The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 827–842 | Cite as

The Use of Amphipols for Solution NMR Studies of Membrane Proteins: Advantages and Constraints as Compared to Other Solubilizing Media

  • Noelya Planchard
  • Élodie Point
  • Tassadite Dahmane
  • Fabrice Giusti
  • Marie Renault
  • Christel Le Bon
  • Grégory Durand
  • Alain Milon
  • Éric Guittet
  • Manuela Zoonens
  • Jean-Luc Popot
  • Laurent J. CatoireEmail author


Solution-state nuclear magnetic resonance studies of membrane proteins are facilitated by the increased stability that trapping with amphipols confers to most of them as compared to detergent solutions. They have yielded information on the state of folding of the proteins, their areas of contact with the polymer, their dynamics, water accessibility, and the structure of protein-bound ligands. They benefit from the diversification of amphipol chemical structures and the availability of deuterated amphipols. The advantages and constraints of working with amphipols are discussed and compared to those associated with other non-conventional environments, such as bicelles and nanodiscs.


Membrane proteins Solution NMR Amphipols 





Polyacrylate-based amphipol A8-35


Low-affinity leukotriene receptor






Cell-free expression


Cross-correlated relaxation-enhanced polarization transfer




A8-35 with perdeuterated octyl and isopropyl chains and a hydrogenated polyacrylate backbone






G protein-coupled receptor


12S-Hydroxyheptadeca-5Z,8E,10E-trienoic acid


Hetero-nuclear Overhauser spectroscopy


12S-Hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid


Hetero-single quantum correlation experiment


Outer membrane protein A from Klebsiella pneumoniae


Leukotriene B4


Molecular dynamics


Membrane protein


Molecular weight


Non-ionic amphipol


Maltose neopentyl glycol


Nuclear magnetic resonance


Nuclear Overhauser effect




Outer membrane protein A from Escherichia coli


Outer membrane protein X from Escherichia coli


Perdeuterated A8-35


Phosphocholine amphipol


Stokes radius


Sulfonated amphipol


Sodium dodecylsulfate


Solid-state NMR


Size exclusion chromatography


Transmembrane domain of OmpA


Transverse relaxation optimized spectroscopy



We are extremely grateful to Sophie Walmé and Marie-Noëlle Rager (ChimieParisTech, Paris), and Carine van Heijenoort (ICSN, Gif/Yvette) for assistance with NMR experiments. We express our gratitude to B. Pucci (Université d’Avignon et des Pays de Vaucluse) for his long-term involvement in the development of non-ionic amphipols. This work was supported by the Centre National de la Recherche Scientifique (CNRS), Paris-7 University (Sorbonne Paris Cité), the “Initiative d’Excellence” program from the French State (Grant “DYNAMO,” ANR-11-LABX-0011-01), Human Frontier Science Program Organization Grant RG00223/2000-M, from the Agence Nationale pour la Recherche ANR-07-BLAN-0092 “Amphipol-assisted folding of GPCRs,” and E.U. Specific Targeted Research Project “Innovative tools for membrane protein structural proteomics.” LJC is a recipient of Projects Exploratoires/Premier Soutien (PEPS, LeukomotiVe project) from the CNRS.


  1. Althoff T, Mills DJ, Popot JL, Kühlbrandt W (2011) Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1. EMBO J 30:4652–4664CrossRefGoogle Scholar
  2. Arnold T, Poynor M, Nussberger S, Lupas AN, Linke D (2007) Gene duplication of the eight-stranded β-barrel OmpX produces a functional pore: a scenario for the evolution of transmembrane β-barrel. J Mol Biol 366:1174–1184CrossRefGoogle Scholar
  3. Arora A, Abildgaard F, Bushweller JH, Tamm LK (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat Struct Biol 8:334–338CrossRefGoogle Scholar
  4. Banères JL, Martin A, Hullot P, Girard JP, Rossi JC, Parello J (2003) Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1. J Mol Biol 329:801–814CrossRefGoogle Scholar
  5. Banères JL, Popot JL, Mouillac B (2011) New advances in production and functional folding of G-protein-coupled receptors. Trends Biotechnol 29:314–322CrossRefGoogle Scholar
  6. Bayburt TH, Grinkova YV, Sligar SG (2002) Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins. Nano Lett 2:853–856CrossRefGoogle Scholar
  7. Bazzacco P, Billon-Denis E, Sharma KS, Catoire LJ, Mary S, Le Bon C, Point E, Banères JL, Durand G, Zito F, Pucci B, Popot JL (2012) Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance. Biochemistry 51:1416–1430CrossRefGoogle Scholar
  8. Catoire LJ, Zoonens M, van Heijenoort C, Giusti F, Popot JL, Guittet E (2009) Inter- and intramolecular contacts in a membrane protein/surfactant complex observed by heteronuclear dipole-to-dipole cross-relaxation. J Magn Reson 197:91–95CrossRefGoogle Scholar
  9. Catoire LJ, Damian M, Giusti F, Martin A, van Heijenoort C, Popot JL, Guittet E, Banères JL (2010a) Structure of a GPCR ligand in its receptor–bound state: leukotriene B4 adopts a highly constrained conformation when associated to human BLT2. J Am Chem Soc 132:9049–9057CrossRefGoogle Scholar
  10. Catoire LJ, Zoonens M, van Heijenoort C, Giusti F, Guittet E, Popot JL (2010b) Solution NMR mapping of water-accessible residues in the transmembrane β-barrel of OmpX. Eur Biophys J 39:623–630CrossRefGoogle Scholar
  11. Catoire LJ, Damian M, Baaden M, Guittet E, Banères JL (2011) Electrostatically-driven fast association and perdeuteration allow detection of transferred cross-relaxation for G protein-coupled receptor ligands with equilibrium dissociation constants in the high-to-low nanomolar range. J Biomol NMR 50:191–195CrossRefGoogle Scholar
  12. Catoire LJ, Warnet XL, Warschawski DE (2014) Micelles, bicelles, amphipols, nanodiscs, liposomes or intact cells: the hitchhiker’s guide to the membrane protein study by NMR. In: Mus-Veteau I (ed) Membrane protein production for structural analysis. Springer, Berlin (in press)Google Scholar
  13. Chae PS, Rasmussen SG, Rana RR, Gotfryd K, Chandra R, Goren MA, Kruse AC, Nurva S, Loland CJ, Pierre Y, Drew D, Popot JL, Picot D, Fox BG, Guan L, Gether U, Byrne B, Kobilka B, Gellman SH (2010) Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat Methods 7:1003–1008CrossRefGoogle Scholar
  14. Champeil P, Menguy T, Tribet C, Popot JL, le Maire M (2000) Interaction of amphipols with the sarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 275:18623–18637CrossRefGoogle Scholar
  15. Charvolin D, Perez JB, Rouvière F, Giusti F, Bazzacco P, Abdine A, Rappaport F, Martinez KL, Popot JL (2009) The use of amphipols as universal molecular adapters to immobilize membrane proteins onto solid supports. Proc Natl Acad Sci USA 106:405–410CrossRefGoogle Scholar
  16. Czerski L, Sanders CR (2000) Functionality of a membrane protein in bicelles. Anal Biochem 284:327–333CrossRefGoogle Scholar
  17. Dahmane T, Damian M, Mary S, Popot JL, Banères JL (2009) Amphipol-assisted in vitro folding of G protein-coupled receptors. Biochemistry 48:6516–6521CrossRefGoogle Scholar
  18. Dahmane T, Giusti F, Catoire LJ, Popot JL (2011) Sulfonated amphipols: synthesis, properties, and applications. Biopolymers 95:811–823CrossRefGoogle Scholar
  19. Dahmane T, Rappaport F, Popot JL (2013) Amphipol-assisted folding of bacteriorhodopsin in the presence and absence of lipids. Functional consequences. Eur Biophys J 42:85–101CrossRefGoogle Scholar
  20. Denisov IG, Grinkova YV, Lazarides AA, Sligar SG (2004) Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size. J Am Chem Soc 126:3477–3478CrossRefGoogle Scholar
  21. Diab C, Tribet C, Gohon Y, Popot JL, Winnik FM (2007) Complexation of integral membrane proteins by phosphorylcholine-based amphipols. Biochim Biophys Acta 1768:2737–2747CrossRefGoogle Scholar
  22. Dupont M, De E, Chollet R, Chevalier J, Pages J-M (2004) Enterobacter aerogenes OmpX, a cation-selective channel mar- and osmo-regulated. FEBS Lett 569:27–30CrossRefGoogle Scholar
  23. Elter S, Raschle T, Arens S, Gelev V, Etzkorn M, Wagner G (2014) The use of amphipols for NMR structural characterization of 7-TM proteins. J Membr Biol (in press)Google Scholar
  24. Etzkorn M, Raschle T, Hagn F, Gelev V, Rice AJ, Walz T, Wagner G (2013) Cell-free expressed bacteriorhodopsin in different soluble membrane mimetics: biophysical properties and NMR accessibility. Structure 21:394–401CrossRefGoogle Scholar
  25. Etzkorn M, Zoonens M, Catoire LJ, Popot JL, Hiller S (2014) How amphipols embed membrane proteins: Global solvent accessibility and interaction with a flexible protein terminus. J Membr Biol (in press)Google Scholar
  26. Fernández C, Adeishvili K, Wüthrich K (2001) Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles. Proc Natl Acad Sci USA 98:2358–2363CrossRefGoogle Scholar
  27. Fiaux J, Bertelsen EB, Horwich AL, Wüthrich K (2002) NMR analysis of a 900 K GroEL GroES complex. Nature 418:207–211CrossRefGoogle Scholar
  28. Giusti F, Popot JL, 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
  29. Giusti F, Rieger J, Catoire L, Qian, S, Calabrese AN, Watkinson TG, Radford S, Ashcroft A, Fradet A, Popot JL (2014) Synthesis, characterization and applications of a perdeuterated amphipol. J Membr Biol (in press)Google Scholar
  30. Gohon Y, Pavlov G, Timmins P, Tribet C, Popot JL, Ebel C (2004) Partial specific volume and solvent interactions of amphipol A8-35. Anal Biochem 334:318–334CrossRefGoogle Scholar
  31. Gohon Y, Giusti F, Prata C, Charvolin D, Timmins P, Ebel C, Tribet C, Popot JL (2006) Well-defined nanoparticles formed by hydrophobic assembly of a short and polydisperse random terpolymer, amphipol A8-35. Langmuir 22:1281–1290CrossRefGoogle Scholar
  32. Gohon Y, Dahmane T, Ruigrok R, Schuck P, Charvolin D, Rappaport F, Timmins P, Engelman DM, Tribet C, Popot JL, Ebel C (2008) Bacteriorhodopsin/amphipol complexes: structural and functional properties. Biophys J 94:3523–3537CrossRefGoogle Scholar
  33. Griesinger C, Bennati M, Vieth HM, Luchinat C, Parigi G, Höfer P, Engelke F, Glaser SJ, Denysenkov V, Prisner TF (2012) Dynamic nuclear polarization at high magnetic fields in liquids. Prog Nucl Magn Reson Spectrosc 64:4–28CrossRefGoogle Scholar
  34. Hagn F, Etzkorn M, Raschle T, Wagner G (2013) Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J Am Chem Soc 135:1919–1925CrossRefGoogle Scholar
  35. Kang CB, Li Q (2011) Solution NMR study of integral membrane proteins. Curr Opin Struct Biol 15:560–569CrossRefGoogle Scholar
  36. Kelly E, Privé GG, Tieleman PD (2005) Molecular models of lipopeptide detergents: large coiled-coils with hydrocarbon interiors. J Am Chem Soc 127:13446–13447CrossRefGoogle Scholar
  37. Koutsopoulos S, Kaiser L, Eriksson HM, Zhang S (2012) Designer peptide surfactants stabilize diverse functional membrane proteins. Chem Soc Rev 41:1721–1728CrossRefGoogle Scholar
  38. Le Bon C, Popot JL, Giusti F (2014a) Labeling and functionalizing amphipols for biological applications. J Membr Biol (in press)Google Scholar
  39. Le Bon C, Della Pia EA, Giusti F, Lloret N, Zoonens M, Martinez KL, Popot JL (2014b) Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins. Nucleic Acids Res (in press)Google Scholar
  40. Lee D, Walter KF, Brückner AK, Hilty C, Becker S, Griesinger C (2008) Bilayer in small bicelles revealed by lipid-protein interactions using NMR spectroscopy. J Am Chem Soc 130:13822–13823CrossRefGoogle Scholar
  41. Martinez KL, Gohon Y, Corringer PJ, Tribet C, Mérola F, Changeux JP, Popot JL (2002) Allosteric transitions of Torpedo acetylcholine receptor in lipids, detergent and amphipols: molecular interactions vs. physical constraints. FEBS Lett 528:251–256CrossRefGoogle Scholar
  42. McGregor CL, Chen L, Pomroy NC, Hwang P, Go S, Chakrabartty A, Privé GG (2003) Lipopeptide detergents designed for the structural study of membrane proteins. Nat Biotechnol 21:171–176CrossRefGoogle Scholar
  43. Mouillac B, Banères JL (2010) Mammalian membrane receptors expression as inclusion bodies in Escherichia coli. Methods Mol Biol 601:39–48CrossRefGoogle Scholar
  44. Nath A, Atkins WM, Sligar SG (2007) Applications of phospholipid bilayer nanodiscs in the study of membranes and membrane proteins. Biochemistry 46:2059–2069CrossRefGoogle Scholar
  45. Pautsch A, Schulz GE (2000) High-resolution structure of the OmpA membrane domain. J Mol Biol 298:273–282CrossRefGoogle Scholar
  46. Perlmutter JD, Drasler WJ, Xie W, Gao J, Popot JL, Sachs JN (2011) All-atom and coarse-grained molecular dynamics simulations of a membrane protein stabilizing polymer. Langmuir 27:10523–10537CrossRefGoogle Scholar
  47. Perlmutter JD, Popot J-L, Sachs, JN (2014) Molecular dynamics simulations of a membrane protein/amphipol complex. Submitted for publicationGoogle Scholar
  48. Picard M, Dahmane T, Garrigos M, Gauron C, Giusti F, le Maire M, Popot JL, 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
  49. Plevin MJ, Boisbouvier J (2012) Isotope-labelling of methyl groups for NMR studies of large proteins In: Clore M, Potts J (eds) Recent developments in biomolecular NMR, RSC biomolecular sciences no. 25. Royal Society of Chemistry. doi: 10.1039/9781849735391 Google Scholar
  50. Pocanschi CL, Dahmane T, Gohon Y et al (2006) Amphipathic polymers: tools to fold integral membrane proteins to their active form. Biochemistry 45:13954–13961CrossRefGoogle Scholar
  51. Poget SF, Girvin ME (2007) Solution NMR of membrane proteins in bilayer mimics: small is beautiful, but sometimes bigger is better. Biochim Biophys Acta 68:3098–3106CrossRefGoogle Scholar
  52. Popot JL (2010) Amphipols, nanodiscs, and fluorinated surfactants: three nonconventional approaches to studying membrane proteins in aqueous solutions. Ann Rev Biochem 79:737–775CrossRefGoogle Scholar
  53. Popot JL, Althoff T, Bagnard D et al (2011) Amphipols from A to Z. Ann Rev Biophys 40:379–408CrossRefGoogle Scholar
  54. Privé G (2009) Lipopeptide detergents for membrane protein studies. Curr Opin Struct Biol 19:1–7CrossRefGoogle Scholar
  55. Raschle T, Hiller S, Etzkorn M, Wagner G (2010) Nonmicellar systems for solution NMR spectroscopy of membrane proteins. Curr Opin Struct Biol 20:471–479CrossRefGoogle Scholar
  56. Renault M (2008) Etudes structurales et dynamiques de la protéine membranaire KpOmpA par RMN en phase liquide et solide. Ph. D. Thesis Université Paul Sabatier, Toulouse, FranceGoogle Scholar
  57. Renault M, Tommassen-van Boxtel R, Bos MP, Post JA, Tommassen J, Baldus M (2012) Cellular solid-state nuclear magnetic resonance spectroscopy. Proc Natl Acad Sci USA 109:4863–4868CrossRefGoogle Scholar
  58. Riek R, Wider G, Pervushin K, Wüthrich K (1999) Polarization transfer by cross-correlated relaxation in solution NMR with very large molecules. Proc Natl Acad Sci USA 96:4918–4923CrossRefGoogle Scholar
  59. Ritchie TK, Grinkova YV, Bayburt TH, Denisov IG, Zolnerciks JK, Atkins WM, Sligar SG (2009) Chapter 11—reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol 464:211–213CrossRefGoogle Scholar
  60. Ruschak AM, Religa TL, Breuer S, Witt S, Kay LE (2010) The proteasome antechamber maintains substrates in an unfolded state. Nature 467:868–871CrossRefGoogle Scholar
  61. Sanders CR 2nd, Landis GC (1995) Reconstitution of membrane proteins into lipid-rich bilayered mixed micelles for NMR studies. Biochemistry 34:4030–4040CrossRefGoogle Scholar
  62. Sharma KS, Durand G, Gabel F, Bazzacco P, Le Bon C, Billon-Denis E, Catoire LJ, Popot JL, Ebel C, Pucci B (2012) Non–ionic amphiphilic homopolymers: synthesis, solution properties, and biochemical validation. Langmuir 28:4625–4639CrossRefGoogle Scholar
  63. Takeda M, Kainosho M (2012) Cell-free protein synthesis using E. coli cell extract for NMR studies. Adv Exp Med Biol 992:167–177CrossRefGoogle Scholar
  64. Tifrea DF, Sun G, Pal S, Zardeneta G, Cocco MJ, Popot JL, de la Maza LM (2011) Amphipols stabilize the Chlamydia major outer membrane protein and enhance its protective ability as a vaccine. Vaccine 29:4623–4631CrossRefGoogle Scholar
  65. Triba MN, Warschawski DE, Devaux PF (2005) Reinvestigation by phosphorus NMR of lipid distribution in bicelles. Biophys J 88:1887–1901CrossRefGoogle Scholar
  66. Tribet C, Audebert R, Popot JL (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci USA 93:15047–15050CrossRefGoogle Scholar
  67. Tribet C, Diab C, Dahmane T, Zoonens M, Popot JL, Winnik FM (2009) Thermodynamic characterization of the exchange of detergents and amphipols at the surfaces of integral membrane proteins. Langmuir 25:12623–12634CrossRefGoogle Scholar
  68. Vogt J, Schulz GE (1999) The structure of the outer membrane protein OmpX from Escherichia coli reveals possible mechanisms of virulence. Structure 7:1301–1309CrossRefGoogle Scholar
  69. Vold RR, Prosser RS, Deese AJ (1997) Isotropic solutions of phospholipid bicelles: a new membrane mimetic for high-resolution NMR studies of polypeptides. J Biomol NMR 9:329–335CrossRefGoogle Scholar
  70. Wang X, Corin K, Baaske P, Wienken CJ, Jerabek-Willemsen M, Duhr S, Braun D, Zhang S (2011) Peptide surfactants for cell-free production of functional G protein-coupled receptors. Proc Natl Acad Sci USA 108:9049–9054CrossRefGoogle Scholar
  71. Warschawski DE, Arnold AA, Beaugrand M, Gravel A, Chartrand E, Marcotte I (2011) Choosing membrane mimetics for NMR structural studies of transmembrane proteins. Biochim Biophys Acta 1808:1957–1974CrossRefGoogle Scholar
  72. Wüthrich K (1986) NMR of proteins and nucleic acids. Wiley, New YorkCrossRefGoogle Scholar
  73. Yokomizo T, Kako K, Terawaki K, Izumi T, Shimizu T (2000) A second leukotriene B(4) receptor, BLT2. A new therapeutic target in inflammation and immunological disorders. J Exp Med 192:421–432CrossRefGoogle Scholar
  74. Zhao X, Nagai Y, Reeves PJ, Kiley P, Khorana HG, Zhang S (2006) Designer short peptide surfactants stabilize G protein-coupled receptor bovine rhodopsin. Proc Natl Acad Sci USA 103:17707–17712CrossRefGoogle Scholar
  75. Zhou HX, Cross TA (2013) Influences of membrane mimetic environments on membrane protein structures. Annu Rev Biophys 42:361–392CrossRefGoogle Scholar
  76. Zoonens M, Catoire LJ, Giusti F, Popot JL (2005) NMR study of a membrane protein in detergent–free aqueous solution. Proc Natl Acad Sci USA 102:8893–8898CrossRefGoogle Scholar
  77. Zoonens M, Giusti F, Zito F, Popot JL (2007) Dynamics of membrane protein/amphipol association studied by Förster resonance energy transfer. Implications for in vitro studies of amphipol-stabilized membrane proteins. Biochemistry 46:10392–10404CrossRefGoogle Scholar
  78. Zoonens M, Zito F, Martinez KL, Popot JL (2014) Amphipols: a general introduction and some protocols. In: Mus-Veteau I (ed) Membrane protein production for structural analysis. Springer, BerlinGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Noelya Planchard
    • 1
    • 2
  • Élodie Point
    • 1
  • Tassadite Dahmane
    • 1
    • 3
  • Fabrice Giusti
    • 1
  • Marie Renault
    • 4
  • Christel Le Bon
    • 1
  • Grégory Durand
    • 5
    • 6
  • Alain Milon
    • 4
  • Éric Guittet
    • 7
  • Manuela Zoonens
    • 1
  • Jean-Luc Popot
    • 1
  • Laurent J. Catoire
    • 1
    Email author
  1. 1.Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, Institut de Biologie Physico-Chimique (FRC 550), UMR 7099, CNRSUniversité Paris 7ParisFrance
  2. 2.Centre de Versailles-Grignon, Institut Jean-Pierre Bourgin, UMR 1318 INRA-AgroParisTech, Bâtiment 7INRAVersailles CedexFrance
  3. 3.Department of Biochemistry and Molecular BiophysicsColumbia UniversityNew YorkUSA
  4. 4.Laboratoire de RMN et des interactions protéines/membranes, CNRSInstitut de Pharmacologie et de Biologie StructuraleToulouseFrance
  5. 5.Equipe Chimie Bioorganique et Systèmes AmphiphilesUniversité d’Avignon et des Pays de VaucluseAvignonFrance
  6. 6.Institut des Biomolécules Max Mousseron (UMR 5247)Montpellier Cedex 05France
  7. 7.Centre de Recherche de Gif, Laboratoire de Chimie et Biologie Structurales, UPR 2301 CNRSICSNGif-sur-YvetteFrance

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