the bilayer (as monitored, e.g., by the lateral proessure profile) through the addition of small molecules which partition into the bilayer can greatly affect the biological functionality of the protein. We report about first steps towards the design of a suitable coarse-grained "toy" protein by which these mechanisms can be studied in DPD simulations.

References:
Ask F. Jakobsen, "Constant-pressure and constant-surface tension simulations in dissipative particle dynamics",
J. Chem. Phys. 122, 124901/1-8 (2005).
Ask F. Jakobsen, Ole G. Mouritsen, and Gerhard Besold, "Artifacts in dynamical simulations of coarse-grained model lipid bilayers", J. Chem. Phys. (2005, in press).
Erik Lindahl and Olle Edholm, "Mesoscopic undulations and thickness fluctuations in lipid bilayers from molecular dynamics simulations", Biophys. J. 79, 426-433 (2000).
Julian C. Shillcock and Reinhard Lipowsky, "Equilibrium structure and lateral stress distribution of amphiphilic bilayers from dissipative particle dynamics simulation", J. Chem. Phys. 117 5048-5061 (2002).


Ask Frode Jakobsen
MEMPHYS–Center for Biomembrane Physics
Physics Department, University of Southern Denmark
Campusvej 55, DK-5230 Odense M, Denmark
Tel: +45 6550 3686
Fax: +45 6615 8760
Email: ask@memphys.sdu.dk
WWW: http://www.memphys.sdu.dk/


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Lipid Influence on Selective Water Transport

Morten Ø. Jensen

MEMPHYS - Center for Biomembrane Physics, University of Southern Denmark

Water transport through highly selective Aquaporin water channels embedded in different lipid bilayers is studied by Molecular Dynamics simulations. We examine the lipid bilayer as a potential determinant for this type of transport.


Morten Østergaard Jensen
Research Assistant Professor
MEMPHYS - Center for Biomembrane Physics
Physics Department, University of Southern Denmark
Campusvej 55, DK-5230 Odense M, Denmark
Tel: +45 6550 3510
Fax: +45 6615 8760
Email: mjensen@memphys.sdu.dk
WWW: http://www.memphys.sdu.dk


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Synthetic Peptides as Models for Intrinsic Membrane Proteins

J. Antoinette Killian

Department Biochemistry of Membranes, Utrecht University

Membrane proteins play an essential role in many life processes. The structural and functional properties of these proteins are sensitive to interactions with lipids in the host membrane. The aim of the research presented here is to determine how and to what extent the lipid environment can influence membrane protein structure and organization by affecting properties of their transmembrane segments. By employing designed model peptides, that mimick transmembrane parts of proteins, and incorporating them into well-defined synthetic lipid bilayers of varying composition, we can analyze in detail the influence of lipids on structural and motional properties of transmembrane protein segments. In particular we focus on the importance of hydrophobic matching and the role of anchoring interactions with the lipid/water interface of amino acids that flank the hydrophobic transmembrane segments. The systems are studied by a range of biophysical methods, including solid state 2H and 31P NMR, infra-red spectroscopy, fluorescence and mass spectrometry. Results of these studies will be discussed.

References:
J.A. Killian, "Synthetic peptides as models for intrinsic membrane proteins" (minireview), FEBS Letters 555 134-138 (2003).
E. van den Brink-van der Laan, V. Chupin, J. A. Killian, and B. de Kruijff, "Stability of KcsA tetramer depends on membrane lateral pressure", Biochemistry 43 4240-4250 (2004).
E. van den Brink-van der Laan, V. Chupin, J. A. Killian, and B. de Kruijff, "Small alcohols destabilize the KcsA tetramer via their effect on the membrane lateral pressure", Biochemistry 43 5937-5942 (2004).
E. Strandberg, S. Özdirekcan, D.T.S. Rijkers, P. van der Wel, R.E. Koeppe II, R.M.J. Liskamp, and J.A. Killian, "Tilt angles of transmembrane model peptides in oriented and non-oriented lipid bilayers as determined by 2H NMR", Biophysical J. 86 3709-3721 (2004).
S. Özdirekcan, D.T.S. Rijkers, and J.A. Killian, "Influence of flanking residues on tilt and rotation angles of transmembrane peptides in lipid bilayers. A solid state 2H NMR study", Biochemistry 44 1004-1012 (2005).


J. Antoinette Killian
Professor
Department of Biochemistry of Membranes
Institute of Biomembranes / Bijvoetcenter, Utrecht University
Padualaan 8, 3584 CH Utrecht, The Netherlands
Tel: +31 30 253-3442
Fax: +31 30 253-3969
Email: j.a.killian@chem.uu.nl
WWW: http://cble.chem.uu.nl/biomem/


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Killing Cells: From Endostatin to Cytochrome c to Aβ Peptides to Antimicrobial Peptides

Paavo K. J. Kinnunen

Helsinki Biophysics & Biomembrane Group, Biomedicum, University of Helsinki, and
MEMPHYS - Center for Biomembrane Physics, University of Southern Denmark

Because of their central roles in maintaining normal functions of all eukaryotic organisms the molecular mechanisms of apoptosis and antimicrobial peptide (AMP) action have been intensively studied. While these two processes are seemingly distant we have recently demonstrated an unprecedented connection. More specifically, several proteins, which trigger apoptosis (e.g. histone H1, cytochrome c, endostatin and glyceraldehyde-3-phosphate- dehydrogenase) all form Congo red staining fibers in the presence of membranes containing acidic phospholipids. Interestingly, this property is shared by all AMPs studied by us so far. These fibers also incorporate phospholipids and thus represent supramolecular lipid-protein assemblies. The light green birefringence upon Congo red staining is a characteristic feature of amyloid. The latter is an insoluble protein aggregate with extensive intermolecular β-sheet structure and is associated with several major disorders, such as Alzheimer’s disease and type 2 diabetes in which cell death is seen in the affected tissues. Studies on amyloid formation have revealed, that their formation in vitro requires acidic pH and lowered medium dielectricity. Our data suggest that in vivo these conditions are met on the surfaces of membranes containing acidic lipids. Moreover, the highly anisotropic nature of these surfaces should facilitate the aggregate formation by orienting the proteins or peptides. Importantly, ‘mature’ amyloid is not toxic to cells and there is now consensus that it is the earlier states in the process, profibrils, which are the actual cytotoxic forms. It is likely that triggering by acidic phospholipids of the formation of these profibrils cause membranes to loose their barrier function, most likely concomitant with severe derangement of the membrane organization.

To conclude, our findings provide a somewhat unexpected link between four major areas of research viz.
(i) mechanisms of triggering apoptosis, (ii) mechanisms of cytotoxicity of amyloid forming proteins and peptides, (iii) mechanisms triggering amyloid formation, and (iv) mechanisms of action antimicrobial peptides. These findings also provide simple explanations for several clinical findings, such as regression of cancer seen in patients with bacterial infection, and the accumulation of amyloid surrounding tumors. Further elucidation of the molecular level details of lipid-induced formation of cytoxic protein fibers should enable to develop novel means to combat cancer as well as the emerging strains of microbes resistant to conventional antibiotics.


Paavo K. J. Kinnunen
Professor
Helsinki Biophysics & Biomembrane Group
Department of Medical Chemistry, Institute of Biomedicine
POB 63, FIN-00014 University of Helsinki, Finland
Tel: +358 9 19125-400
Fax: +358 9 19125-444
Email: paavo.kinnunen@helsinki.fi
WWW: http://www.biophysics.helsinki.fi


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Striated Domains. An Intriguing Model System for Biomembranes

Ben de Kruijff

Department Biochemistry of Membranes, Institute of Biomembranes / Bijvoetcenter, Utrecht University

Transmembrane peptides with uncharged flanking residues incorporated in DPPC bilayers under gel state conditions assembly together with lipid into striated domains in which the peptides are organized in regularly spaced rows [1,2]. The molecular architecture of these striated domains was recently established [3]. The peptides are largely oriented in an antiparallel manner such that the peptides directly interact with each other and with DPPC that has melted by the presence of the peptides. We used these striated domains, made of thio group containing peptides and gold-coated AFM tips, for determining by force spectroscopy the strength of integration of peptides into the bilayer [4]. We established that transmembrane peptides are extremely stably anchored into the bilayer. The interfacial region was found to be the barrier that resists mechanical removal of these peptides from the bilayer.

References:
[1] H. A. Rinia, R. A. Kik, R.A. Demel, M. M. E. Snel, J. A. Killian, J. P. J. M. Van der Eerden, and B de Kruijff, "Visualization of highly ordered striated domains induced by transmembrane peptides in supported phosphatidylcholine bilayers", Biochemistry 39 (2000) 5852-5858.
[2] H. A. Rinia, J.-W. P. Boots, D. T. S. Rijkers, R. A. Kik, M. M. E. Snel, R. A. Demel, J. A. Killian, J. P. J. M. van der Eerden, and B. de Kruijff, "Domain formation in phosphatidylcholine bilayers containing transmembrane peptides: specific effects of flanking residues", Biochemistry 41 (2002) 2814-2824.
[3] E. Sparr, D. N. Ganchev, M. M. E. Snel, A. N. J. A. Ridder, L. M. J. Kroon-Batenburg, V. Chupin, D. T. N. Rijkers, J. A. Killian, and B. de Kruijff, "Molecular organization in striated domains induced by transmembrane a-helical peptides in dipalmitoyl phosphatidylcholine bilayers", Biochemistry 44 (2005) 2-10.
[4] D. N. Ganchev, D. T. S. Rijkers, M. M. E. Snel, J. A. Killian, and B. de Kruijff, "Strength of integration of transmembrane a-helical peptides in lipid bilayers as determined by Atomic Force Spectroscopy", Biochemistry 43 (2004) 14987-14993.


Ben de Kruijff
Professor
Department Biochemistry of Membranes
Institute of Biomembranes / Bijvoetcenter, Utrecht University
Padualaan 8, 3584 CH Utrecht, The Netherlands
Tel: +31 30 253-1607
Fax: +31 30 253-3969
Email: mailto:b.dekruijff@chem.uu.nl
WWW: http://cble.chem.uu.nl/biomem/


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Simulating Phase Transformations of Lipid Membranes

Siewert-Jan Marrink

University of Groningen, The Netherlands

The phase behaviour of lipid systems is very rich and complex. The cell uses the ability of lipid membranes to undergo phase separation or to adopt non-lamellar geometries in a number of fundamental biophysical processes such as raft-formation, vesicle fusion, or membrane poration and lysis. Over the past few years it has become feasible to study a variety of such phase transformation processes using molecular dynamics simulation techniques. I will give some recent examples, using coarse grained models as well as atomistic force fields.


Dr. Jan-Siewert Marrink
Dept. of Biophysical Chemistry, University of Groningen
Nijenborgh 4, NL-9747 AG Groningen, The Netherlands
Tel: +31 50 363-4339
Fax: +31 50 363-4800
Email: S.J.Marrink@rug.nl
WWW: http://md.chem.rug.nl/~marrink/science.html


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Bio-inspired Computer Simulations of Self-Assembling Amphiphilic Systems

David Michel, Doug Cleaver, and Chris Care

Sheffield Hallam University, Sheffield, UK

This study is aimed at developing a novel coarse-grained model for a lipid molecule, with which free self-assembly of biological membranes can be achieved at moderate computing cost. The computer model is based on (rod-like) Gay-Berne and (spherical) Lennard-Jones mixtures considering the rod particles as single site models for the lipid molecules and the spheres as solvent molecules.

By giving the rod-sphere interaction a dipolar symmetry, the hydrophobic effect, believed to be the main driver of amphiphilic self-assembly, is incorporated. Results obtained thus far indicate that free self-assembly of micellar, bilayer and inverse micelle arrangements can be readily achieved. From this, the effect of the molecular interactions parameters governing the self-assembly process is studied. Modifying the amphiphile characteristics such as the hydrophobic strength and the hydrophilic-to-lipophilic balance, provides insight into the relation between molecular interactions and the self-assembling mesoscopic structures to which they lead.

From these preliminary simulations, lipid mixtures are also studied for the micellar and the bilayer phases as well as ternary oil/water/surfactant systems, in order to link the new phase behaviour with the molecular composition of the mixture.


David Michel
Modelling Research Centre, Materials Modelling Group
Materials & Engineering Research Institute
Sheffield Hallam University, City Campus
Sheffield S1 1WB, United Kingdom
Tel: +44 114 225-3068
Fax: +44 114 225-3501
Email: dmichel@hera.shu.ac.uk
WWW: http://www.shu.ac.uk/research/meri/model/david_m/david_m.htm


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Computer Simulations of Model Membranes with Drug Penetration Enhancer Molecules

Rebecca Notman, Jamshed Anwar, Massimo Noro and Brendan O'Malley

Molecular Biophysics, Division of Pharmaceutical Science, King's College London, and Statistics and Modelling, Physical Sciences Group, Unilever R&D Port Sunlight, UK

Chemical penetration enhancers are molecules that increase the permeability of the skin lipid membranes to drug molecules. We are currently investigating model membranes in the presence and absence of drug penetration enhancers using molecular dynamics simulations. A coarse-grained model has been employed that enables these systems to be investigated on larger time and length scales. We have introduced model penetration enhancers into the system in the form of small amphiphilic dimers and are calculating the effects of these molecules on the structural and physical properties of the membranes. Using chemical potential calculations we aim to characterise the barrier to penetration of molecules in the presence and absence of penetration enhancers. We have also observed that these amphiphiles introduce curvature into the bilayers. Membrane curvature is important in a range of cell processes including endocytosis, membrane fusion and cell signalling via membrane-bound proteins. The ability of these molecules to modulate curvature suggests a mechanism of action by which small amphiphilic molecules can modulate such processes.


Rebecca Notman
Department of Pharmacy, King's College London
Franklin Wilkins Building, Stamford Street
London SE1 9NN, United Kingdom
WWW: http://www.kcl.ac.uk/depsta/healifsci/lifsci/molisc/srm/testnewmbp/


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Coarse-Grained Simulations of Transmembrane Proteins in Membrane Fusion

Julian C. Shillcock and Reinhard Lipowsky

Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

Computer simulations have long been used to study molecular scale processes, particularly the kinetics and equilibrium structure of proteins and small molecular assemblies. We use a mesoscopic simulation technique, Dissipative Particle Dynamics (DPD), to study the the fusion of a 28 nm diameter vesicle to a planar bilayer. Three distinct force protocols are used. In the first, fusion is driven solely by the initial tensions in the vesicle and planar membrane. The second protocol fuses the relaxed membranes by using an external force applied to an annulus of lipids to stretch the contact zone until it ruptures. In the last protocol, fusion is induced by applying a bending moment to barrel-shaped proteins embedded in the the membranes followed by a local stretching force as a model of the action of SNARE proteins. The effect of the transmembrane proteins on the membrane’s equilibrium structure is also investigated. Vesicle fusion requires that the natural protective properties of lipid membranes be disrupted in a controlled fashion for the benefit of a cell. DPD simulations allow us to probe putative fusion mechanisms in a systematic way on realistic length and time scales.


Dr. Julian C. Shillcock
Group Leader
Max Planck Institute of Colloids and Interfaces, Theory Divison
D-14424 Potsdam, Germany
Tel: +49 331 567-9618
Fax: +49 331 567-9612
Email: julian.shillcock@mpikg-golm.mpg.de
WWW: http://www.mpikg-golm.mpg.de/th/people/julian/


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Mesoscopic Simulations of Phase Transitions in Biological Membranes

Marieke Kranenburg¹, Maddalena Venturoli¹, Maria Sperotto², and Berend Smit^1,3

¹ University of Amsterdam, The Netherlands, ² Technical University of Denmark, Kgs. Lyngby, Denmark, and ³ CECAM, Lyon, France

In this contribution we demonstrate how one can use mesoscopic simulations to study the phase behavior of membranes. Starting from all-atom simulations we introduce a mapping procedure to arrive at a mesoscopic model of a water-lipid system. We demonstrate the effect of various coarse graining assumptions on the phase behaviour. Depending on the lipid structure and head group interactions we reproduce the experimental phase diagrams.

In addition, we investigate the effect of alcohol on the structure and phase behaviour of a membrane. We find that alcohol can induce an interdigitated structure in which the normal bilayer structure changes into monolayer in which the alcohol molecules screen the hydrophobic tails from the water phase. We compute the effect of the chain length of the alcohol on the phase behaviour of the membrane. At low concentrations of alcohol the membrane has domains of the interdigitated phase that are in coexistence with the normal membrane phase. We use our model to clarify some of the experimental questions related to the structure of the interdigitated phase and put forward a simple model that explains the alcohol chain length dependence of the stability of this interdigitated phase.


Berend Smit
Professor
Computational Chemistry and Physics
Van 't Hoff Institute for Molecular Sciences (HIMS)
University of Amsterdam
Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands
Tel: +31 20 525-5265
Fax: +31 20 525-5604
Email: b.smit@science.uva.nl
WWW: http://molsim.chem.uva.nl/

and

Director
CECAM (Centre Européen de Calcul Atomique Moléculaire)
Ecole Normale Supérieure
Allée d’Italie, 69364 Lyon Cedex 7, France
Tel: +33 4 7272-8638
Fax: +33 4 7272-8636
Email: bsmit@cecam.fr
WWW: http://www.cecam.fr


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To Tilt or not to Tilt? - A Dilemma for Proteins Embedded in a Mismatched Lipid Bilayer

Maddalena Venturoli^1,2, Berend Smit¹, and Maria Maddalena Sperotto³

¹ University of Amsterdam, The Netherlands, ² University College, London, United Kingdom, and
³ Technical University of Denmark, Kgs. Lyngby, Denmark

The hydrophobic matching between the lipid bilayer hydrophobic thickness and the hydrophobic length of integral membrane proteins has been proposed as one of the main physical mechanisms that regulate the lipid-protein interaction in biomembranes. It is now recognized that hydrophobic matching is used in cell membrane organization.

Biological membranes have at their disposal a number of ways to compensate for hydrophobic mismatch. To adjust to a mismatched membrane, a membrane protein may cause a change of the lipid bilayer hydrophobic thickness in its vicinity. Another way that a protein may have to adapt to a too thin lipid bilayer is to tilt. In addition to the protein as a whole, the individual helices of which a protein might be composed may also experience a tilt; there is indeed experimental evidence that the latter phenomenon may occur in channel proteins, and that a change of the tilt-angle of the individual helices could be the cause of a changed in protein activity.

We present a mesoscopic model for phospholipid bilayers (DMPC) with embedded proteins, which we have studied with the help of the Dissipative Particle Dynamics simulation technique (Venturoli et al., Biophys. J. 88, 2005). We consider proteins of different hydrophobic length, as well as different sizes. We make predictions about the physical hydrophobic-mismatch condition that induces a protein to tilt in the lipid bilayer, rather than (or, at the same time) inducing a bilayer deformation in the vicinity of the protein. We quantify the perturbation in terms of a coherence length, ξ_P, of the lipid bilayer hydrophobic thickness profile around the protein. The dependence on temperature of ξ_P, and the protein tilt-angle, were studied above the main-transition temperature of the pure system, i.e., in the fluid phase. We found that ξ_P depends on mismatch, i.e., the higher the mismatch is, the longer ξ_P becomes, at least for positive values of mismatch; a dependence on the protein size appears as well. In the case of large model proteins experiencing extreme mismatch conditions, in the region next to the so-called lipid annulus, there appears an undershooting (or overshooting) region where the bilayer hydrophobic thickness is locally lower (or higher) than in the unperturbed bilayer, depending on whether the protein hydrophobic length is longer (or shorter) than the pure lipid bilayer hydrophobic thickness.

Proteins may tilt when embedded in a too-thin bilayer. Our simulation data suggest that, when the embedded protein has a small size, the main mechanism to compensate for a large hydrophobic mismatch is the tilt; whereas large proteins react to negative mismatch by causing an increase of the hydrophobic thickness of the nearby bilayer. Furthermore, for the case of small, peptidelike proteins, we found the same type of functional dependence of the protein tilt-angle on mismatch, as was recently detected by fluorescence spectroscopy measurements.


Dr. Maria Maddalena Sperotto
Guest scientist
Biochemistry and Nutrition Group, Biocentrum-DTU
The Technical University of Denmark
Building 224, DK-2800 Kgs. Lyngby, Denmark
Email: maria@cbs.dtu.dk



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Exploring the Effect of Anaesthetic Gases on Biomembranes

Lorna M. Stimson, Ilpo Vattulainen, Tomek Rog, and Mikko Karttunen

Helsinki University of Technology, Finland

A general anaesthetic is a substance which is able to induce a reversible loss of consciousness associated with a lack of response to painful stimuli. A great range of molecules have been found to be active as general anaesthetics. This range includes molecular species from simple mono-atomic xenon gas to much larger and more complex structures. Many hypotheses have been suggested to explain the effect of such anaesthetic gases on the body. In this work, we aim to investigate the hypothesis that anaesthetics cause disruptions in the cellular membrane which are responsible for the anaesthetic effect. [Eckenhoff, 2001]

It has been shown that changes in the lipid bilayer cause alterations in the structure and function on embedded proteins. Of particular interest are mechanosensitive channel proteins, for example MscL [Gullingsrud and Schulten, 2004], which in an open state, allow diffusion of small cations such as Na⁺, K⁺ and Ca²⁺. The effect of pressure on the gating of these proteins has recently been elaborated [Cantor, 2004]. The diffusion of these ions is fundamental for the control of the potential across the membrane and therefore to the conductance of nerve impulses. [Cantor, 1998]

The initial investigations centre on the effect of xenon gas on the model membranes. We have carried out molecular dynamics simulations of fully hydrated phospholipid bilayers consisting of 128 lipid molecules and between 3000 and 4000 water molecules. The different lipid compositions that have been studied are pure dipalmitoyl-
phosphatidylcholine, pure dioleoylphosphatidylcholine and combinations of these with cholesterol. For each system xenon is then introduced into the solvent phase. We observe the rapid penetration of the bilayer by the gas and find that the xenon preferentially occupies the tail region of the membrane. There is an increase in the area per lipid associated with the accommodation of the anaesthetic and for highly saturated systems we observe a slight increase in the order of the hydrocarbon chains.

Here, we present some initial results of these investigations and attempt to compare the findings with simulations of pure membranes in the absence of anaesthetic agents and with previous simulations in which the effect of alcohols on membranes were investigated [Patra et al., 2004].

References:
[Cantor, 1998] Cantor, R., 1998. "The lateral pressure profile in membranes: a physical mechanism of general anesthesia". Toxicol. Lett. 101:451-458.
[Cantor, 2004] Cantor, R., 2004. "Modulation of membrane protein activity: bilayer composition and the lateral pressure profile". Biophys. J. 86(1):330A-330A.
[Eckenhoff, 2001] Eckenhoff, R. G., 2001. "Promiscous ligands and attractive cavities". Mol. Interventions 1(5):258.
[Gullingsrud and Schulten, 2004] Gullingsrud, J. and K. Schulten, 2004. "Lipid bilayer pressure profiles and mechanosensitive channel gating". Biophys. J. 86(6):3496-3509.
[Patra et al., 2004] Patra, M., E. Salonen, E. Terama, R. Faller, B. W. Lee, J. Holopainen, and M. Karttunen, 2004. "Under the influence of alcohol: The effect of ethanol and methanol on lipid bilayers". submitted.


Dr. Lorna M. Stimson
Biophysics and Statistical Mechanics Group
Laboratory of Computational Engineering
PO Box 9203, FIN-02015 Helsinki University of Technology, Finland
Tel: +358 945 15732 / +358 445 676267
Fax: +358 945 14830
Email: lorna@lce.hut.fi
WWW: http://www.softsimu.org


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Membrane Electroporation

Mounir Tarek

University Henri-Poincaré, Nancy, France

The application of high electric fields to cells or tissues permeabilizes the cell membrane and is thought to produce aqueous-filled pores in the lipid bilayer. This process is referred to as electroporation. It finds today numerous applications since, under certain conditions, it is reversible, and hence permits efficient transmembrane transfer of small molecules. We present here recent results from all atomistic MD simulations of the action of an external electric field on membranes (lipid bilayers, bilayers with transmembrane channels, DNA translocation, ...), aimed at investigating the electroporation phenomena at the molecular level. We discuss the membrane stability and its intrinsic properties under the external stress.


Dr. Mounir Tarek
Equipe de dynamique des assemblages membranaires
Unité Mixte de Recherches CNRS UHP 7565
Université Henri-Poincaré, Nancy I
BP 239, 54506 Vandoeuvre-lès-Nancy, cedex France
Tel: +33 3 83 68 40 95
Fax: +33 3 83 68 43 87
Email: Mounir.Tarek@edam.uhp-nancy.fr
WWW: http://www.edam.uhp-nancy.fr/


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Making Sense of Systems Biology: Uncovering Natures Valves and Sensors

Richard Templer¹, Oscar Ces¹, Stephen Alley¹, Mauricio Barahona¹, Paula Booth², Suzy Jackowski³,
and George Attard⁴


¹Imperial College London, ²Bristol University, ³University of Tennessee, and ⁴Southampton University

In the spirit of this workshop I will discuss some of our work on the interaction of proteins with lipid membranes in the context of modelling the behaviour of an entire cell system. Work on the membrane binding and activity of CTP:phosphocholine cytidylyl transferase and of the refolding of bacteriorhodopsin give compelling evidence for the idea that stored curvature elasticity affect both the partitioning and dynamics of membrane proteins. It appears that stored curvature elastic energy is biologically significant and that cells may indeed actively control lipid composition around a preferred value. I will present some modelling that has been done on lipid homeostasis and will discuss to what level of detail modelling will need to be taken in order to unravel the system biology of cellular processes.


Richard Templer
Professor
The Department of Chemistry
Imperial College London
Exhibition Road, London SW7 2AZ, United Kingdom
Tel: +44 207 594-5855
Fax: +44 207 594-5801
Email: r.templer@ic.ac.uk
WWW: http://www.ch.ic.ac.uk/liquid_crystal/templer%20web%20page.htm


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Lipid - Small Molecule Interactions in Lipid Bilayers

D. Peter Tieleman

Department of Biological Sciences, University of Calgary, Calgary, AB, Canada

A key function of biological membranes is to provide mechanisms for controlled transport of ions, nutrients, metabolites, peptides and proteins across the membrane. We are using computer simulations to study several processes involved in transport. The simplest case is passive transport of small molecules that partition to the membrane interior and are able to diffuse across the membrane. In model membranes, the distribution of small molecules can be accurately calculated and we are making progress toward understanding the factors that determine the partitioning behavior in the inhomogeneous lipid environment. Based on simulations we can determine the distribution of pyrene, halothane, hexane, and lipids in a bilayer and investigate their effect on lipid properties. These distributions are of interest themselves but also provide a way to improve interaction parameters with lipids and to investigate subtle modifications to bilayer structure that may affect proteins.


D. Peter Tieleman
Associate Professor, AHFMR Scholar
Department of Biological Sciences, University of Calgary
2500 University Dr., NW Calgary, AB T2N1N4 Canada
Tel: +1 403 220-2966
Fax: +1 403 289-9311
Email: tieleman@ucalgary.ca
WWW: http://moose.bio.ucalgary.ca


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From Atomic to Coarse-Grained Descriptions of Membranes

Ilpo Vattulainen

Laboratory of Physics and Helsinki Institute of Physics, Helsinki University of Technology

We discuss how the structure and dynamics of lipid bilayers can be modeled by a variety of different methods, ranging from atomistic simulation techniques with full-atom descriptions to coarse-grained models in which a great number of microscopic degrees of freedom has been replaced with stochastic noise. As an example of coarse graining, we apply the inverse Monte Carlo (IMC) method [1,2] to atomic-level studies of multicomponent lipid membranes including cholesterol [3]. The IMC allows systematic coarse graining in the sense that the effective interactions used in the coarse-grained membrane system are systematically derived from detailed atomic-scale MD simulations, which guarantees that the radial distribution properties of the coarse-grained model are consistent with those given by the corresponding atomistic system. The coarse-grained model allows us to explore large-scale properties of membranes, in this case the formation of cholesterol-rich and cholesterol-poor domains at intermediate cholesterol concentrations [2], in agreement with experiments. This approach provides a speed-up of approximately eight orders of magnitude compared to atomistic simulations.

References:
[1] A. Lyubartsev and A. Laaksonen, Phys. Rev. E 52, 3730 (1995).
[2] T. Murtola, E. Falck, M. Patra, M. Karttunen, and I. Vattulainen, J. Chem. Phys. 121, 9156 (2004).
[3] E. Falck, M. Patra, M. Karttunen, M.T. Hyvonen, and I. Vattulainen, Biophys. J. 87, 1076 (2004).


Dr. Ilpo Vattulainen
Researcher
Group Leader, Biological Physics & Soft Matter Group
Laboratory of Physics and Helsinki Institute of Physics
Helsinki University of Technology
PO Box 1100, FI-02015 HUT, Finland
Tel: +358 9 451-5805
Fax: +358 9 451-3116
Email: ilpo.vattulainen@hut.fi
WWW: http://www.fyslab.hut.fi/bio/


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Last update: 31 May 2005 / Gerhard Besold