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The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 981–996 | Cite as

Solution Behavior and Crystallization of Cytochrome bc1 in the Presence of Amphipols

  • Delphine Charvolin
  • Martin PicardEmail author
  • Li-Shar Huang
  • Edward A. Berry
  • Jean-Luc Popot
Article

Abstract

Detergents classically are used to keep membrane proteins soluble in aqueous solutions, but they tend to destabilize them. This problem can be largely alleviated thanks to the use of amphipols (APols), small amphipathic polymers designed to substitute for detergents. APols adsorb at the surface of the transmembrane region of membrane proteins, keeping them water-soluble while stabilizing them bio-chemically. Membrane protein/APol complexes have proven, however, difficult to crystallize. In this study, the composition and solution properties of complexes formed between mitochondrial cytochrome bc1 and A8-35, the most extensively used APol to date, have been studied by means of size exclusion chromatography, sucrose gradient sedimentation, and small-angle neutron scattering. Stable, monodisperse preparations of bc1/A8-35 complexes can be obtained, which, depending on the medium, undergo either repulsive or attractive interactions. Under crystallization conditions, diffracting three-dimensional crystals of A8-35-stabilized cytochrome bc1 formed, but only in the concomitant presence of APol and detergent.

Keywords

Membrane proteins Surfactants Amphipathic polymers Stability Small-angle neutron scattering Size exclusion chromatography 

Abbreviations

A8-35

A specific type of amphipol (Tribet et al. 1996)

APol

Amphipol

AUC

Analytical ultracentrifugation

BR

Bacteriorhodopsin

C8E4

Tetraethylene glycol monooctyl ether

Cmc

Critical micellar concentration

CMP

Contrast match point

DDM

Dodecyl-β-d-maltoside

EDTA

Ethylene-diamine-tetra-acetic acid

EM

Electron microscopy

FAPolNBD

Fluorescent (NBD-labeled) A8-35

KMES

2-Morpholino-ethane sulfonic acid potassium salt

MD

Molecular dynamics

MW

Molecular weight

MWCO

Molecular weight cutoff

NBD

7-nitrobenz-2-oxa-1,3-diazol-4-yl

NMR

Nuclear magnetic resonance

βog

Octyl-β-d-glucopyranoside

OmpX

Outer membrane protein X from Escherichia coli

PEG4k

4 kDa polyethylene glycol

SANS and SAXS

Small-angle neutron and X-ray scattering, respectively

SEC

Size exclusion chromatography

SERCA1a

Sarcoplasmic reticulum calcium pump from fast twitch muscle

tOmpA

Transmembrane domain of outer membrane protein A from E. coli

Tris

Tris-hydroxymethyl-amino-methane

Notes

Acknowledgments

Particular thanks are due to A.-N. Galatanu and J.-C. Courant for participating in some of these experiments, to P. Hervé for the synthesis of [3H]A8-35, to P. Timmins for his participation in the SANS measurements, to D. Picot and L. Barucq for testing crystals at the ESRF, to I. Gallay for help with the X-ray equipment at the IBPC, to J. Barra and L.J. Catoire for their precious help with the figures, and to Y. Gohon and M. Zoonens for useful comments on the manuscript. This study was supported by the Centre National de la Recherche Scientifique, Université Paris-7, the Human Frontier Science Program Organization (Grant RG00223-2000-M), E.U. Specific Targeted Research Project LSHG-CT-2005-513770 IMPS (Innovative tools for membrane protein structural proteomics), and NIH grant R01DK44842. The stays of EAB in France were subsidized by awards from the France-Berkeley Fund and the French Ministère de l’Education Nationale et de la Recherche.

References

  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. Arunmanee W, Harris JR, Lakey JH (2014) Outer membrane protein F stabilised with minimal amphipol forms linear arrays and LPS-dependent 2D crystals. J Membr Biol. doi: 10.1007/s00232-014-9640-5 CrossRefGoogle Scholar
  3. Bazzacco P, Billon-Denis E, Sharma KS, Catoire LJ, Mary S, Le Bon C, Point E, Banères J-L, Durand G, Zito F, Pucci B, Popot J-L (2012) Non-ionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution NMR. Biochemistry 51:1416–1430CrossRefGoogle Scholar
  4. Berry EA, Huang L-S, DeRose V (1991) Ubiquinol–cytochrome c oxidoreductase from higher plants. Isolation and characterization of the bc 1 complex from potato tuber mitochondria. J Biol Chem 266:9064–9077PubMedGoogle Scholar
  5. Berry EA, Guergova-Kuras M, Huang L-S, Crofts AR (2000) Structure and function of cytochrome bc 1 complexes. Annu Rev Biochem 69:1005–1075CrossRefGoogle Scholar
  6. Breyton C, Tribet C, Olive J, Dubacq J-P, Popot J-L (1997) Dimer to monomer conversion of the cytochrome b 6 f complex: causes and consequences. J Biol Chem 272:21892–21900CrossRefGoogle Scholar
  7. Cao E, Liao M, Cheng Y, Julius D (2013) TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 504:113–118CrossRefGoogle Scholar
  8. Catoire LJ, Zoonens M, van Heijenoort C, Giusti F, Popot J-L, Guittet E (2009) Inter- and intramolecular contacts in a membrane protein/surfactant complex observed by heteronuclear dipole-to-dipole cross-relaxation. J Magn Res 197:91–95CrossRefGoogle Scholar
  9. Catoire LJ, Zoonens M, van Heijenoort C, Giusti F, Guittet E, Popot J-L (2010) Solution NMR mapping of water-accessible residues in the transmembrane β-barrel of OmpX. Eur Biophys J 39:623–630CrossRefGoogle Scholar
  10. 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
  11. Cvetkov TL, Huynh KW, Cohen MR, Moiseenkova-Bell VY (2011) Molecular architecture and subunit organization of TRPA1 ion channel revealed by electron microscopy. J Biol Chem 286:38168–38176CrossRefGoogle Scholar
  12. Dahmane T, Damian M, Mary S, Popot J-L, Banères J-L (2009) Amphipol-assisted in vitro folding of G protein-coupled receptors. Biochemistry 48:6516–6521CrossRefGoogle Scholar
  13. Dahmane T, Giusti F, Catoire LJ, Popot J-L (2011) Sulfonated amphipols: synthesis, properties and applications. Biopolymers 95:811–823CrossRefGoogle Scholar
  14. Dahmane T, Rappaport F, Popot J-L (2013) Amphipol-assisted folding of bacteriorhodopsin in the presence and absence of lipids. Functional consequences. Eur Biophys J 42:85–101CrossRefGoogle Scholar
  15. Diab C, Tribet C, Gohon Y, Popot J-L, Winnik FM (2007a) Complexation of integral membrane proteins by phosphorylcholine-based amphipols. Biochim Biophys Acta 1768:2737–2747CrossRefGoogle Scholar
  16. Diab C, Winnik FM, Tribet C (2007b) Enthalpy of interaction and binding isotherms of non-ionic surfactants onto micellar amphiphilic polymers (amphipols). Langmuir 23:3025–3035CrossRefGoogle Scholar
  17. 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
  18. 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
  19. 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
  20. Flötenmeyer M, Weiss H, Tribet C, Popot J-L, Leonard K (2007) The use of amphipathic polymers for cryo-electron microscopy of NADH: ubiquinone oxidoreductase (complex I). J Microsc 227:229–235CrossRefGoogle Scholar
  21. 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
  22. 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 perdeuterated amphipol. J Membr Biol. doi: 10.1007/s00232-014-9656-x CrossRefGoogle Scholar
  23. 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
  24. 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
  25. 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
  26. Guinier A, Fournet G (1955) Small-angle scattering of X-rays. Wiley, New YorkGoogle Scholar
  27. Huynh KW, Cohen MR, Moiseenkova-Bell VY (2014) Application of amphipols for structure–functional analysis of TRP channels. J Membrane Biol. doi: 10.1007/s00232-014-9684-6 CrossRefGoogle Scholar
  28. Leney AC, McMorran LM, Radford SE, Ashcroft AE (2012) Amphipathic polymers enable the study of functional membrane proteins in the gas phase. Anal Chem 84:9841–9847CrossRefGoogle Scholar
  29. 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
  30. 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
  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 Membrane 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 CL, Dahmane T, Gohon Y, Rappaport F, Apell H-J, Kleinschmidt JH, Popot J-L (2006) Amphipathic polymers: tools to fold integral membrane proteins to their active form. Biochemistry 45:13954–13961CrossRefGoogle Scholar
  36. 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
  37. Polovinkin V, Gushchin I, Balandin T, Chervakov P, Round E, Schevchenko V, Popov A, Borshchevskiy V, Popot J-L, Gordeliy V (2014) High-resolution structure of a membrane protein transferred from amphipol to a lipidic mesophase (under review)Google Scholar
  38. Popot J-L (2010) Amphipols, nanodiscs, and fluorinated surfactants: three non-conventional approaches to studying membrane proteins in aqueous solutions. Annu Rev Biochem 79:737–775CrossRefGoogle Scholar
  39. 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
  40. 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
  41. Privé GG (2007) Detergents for the stabilization and crystallization of membrane proteins. Methods 41:388–397CrossRefGoogle Scholar
  42. Sharma KS, Durand G, Gabel F, Bazzacco P, Le Bon C, Billon-Denis E, Catoire LJ, Popot J-L, Ebel C, Pucci B (2012) Non-ionic amphiphilic homopolymers: synthesis, solution properties, and biochemical validation. Langmuir 28:4625–4639CrossRefGoogle Scholar
  43. Smith AL (1967) Preparation, properties, and conditions for assay of mitochondria: slaughterhouse material, small scale. Methods Enzymol 10:81–86CrossRefGoogle Scholar
  44. Stroebel D, Choquet Y, Popot J-L, Picot D (2003) An atypical haem in the cytochrome b 6 f complex. Nature 426:413–418CrossRefGoogle Scholar
  45. Tifrea DF, Sun G, Pal S, Zardeneta G, Cocco MJ, Popot J-L, 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
  46. Timmins P, Pebay-Peyroula E, Welte W (1994) Detergent organisation in solutions and in crystals of membrane proteins. Biophys Chem 53:27–36CrossRefGoogle 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. Tribet C, Diab C, Dahmane T, Zoonens M, Popot J-L, Winnik FM (2009) Thermodynamic characterization of the exchange of detergents and amphipols at the surfaces of integral membrane proteins. Langmuir 25:12623–12634CrossRefGoogle Scholar
  49. Trumpower BL, Edwards CA (1979) Purification of a reconstitutively active iron–sulfur protein (oxidation factor) from succinate–cytochrome c reductase complex of bovine heart mitochondria. J Biol Chem 254:8697–8706PubMedGoogle Scholar
  50. Zhang Z, Huang L-S, Shulmeister VM, Chi Y-I, Kim KK, Hung L-W, Crofts AR, Berry EA, Kim S-H (1998) Electron transfer by domain movement in cytochrome bc 1. Nature 392:677–684CrossRefGoogle Scholar
  51. Zoonens M, Popot J-L (2014) Amphipols for each season. J Membrane Biol. doi: 10.1007/s00232-014-9666-8 CrossRefGoogle Scholar
  52. 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
  53. Zoonens M, Giusti F, Zito F, Popot J-L (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
  54. Zoonens M, Zito F, Martinez KL, Popot J-L (2014) Amphipols: a general introduction and some protocols. In: Mus-Veteau I (ed) Membrane protein production for structural analysis. Springer, New YorkGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Delphine Charvolin
    • 1
  • Martin Picard
    • 1
    • 2
    Email author
  • Li-Shar Huang
    • 3
  • Edward A. Berry
    • 3
  • Jean-Luc Popot
    • 1
  1. 1.UMR 7099, Centre National de la Recherche Scientifique/Université Paris-7, Institut de Biologie Physico-Chimique, FRC 550ParisFrance
  2. 2.Faculté de Pharmacie, Laboratoire de Cristallographie et RMN BiologiquesCentre National de la Recherche Scientifique/Université Paris Descartes UMR 8015ParisFrance
  3. 3.Department of Biochemistry and Molecular BiologySUNY Upstate Medical UniversitySyracuseUSA

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