, Volume 41, Issue 4, pp 349-360

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

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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.