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

• Published in: Journal of bioenergetics and biomembranes 2009-08, Vol.41 (4), p.349-360
• Main Authors: Talbot, Jean-Claude, Dautant, Alain, Polidori, Ange, Pucci, Bernard, Cohen-Bouhacina, Touria, Maali, Abdelhamid, Salin, Bénédicte, Brèthes, Daniel, Velours, Jean, Giraud, Marie-France
• Format: Article
• Language: English
• Subjects: Acrylamides - chemistry; Animal Anatomy; Animal Biochemistry; ATP; Biochemistry; Bioorganic Chemistry; Cellular Biology; Chemical Fractionation - methods; Chemistry; Chemistry and Materials Science; Detergents; Enzymatic activity; Fluorine - chemistry; Histology; Hydrogen - chemistry; Inhibitors; Life Sciences; Lipids; Membranes; Mitochondrial Membranes - chemistry; Mitochondrial Membranes - enzymology; Mitochondrial Proton-Translocating ATPases - chemistry; Mitochondrial Proton-Translocating ATPases - isolation & purification; Morphology; Organic Chemistry; Proteins; Studies; Surface-Active Agents - chemistry; Surfactants; Yeast; Yeasts
• ISSN: 0145-479X; 1573-6881
• DOI: 10.1007/s10863-009-9235-5

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.