The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 897–908 | Cite as

Thermal Fluctuations in Amphipol A8-35 Particles: A Neutron Scattering and Molecular Dynamics Study

  • Moeava Tehei
  • Jason D. Perlmutter
  • Fabrice Giusti
  • Jonathan N. Sachs
  • Giuseppe ZaccaiEmail author
  • Jean-Luc PopotEmail author


Amphipols are a class of polymeric surfactants that can stabilize membrane proteins in aqueous solutions as compared to detergents. A8-35, the best-characterized amphipol to date, is composed of a polyacrylate backbone with ~35 % of the carboxylates free, ~25 % grafted with octyl side-chains, and ~40 % with isopropyl ones. In aqueous solutions, A8-35 self-organizes into globular particles with a molecular mass of ~40 kDa. The thermal dynamics of A8-35 particles was measured by neutron scattering in the 10-picosecond, 18-picosecond, and 1-nanosecond time-scales on natural abundance and deuterium-labeled molecules, which permitted to separate backbone and side-chain motions. A parallel analysis was performed on molecular dynamics trajectories (Perlmutter et al., Langmuir 27:10523–10537, 2011). Experimental results and simulations converge, from their respective time-scales, to show that A8-35 particles feature a more fluid hydrophobic core, predominantly containing the octyl chains, and a more rigid solvent-exposed surface, made up predominantly of the hydrophilic polymer backbone. The fluidity of the core is comparable to that of the lipid environment around proteins in the center of biological membranes, as also measured by neutron scattering. The biological activity of proteins depends sensitively on molecular dynamics, which itself is strongly dependent on the immediate macromolecular environment. In this context, the characterization of A8-35 particle dynamics constitutes a step toward understanding the effect of amphipols on membrane protein stability and function.


Membrane proteins Surfactants Polymers Molecular dynamics QENS 



An anionic amphipol of average molecular mass ~4.3 kDa, containing ~35 % free carboxylates, ~25 % octyl side-chains, and ~40 % isopropyl ones




Critical association concentration


A8-35 with per-deuterated side-chains


Elastic incoherent neutron scattering


Natural abundance A8-35


Inelastic neutron scattering


Molecular dynamics


Number-averaged molecular mass

mQ water

Water purified on a A10 Advantage Millipore System


Mean square displacement

OmpA, OmpX

Respectively outer membrane proteins A and X from Escherichia coli


Quasi-elastic neutron scattering


Stokes radius


Small-angle neutron scattering


Size exclusion chromatography



Particular thanks are due to Michael Marek Koza and Bernhard Frick, ILL local contacts on IN6 and IN16, respectively. This work was supported by the French Centre National de la Recherche Scientifique (CNRS), by Université Paris–7 Denis Diderot, and by Grant “DYNAMO”, ANR-11-LABX-0011-01 from the French “Initiative d’Excellence” Program. ‘Computational resources were provided by the Minnesota Supercomputing Institute (MSI).


  1. Althoff T, Mills DJ, Popot J-L, Kühlbrandt W (2011) Assembly of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1. EMBO J 30:4652–4664CrossRefGoogle Scholar
  2. Bée M (1988) Quasielastic neutron scattering: principles and applications in solid state chemistry. Biology and Materials Science Adam Hilger, PhiladelphiaGoogle Scholar
  3. Bowie JU (2001) Stabilizing membrane proteins. Curr Opin Struct Biol 11:397–402CrossRefGoogle Scholar
  4. Champeil P, Menguy T, Tribet C, Popot J-L, le Maire M (2000) Interaction of amphipols with the sarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 275:18623–18637CrossRefGoogle Scholar
  5. Charvolin D, Picard M, Huang L-S, Berry EA, Popot J-L (2014) Solution behavior and crystallization of cytochrome bc 1 in the presence of amphipols. J Membr Biol. doi: 10.1007/s00232-014-9694-4 CrossRefGoogle Scholar
  6. Etzkorn M, Zoonens M, Catoire LJ, Popot J-L, Hiller S (2014) How amphipols embed membrane proteins: global solvent accessibility and interaction with a flexible protein terminus. J Membr Biol. doi: 10.1007/s00232-014-9657-9 CrossRefGoogle Scholar
  7. Feinstein HE, Tifrea D, Sun G, Popot J-L, de la Maza LM, Cocco MJ (2014) Long-term stability of a vaccine formulated with the amphipol-trapped major outer membrane protein from Chlamydia trachomatis. J Membr Biol. doi: 10.1007/s00232-014-9693-5 CrossRefGoogle Scholar
  8. Ferrand M, Dianoux AJ, Petry W, Zaccai G (1993) Thermal motions and function of bacte-rio-rhod-opsin in purple membranes: effects of temperature and hydration studied by neutron scattering. Proc Natl Acad Sci USA 90:9668–9672CrossRefGoogle Scholar
  9. Fitter J, Lechner RE, Büldt G, Dencher NA (1996) Internal molecular motions of bacteriorhodopsin: hydration-induced flexibility studied by quasielastic incoherent neutron scattering using oriented purple membranes. Proc Natl Acad Sci USA 193:7600–7605CrossRefGoogle Scholar
  10. Fitter J, Lechner RE, Dencher NA (1997) Picosecond molecular motions in bacteriorhodopsin from neutron scattering. Biophys J 73:2126–2137CrossRefGoogle Scholar
  11. Frölich A, Gabel F, Jasnin M, Lehnert U, Oesterhelt D, Stadler AM, Tehei M, Weik M, Wood K, Zaccai G (2009) From shell to cell: neutron scattering studies of biological water dynamics and coupling to activity. Faraday Discuss 41:117–130 discussion 175-207CrossRefGoogle Scholar
  12. Garavito RM, Ferguson-Miller S (2001) Detergents as tools in membrane biochemistry. J Biol Chem 276:32403–32406CrossRefGoogle Scholar
  13. Giusti F, Popot J-L, Tribet C (2012) Well-defined critical association concentration and rapid adsorption at the air/water interface of a short amphiphilic polymer, amphipol A8-35: a study by Förster resonance energy transfer and dynamic surface tension measurements. Langmuir 28:10372–10380CrossRefGoogle Scholar
  14. Giusti F, Rieger J, Catoire L, Qian S, Calabrese AN, Watkinson TG, Casiraghi M, Radford SE, Ashcroft AE, Popot J-L (2014) Synthesis, characterization and applications of a per-deuterated amphipol. J Membr Biol. doi: 10.1007/s00232-014-9656-x CrossRefGoogle Scholar
  15. Gohon Y, Popot J-L (2003) Membrane protein-surfactant complexes. Curr Opin Colloid Interface Sci 8:15–22CrossRefGoogle Scholar
  16. Gohon Y, Pavlov G, Timmins P, Tribet C, Popot J-L, Ebel C (2004) Partial specific volume and solvent interactions of amphipol A8-35. Anal Biochem 334:318–334CrossRefGoogle Scholar
  17. Gohon Y, Giusti F, Prata C, Charvolin D, Timmins P, Ebel C, Tribet C, Popot J-L (2006) Well-defined nanoparticles formed by hydrophobic assembly of a short and polydisperse random terpolymer, amphipol A8-35. Langmuir 22:1281–1290CrossRefGoogle Scholar
  18. Gohon Y, Dahmane T, Ruigrok R, Schuck P, Charvolin D, Rappaport F, Timmins P, Engelman DM, Tribet C, Popot J-L, Ebel C (2008) Bacteriorhodopsin/amphipol complexes: structural and functional properties. Biophys J. 94:3523–3537CrossRefGoogle Scholar
  19. Huynh KW, Cohen MR, Moiseenkova-Bell VY (2014) Application of amphipols for structu-re-functional analysis of TRP channels. J Membr Biol. doi: 10.1007/s00232-014-9684-6 CrossRefGoogle Scholar
  20. Jasnin M, van Eijck L, Koza MM, Peters J, Laguri C, Lortat-Jacob H, Zaccai G (2010) Dynamics of heparan sulfate explored by neutron scattering. Phys Chem Chem Phys 12:3360–3362CrossRefGoogle Scholar
  21. Jorgensen WL, Jenson C (1998) Temperature dependence of TIP3P, SPC, and TIP4P water from NPT Monte Carlo simulations: seeking a temperature of maximum density. J Comp Chem 19:1179–1186CrossRefGoogle Scholar
  22. Klauda JB, Kucerka N, Brooks BR, Pastor RW, Nagle JF (2006) Simulation-based methods for interpreting X-ray data from lipid bilayers. Biophys J 90:2796–2807CrossRefGoogle Scholar
  23. Kleinschmidt JH, Popot J-L (2014) Folding and stability of integral membrane proteins in amphipols. Arch Biochem Biophys (in press)Google Scholar
  24. König S, Sackmann E (1996) Molecular and collective dynamics of lipid bilayers. Curr Opin Colloid Interface Sci 1:78–82CrossRefGoogle Scholar
  25. Liao M, Cao E, Julius D, Cheng Y (2013) Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504:107–112CrossRefGoogle Scholar
  26. Liao M, Cao E, Julius D, Cheng Y (2014) Single particle electron cryo-microscopy of a mammalian ion channel. Curr Opin Struct Biol 27:1–7CrossRefGoogle Scholar
  27. MacKerell AD Jr, Bashford D, Bellott M, Dunbrack RL Jr, Evanseck JD, Field MJ, Fischer S, Gao J, Guo H, Ha S, Joseph-McCarthy D, Kuchnir L, Kuczera K, Lau FTK, Mattos C, Michnick S, Ngo T, Nguyen DT, Prodhom B, Reiher WR III, Roux B, Schlenkrich M, Smith JC, Stote R, Straub J, Watanabe M, Wiórkiewicz-Kuczera J, Yin D, Karplus M (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B 102:3586–3616CrossRefGoogle Scholar
  28. Marrink SJ, Risselada HJ, Yefimov S, Tieleman DP, de Vries AH (2007) The MARTINI force field: coarse grained model for biomolecular simulations. J Phys Chem B 111:7812–7824CrossRefGoogle Scholar
  29. Natali F, Castellano C, Pozzi D, Congiu-Castellano A (2005) Dynamic properties of an orient-ed lipid/DNA complex studied by neutron scattering. Biophys J 88:1081–1090CrossRefGoogle Scholar
  30. Perez J, Zanotti JM, Durand D (1999) Evolution of the internal dynamics of two globular proteins from dry powder to solution. Biophys J 77:454–469CrossRefGoogle Scholar
  31. Perlmutter JD, Drasler WJ, Xie W, Gao J, Popot J-L, Sachs JN (2011) All-atom and coarse-grained molecular dynamics simulations of a membrane protein stabilizing polymer. Langmuir 27:10523–10537CrossRefGoogle Scholar
  32. Perlmutter JD, Popot J-L, Sachs JN (2014) Molecular dynamics simulations of a membrane protein/amphipol complex. J Membr Biol. doi: 10.1007/s00232-014-9690-8 CrossRefGoogle Scholar
  33. Picard M, Dahmane T, Garrigos M, Gauron C, Giusti F, le Maire M, Popot J-L, Champeil P (2006) Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study. Biochemistry 45:1861–1869CrossRefGoogle Scholar
  34. Planchard N, Point E, Dahmane T, Giusti F, Renault M, Le Bon C, Durand G, Milon A, Guittet E, Zoonens M, Popot J-L, Catoire LJ (2014) The use of amphipols for solution NMR studies of membrane proteins: advantages and limitations as compared to other solubilizing media. J Membr Biol. doi: 10.1007/s00232-014-9654-z CrossRefGoogle Scholar
  35. Pocanschi C, Popot J-L, Kleinschmidt JH (2013) Folding and stability of outer membrane protein A (OmpA) from Escherichia coli in an amphipathic polymer, amphipol A8-35. Eur Biophys J 42:103–118CrossRefGoogle Scholar
  36. Polovinkin V, Balandin T, Volkov O, Round E, Borshchevskiy V, Utrobin P, von Stetten D, Royant A, Willbold D, Arzumanyan A, Popot J-L, Gordeliy V (2014) Nanoparticle surface enhanced Raman scattering of bacteriorhodopsin stabilized by amphipol A8-35. J Membr Biol. doi: 10.1007/s00232-014-9701-9 CrossRefGoogle Scholar
  37. Popot JL (2010) Amphipols, nanodiscs, and fluorinated surfactants: three non-conventional approaches to studying membrane proteins in aqueous solutions. Annu Rev Biochem 79:737–775CrossRefGoogle Scholar
  38. Popot J-L, Berry EA, Charvolin D, Creuzenet C, Ebel C, Engelman DM, Flötenmeyer M, Giusti F, Gohon Y, Hervé P, Hong Q, Lakey JH, Leonard K, Shuman HA, Timmins P, Warschawski DE, Zito F, Zoonens M, Pucci B, Tribet C (2003) Amphipols: polymeric surfactants for membrane biology research. Cell Mol Life Sci 60:1559–1574CrossRefGoogle Scholar
  39. Popot J-L, Althoff T, Bagnard D, Banères J-L, Bazzacco P, Billon-Denis E, Catoire LJ, Champeil P, Charvolin D, Cocco MJ, Crémel G, Dahmane T, de la Maza LM, Ebel C, Gabel F, Giusti F, Gohon Y, Goormaghtigh E, Guittet E, Kleinschmidt JH, Kühlbrandt W, Le Bon C, Martinez KL, Picard M, Pucci B, Rappaport F, Sachs JN, Tribet C, van Heijenoort C, Wien F, Zito F, Zoonens M (2011) Amphipols from A to Z. Annu Rev Biophys 40:379–408CrossRefGoogle Scholar
  40. Rogan PK, Zaccai G (1981) Hydration of purple membrane as a function of relative humidity. J Mol Biol 145:281–284CrossRefGoogle Scholar
  41. Rosenbusch JP (2001) Stability of membrane proteins: relevance for the selection of appropriate methods for high-resolution structure determinations. J Struct Biol 136:144–157CrossRefGoogle Scholar
  42. Stansfeld PJ, Jeffreys EE, Sansom MSP (2013) Multiscale simulations reveal conserved patterns of lipid interactions with aquaporins. Structure 21:810–819CrossRefGoogle Scholar
  43. Tehei M, Zaccai G (2005) Adaptation to extreme environments: macromolecular dynamics in complex systems. Biochim Biophys Acta 1724:404–410CrossRefGoogle Scholar
  44. Tehei M, Madern D, Pfister C, Zaccai G (2001) Fast dynamics of halophilic malate dehydrogenase and BSA measured by neutron scattering under various solvent conditions influencing protein stability. Proc Natl Acad Sci USA 98:14356–14361CrossRefGoogle Scholar
  45. Tehei M, Madern D, Franzetti B, Zaccai G (2005) Neutron scattering reveals the dynamic basis of protein adaptation to extreme temperature. J Biol Chem 280:40974–40979CrossRefGoogle Scholar
  46. Trapp M, Gutberlet T, Juranyi F, Unruh T, Demé B, Tehei M, Peters J (2010) Hydration dependent studies of highly aligned multilayer lipid membranes by neutron scattering. J Chem Phys 133:164505CrossRefGoogle Scholar
  47. Tribet C, Audebert R, Popot J-L (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci USA 93:15047–15050CrossRefGoogle Scholar
  48. Váró G, Lanyi JK (1991) Distortions in the photocycle of bacteriorhodopsin at moderate dehydration. Biophys J 59:313–322CrossRefGoogle Scholar
  49. Venkatesan M, Hirtzel CS, Rajagopalan R (1985) The effect of colloidal forces on the self-diffusion coefficients in strongly interacting dispersions. J Chem Phys 82:5685–5695CrossRefGoogle Scholar
  50. Weik M, Patzelt H, Zaccai G, Oesterhelt D (1998) Localization of glycolipids in membranes by in vivo labeling and neutron diffraction. Mol Cell 1:411–419CrossRefGoogle Scholar
  51. Zaccai G (1987) Structure and hydration of purple membranes in different conditions. J Mol Biol 194:569–572CrossRefGoogle Scholar
  52. Zaccai G (2011) Neutron scattering perspectives for protein dynamics. J Non-Cryst Solids 357:615–621CrossRefGoogle Scholar
  53. Zaccai G (2013) The ecology of protein dynamics. Curr Phys Chem 3:9–16CrossRefGoogle Scholar
  54. Zoonens M, Popot J-L (2014) Amphipols for each season. J Membr Biol. doi: 10.1007/s00232-014-9666-8 CrossRefGoogle Scholar
  55. Zoonens M, Catoire LJ, Giusti F, Popot J-L (2005) NMR study of a membrane protein in detergent-free aqueous solution. Proc Natl Acad Sci USA 102:8893–8898CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Moeava Tehei
    • 1
  • Jason D. Perlmutter
    • 2
  • Fabrice Giusti
    • 3
  • Jonathan N. Sachs
    • 4
  • Giuseppe Zaccai
    • 5
    • 6
    Email author
  • Jean-Luc Popot
    • 3
    Email author
  1. 1.Centre for Medical Radiation Physics and Centre for Medical and Molecular Bioscience, University of WollongongWollongongAustralia
  2. 2.Department of PhysicsBrandeis UniversityWalthamUSA
  3. 3.UMR 7099, Centre National de la Recherche Scientifique/Université Paris-7 Institut de Biologie Physico-Chimique (FRC 550)ParisFrance
  4. 4.Department of Biomedical EngineeringUniversity of MinnesotaMinneapolisUSA
  5. 5.Institut de Biologie Structurale, CEA/CNRS/UJF UMR5075GrenobleFrance
  6. 6.Institut Laue LangevinGrenobleFrance

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