Journal of Bioenergetics and Biomembranes

, 41:349

Hydrogenated and fluorinated surfactants derived from Tris(hydroxymethyl)-acrylamidomethane allow the purification of a highly active yeast F1-F0 ATP-synthase with an enhanced stability

  • Jean-Claude Talbot
  • Alain Dautant
  • Ange Polidori
  • Bernard Pucci
  • Touria Cohen-Bouhacina
  • Abdelhamid Maali
  • Bénédicte Salin
  • Daniel Brèthes
  • Jean Velours
  • Marie-France Giraud
Article

DOI: 10.1007/s10863-009-9235-5

Cite this article as:
Talbot, JC., Dautant, A., Polidori, A. et al. J Bioenerg Biomembr (2009) 41: 349. doi:10.1007/s10863-009-9235-5

Abstract

Loss of stability and integrity of large membrane protein complexes as well as their aggregation in a non-lipidic environment are the major bottlenecks to their structural studies. We have tested C12H25-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H12-TAC) among many other detergents for extracting the yeast F1F0 ATP-synthase. H12-TAC was found to be a very efficient detergent for removing the enzyme from mitochondrial membranes without altering its sensitivity towards specific ATP-synthase inhibitors. This extracted enzyme was then solubilized by either dodecyl maltoside (DDM), H12-TAC or fluorinated surfactants such as C2H5-C6F12-C2H4-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H2F6-TAC) or C6F13-C2H4-S-poly-Tris-(hydroxymethyl)acrylamidomethane (F6-TAC), two surfactants exhibiting a comparable polar head to H12-TAC but bearing a fluorinated hydrophobic tail. Preparations from enzymes purified in the presence of H12-TAC were found to be more adapted for AFM imaging than ATP-synthase purified with DDM. Keeping H12-TAC during the Ni-NTA IMAC purification step or replacing it by DDM at low concentrations did not however allow preserving enzyme activity, while fluorinated surfactants H2F6-TAC and F6-TAC were found to enhance enzyme stability and integrity as indicated by sensitivity towards inhibitors. ATPase specific activity was higher with F6-TAC than with H2F6-TAC. When enzymes were mixed with egg phosphatidylcholine, ATP-synthases purified in the presence of H2F6-TAC or F6-TAC were more stable upon time than the DDM purified enzyme. Furthermore, in the presence of lipids, an activation of ATP-synthases was observed that was transitory for enzymes purified with DDM, but lasted for weeks for ATP-synthases isolated in the presence of molecules with Tris polyalcoholic moieties. Relipidated enzymes prepared with fluorinated surfactants remained highly sensitive towards inhibitors, even after 6 weeks.

Keywords

Membrane protein complexesF1F0 ATP-synthaseDetergentFluorinated surfactants

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Jean-Claude Talbot
    • 1
  • Alain Dautant
    • 1
  • Ange Polidori
    • 2
  • Bernard Pucci
    • 2
  • Touria Cohen-Bouhacina
    • 3
  • Abdelhamid Maali
    • 3
  • Bénédicte Salin
    • 1
  • Daniel Brèthes
    • 1
  • Jean Velours
    • 1
  • Marie-France Giraud
    • 1
  1. 1.CNRS, Institut de Biochimie et Génétique CellulairesUniversité Bordeaux 2Bordeaux cedexFrance
  2. 2.Faculté des Sciences, Laboratoire de Chimie Bioorganique et des systèmes moléculaires vectoriels (LCBOSMV)Université d’AvignonAvignonFrance
  3. 3.Centre de Physique Moléculaire Optique et Hertzienne (CPMOH)Université Bordeaux 1Talence cedexFrance