Sedimentation Velocity Analytical Ultracentrifugation in Hydrogenated and Deuterated Solvents for the Characterization of Membrane Proteins

  • Aline Le Roy
  • Hugues Nury
  • Benjamin Wiseman
  • Jonathan Sarwan
  • Jean-Michel Jault
  • Christine Ebel
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1033)

Abstract

This chapter is a step-by-step protocol for setting up, realizing, and analyzing sedimentation velocity experiments in hydrogenated and deuterated solvents, in the context of the characterization of membrane protein, in terms of homogeneity, association state, and amount of bound detergent, based on a real case study of the membrane protein BmrA solubilized in n-Dodecyl-β-d-Maltopyranoside) detergent.

Key words

Sedimentation velocity Analytical ultracentrifugation Membrane proteins Homogeneity Association state Bound detergent BmrA Detergent Heavy water D2SEDFIT 

Notes

Acknowledgments

This work used the AUC platform of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB).

References

  1. 1.
  2. 2.
    Musatov A, Robinson NC (2002) Cholate-induced dimerization of detergent- or phospholipid-solubilized bovine cytochrome C oxidase. Biochemistry 41:4371–4376PubMedCrossRefGoogle Scholar
  3. 3.
    Fisher LE, Engelman DM, Sturgis JN (2003) Effect of detergents on the association of the glycophorin a transmembrane helix. Biophys J 85:3097–3105PubMedCrossRefGoogle Scholar
  4. 4.
    Josse D, Ebel C, Stroebel D, Fontaine A, Borges F, Echalier A, Baud D, Renault F, Le Maire M, Chabrieres E, Masson P (2002) Oligomeric states of the detergent-solubilized human serum paraoxonase (PON1). J Biol Chem 277:33386–33397PubMedCrossRefGoogle Scholar
  5. 5.
    le Maire M, Champeil P, Møller JV (2000) Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 1508:86–111PubMedCrossRefGoogle Scholar
  6. 6.
    Lebowitz J, Lewis MS, Schuck P (2002) Modern analytical ultracentrifugation in protein science: a tutorial review. Protein Sci 11:2067–2079PubMedCrossRefGoogle Scholar
  7. 7.
    Ebel C (2004) Analytical ultracentrifugation for the study of biological macromolecules. Prog Colloid Polym Sci 127:73–82Google Scholar
  8. 8.
    Howlett GJ, Minton AP, Rivas G (2006) Analytical ultracentrifugation for the study of protein association and assembly. Curr Opin Chem Biol 10:430–436PubMedCrossRefGoogle Scholar
  9. 9.
    Ebel C (2007) Analytical ultracentrifugation. State of the art and perspectives. In: Uversky VN, Permyakov EA (eds) Protein structures: methods in protein structure and stability analysis. Nova, New York, pp 229–260Google Scholar
  10. 10.
    Salvay AG, Ebel C (2006) Analytical ultracentrifuge for the characterization of detergent in solution. Prog Colloid Polym Sci 131:74–82CrossRefGoogle Scholar
  11. 11.
    Salvay AG, Santamaria M, le Maire M, Ebel C (2007) Analytical ultracentrifugation sedimentation velocity for the characterization of detergent–solubilized membrane proteins Ca++-ATPase and ExbB. J Biol Phys 33:399–419PubMedCrossRefGoogle Scholar
  12. 12.
    le Maire M, Arnou B, Olesen C, Georgin D, Ebel C, Moller JV (2008) Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and stokes radius of membrane proteins using sarcoplasmic reticulum Ca2+-ATPase as an example. Nat Protoc 3:1782–1795PubMedCrossRefGoogle Scholar
  13. 13.
    Ebel C (2011) Sedimentation velocity to characterize surfactants and solubilized membrane proteins. Methods 54:56–66PubMedCrossRefGoogle Scholar
  14. 14.
    Nury H, Manon F, Arnou B, le Maire M, Pebay-Peyroula E, Ebel C (2008) Mitochondrial bovine ADP/ATP carrier in detergent is predominantly monomeric but also forms multimeric species. Biochemistry 47:12319–12331PubMedCrossRefGoogle Scholar
  15. 15.
    Dach I, Olesen C, Signor L, Nissen P, le Maire M, Møller JV, Ebel C (2012) Active detergent solubilized H+, K+-ATPase is a monomer. J Biol Chem 287:41963–41978PubMedCrossRefGoogle Scholar
  16. 16.
    Peter Schuck’s Software Home Page. http://www.analyticalultracentrifugation.com, p Download Sedfit and Sedphat and description
  17. 17.
    Borries Demeler’s Ultrascan Software Page. http://www.ultrascan.uthscsa.edu/
  18. 18.
    Walter Stafford’s Programm Sedanal. http://rasmb.bbri.org/sedanal.html.
  19. 19.
    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 334:318–334PubMedCrossRefGoogle Scholar
  20. 20.
    Pavlov G, Finet S, Tatarenko K, Korneeva E, Ebel C (2003) Conformation of heparin studied with macromolecular hydrodynamic methods and X-ray scattering. Eur Biophys J 32:437–449PubMedCrossRefGoogle Scholar
  21. 21.
    Web Site of Spin Analytical. http://www.spinanalytical.com/index.php
  22. 22.
    Schuck P (2004) A model for sedimentation in inhomogeneous media. II. Compressibility of aqueous and organic solvents. Biophys Chem 108:201–214PubMedCrossRefGoogle Scholar
  23. 23.
    Schuck P (2004) A model for sedimentation in inhomogeneous media. I. Dynamic density gradients from sedimenting co-solutes. Biophys Chem 108:187–200PubMedCrossRefGoogle Scholar
  24. 24.
    Steinfels E, Orelle C, Fantino JR, Dalmas O, Rigaud JL, Denizot F, Di Pietro A, Jault JM (2004) Characterization of YvcC (BmrA), a multidrug ABC transporter constitutively expressed in Bacillus subtilis. Biochemistry 43:7491–7502PubMedCrossRefGoogle Scholar
  25. 25.
    Ravaud S, Do Cao MA, Jidenko M, Ebel C, Le Maire M, Jault JM, Di Pietro A, Haser R, Aghajari N (2006) The ABC transporter BmrA from Bacillus subtilis is a functional dimer when in a detergent-solubilized state. Biochem J 395:345–353PubMedCrossRefGoogle Scholar
  26. 26.
    Solovyova A, Schuck P, Costenaro L, Ebel C (2001) Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents. Biophys J 81:1868–1880PubMedCrossRefGoogle Scholar
  27. 27.
    Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J 78:1606–1619PubMedCrossRefGoogle Scholar
  28. 28.
    Buisson M, Valette E, Hernandez JF, Baudin F, Ebel C, Morand P, Seigneurin JM, Arlaud GJ, Ruigrok RW (2001) Functional determinants of the Epstein-Barr virus protease. J Mol Biol 311:217–228PubMedCrossRefGoogle Scholar
  29. 29.
    Schuck P (2010) Sedimentation patterns of rapidly reversible protein interactions. Biophys J 98:2005–2013PubMedCrossRefGoogle Scholar
  30. 30.
    Cole JL, Correia JJ, Stafford WF (2011) The use of analytical sedimentation velocity to extract thermodynamic linkage. Biophys Chem 159:120–128PubMedCrossRefGoogle Scholar
  31. 31.
    Balbo A, Minor KH, Velikovsky CA, Mariuzza RA, Peterson CB, Schuck P (2005) Studying multiprotein complexes by multisignal sedimentation velocity analytical ultracentrifugation. Proc Natl Acad Sci USA 102:81–86PubMedCrossRefGoogle Scholar
  32. 32.
    Hayashi Y, Matsui H, Takagi T (1989) Membrane protein molecular weight determined by low-angle laser light-scattering photometry coupled with high-performance gel chromatography. Methods Enzymol 172:514–528PubMedCrossRefGoogle Scholar
  33. 33.
    Edelstein SJ, Schachman HK (1967) The simultaneous determination of partial specific volumes and molecular weights with microgram quantities. J Biol Chem 242:306–311PubMedGoogle Scholar
  34. 34.
    Edelstein SJ, Schachman HK (1973) Measurement of partial specific volume by sedimentation equilibrium in H2O-D2O solutions. Methods Enzymol 27:82–98PubMedCrossRefGoogle Scholar
  35. 35.
    Brown PH, Balbo A, Zhao H, Ebel C, Schuck P (2011) Density contrast sedimentation velocity for the determination of protein partial-specific volumes. PLoS One 6:e26221PubMedCrossRefGoogle Scholar
  36. 36.
    John Philo’s Software Home Page. http://www.jphilo.mailway.com/, p Download Sednterp
  37. 37.
    Philo J, Talvenheimo J, Wen J, Rosenfeld R, Welcher A, Arakawa T (1994) Interactions of neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), and the NT-3.BDNF heterodimer with the extracellular domains of the TrkB and TrkC receptors. J Biol Chem 269:27840–27846PubMedGoogle Scholar
  38. 38.
    Tanford C, Reynolds JA (1976) Characterization of membrane proteins in detergent solutions. Biochim Biophys Acta 457:133–170PubMedCrossRefGoogle Scholar
  39. 39.
    Tiefenbach K-J, Durchschlag H, Jaenicke R (1999) Spectoscopic and hydrodynamic investigations of nonionic and zwitterionic detergents. Prog Colloid Polym Sci 113:135–141CrossRefGoogle Scholar
  40. 40.
    Breyton C, Gabel F, Abla M, Pierre Y, Lebaupain F, Durand G, Popot J-L, Ebel C, Pucci B (2009) Micellar and biochemical properties of (hemi)fluorinated surfactants are controlled by the size of the polar head. Biophys J 97:1077–1086PubMedCrossRefGoogle Scholar
  41. 41.
    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 28:4625–4639PubMedCrossRefGoogle Scholar
  42. 42.
    Gohon Y, Dahmane T, Ruigrok RW, 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 94:3523–3537PubMedCrossRefGoogle Scholar
  43. 43.
    Durchschlag H, Zipper P (1994) Calculation of the partial volume of organic compounds and polymers. Prog Colloid Polym Sci 94:20–39CrossRefGoogle Scholar
  44. 44.
    Durchschlag H, Zipper P (1997) Calculation of partial specific volumes and other volumetric properties of small molecules and polymers. J Appl Crystallogr 30:803–807CrossRefGoogle Scholar
  45. 45.
    Zhao H, Brown PH, Schuck P (2011) On the distribution of protein refractive index increments. Biophys J 100:2309–2317PubMedCrossRefGoogle Scholar
  46. 46.
    Beri RG, Walker J, Reese ET, Rollings JE (1993) Characterization of chitosans via coupled size-exclusion chromatography and multiple-angle laser light-scattering technique. Carbohydr Res 238:11–26CrossRefGoogle Scholar
  47. 47.
  48. 48.
    Csucs G, Ramsden JJ (1998) Solubilization of planar bilayers with detergent. Biochim Biophys Acta 1369:304–308PubMedCrossRefGoogle Scholar
  49. 49.
    Arnaud N, Georges J (2001) On the analytical use of the Soret-enhanced thermal lens signal in aqueous solutions. Anal Chim Acta 445:239–244CrossRefGoogle Scholar
  50. 50.
    Strop P, Brunger AT (2005) Refractive index-based determination of detergent concentration and its application to the study of membrane proteins. Protein Sci 14:2207–2211PubMedCrossRefGoogle Scholar
  51. 51.
    Abla M, Durand G, Breyton C, Raynal S, Ebel C, Pucci B (2012) A diglucosylated fluorinated surfactant to handle integral membrane proteins in aqueous solution. J Fluor Chem 134:63–71CrossRefGoogle Scholar
  52. 52.
    Bazzacco P, Sharma KS, Durand G, Giusti F, Ebel C, Popot J-L, Pucci B (2009) Trapping and stabilization of integral membrane proteins by hydrophobically grafted glucose-based telomers. Biomacromolecules 10:3317–3326PubMedCrossRefGoogle Scholar
  53. 53.
    Riske KA, Politi MJ, Reed WF, Lamy-Freund MT (1997) Temperature and ionic strength dependent light scattering of DMPG dispersions. Chem Phys Lipids 89:31–44CrossRefGoogle Scholar
  54. 54.
    Salvay AG, Communie G, Ebel C (2012) Sedimentation velocity analytical ultracentrifugation for intrinsically disordered proteins. In: Intrinsically disordered protein analysis, vol 2, Methods and experimental tools. Springer, New York, pp 91–104CrossRefGoogle Scholar
  55. 55.
    Tabarani G, Reina JJ, Ebel C, Vives C, Lortat-Jacob H, Rojo J, Fieschi F (2006) Mannose hyperbranched dendritic polymers interact with clustered organization of DC-SIGN and inhibit gp120 binding. FEBS Lett 580: 2402–2408PubMedCrossRefGoogle Scholar
  56. 56.
    Balbo A, Zhao H, Brown PH, Schuck P (2009) Assembly, loading, and alignment of an analytical ultracentrifuge sample cell. J Vis Exp (33):e1530Google Scholar
  57. 57.
  58. 58.
    Boncoeur E, Durmort C, Bernay B, Ebel C, Di Guilmi A-M, Croizé J, Vernet T, Jault JM (2012) PatA and PatB form a functional heterodimeric ABC multidrug efflux transporter responsible for the resistance of Streptococcus pneumoniae to fluoroquinolones. Biochemistry 51:7755–65PubMedCrossRefGoogle Scholar
  59. 59.
    Schuck P (2003) On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Anal Biochem 320:104–124PubMedCrossRefGoogle Scholar
  60. 60.
    Zhao H, Ghirlando R, Piszczek G, Curth U, Brautigam CA, Schuck P (2013) Recorded Scan Times Can Limit the Accuracy of Sedimentation Coefficients in Analytical Ultracentrifugation. Anal Biochem. 437:104–108PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Aline Le Roy
    • 1
  • Hugues Nury
    • 1
  • Benjamin Wiseman
    • 1
  • Jonathan Sarwan
    • 1
  • Jean-Michel Jault
    • 1
  • Christine Ebel
    • 1
  1. 1.Institut de Biologie Structurale, CEAGrenobleFrance

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