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.
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 C(12)H(25)-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H(12)-TAC) among many other detergents for extracting the yeast F(1)F(0) ATP-synthase. H(12)-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), H(12)-TAC or fluorinated surfactants such as C(2)H(5)-C(6)F(12)-C(2)H(4)-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H(2)F(6)-TAC) or C(6)F(13)-C(2)H(4)-S-poly-Tris-(hydroxymethyl)acrylamidomethane (F(6)-TAC), two surfactants exhibiting a comparable polar head to H(12)-TAC but bearing a fluorinated hydrophobic tail. Preparations from enzymes purified in the presence of H(12)-TAC were found to be more adapted for AFM imaging than ATP-synthase purified with DDM. Keeping H(12)-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 H(2)F(6)-TAC and F(6)-TAC were found to enhance enzyme stability and integrity as indicated by sensitivity towards inhibitors. ATPase specific activity was higher with F(6)-TAC than with H(2)F(6)-TAC. When enzymes were mixed with egg phosphatidylcholine, ATP-synthases purified in the presence of H(2)F(6)-TAC or F(6)-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.
Talbot
Jean-Claude
JC
CNRS, Institut de Biochimie et Génétique Cellulaires, Université Bordeaux 2, 1 rue Camille Saint-Saëns, 33077, Bordeaux cedex, France.
Dautant
Alain
A
Polidori
Ange
A
Pucci
Bernard
B
Cohen-Bouhacina
Touria
T
Maali
Abdelhamid
A
Salin
Bénédicte
B
Brèthes
Daniel
D
Velours
Jean
J
Giraud
Marie-France
MF
D000178:Acrylamides / Q000737:chemistry*; D005591:Chemical Fractionation / Q000379:methods*; D005461:Fluorine / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D051336:Mitochondrial Membranes / Q000737:chemistry* / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification*; D013501:Surface-Active Agents / Q000737:chemistry*
D000178:Acrylamides; D013501:Surface-Active Agents; C445419:tris(hydroxymethyl)-acrylamidomethane; D005461:Fluorine; D006859:Hydrogen; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases