CNRS, Institut de Biochimie et Génétique Cellulaires, Université Bordeaux 2, 1 rue Camille Saint-Saëns, 33077, Bordeaux cedex, France.
J Bioenerg Biomembr. 2009 Aug;41(4):349-60. doi: 10.1007/s10863-009-9235-5. Epub 2009 Oct 10.
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.
稳定性和完整性的丧失以及大型膜蛋白复合物在非脂环境中的聚集，是其结构研究的主要瓶颈。我们已经在许多其他去污剂中测试了 C(12)H(25)-S-聚三-(羟甲基)丙烯酰胺甲烷 (H(12)-TAC)，以提取酵母 F(1)F(0)ATP 合酶。发现 H(12)-TAC 是一种非常有效的去污剂，可以从线粒体膜中提取酶，而不会改变其对特定 ATP 合酶抑制剂的敏感性。然后，通过十二烷基麦芽糖苷 (DDM)、H(12)-TAC 或氟化表面活性剂（如 C(2)H(5)-C(6)F(12)-C(2)H(4)-S-聚三-(羟甲基)丙烯酰胺甲烷 (H(2)F(6)-TAC)或 C(6)F(13)-C(2)H(4)-S-聚三-(羟甲基)丙烯酰胺甲烷 (F(6)-TAC)将提取的酶溶解，这两种表面活性剂的极性头与 H(12)-TAC 相当，但具有氟化疏水尾。与用 DDM 纯化的 ATP 合酶相比，用 H(12)-TAC 纯化的酶制备物更适合 AFM 成像。然而，在 Ni-NTA IMAC 纯化步骤中保留 H(12)-TAC 或用低浓度的 DDM 代替它并不能保持酶的活性，而氟化表面活性剂 H(2)F(6)-TAC 和 F(6)-TAC 被发现增强了酶的稳定性和完整性，这表明它们对抑制剂的敏感性更高。与 H(2)F(6)-TAC 相比，F(6)-TAC 的 ATP 酶比活性更高。当酶与卵磷酯混合时，在 H(2)F(6)-TAC 或 F(6)-TAC 存在下纯化的 ATP 合酶在时间上比用 DDM 纯化的酶更稳定。此外，在存在脂质的情况下，观察到用 DDM 纯化的酶的 ATP 合酶的短暂激活，但在具有 Tris 多醇部分的分子存在下分离的 ATP 合酶持续数周。用氟化表面活性剂重新酯化的酶仍然对抑制剂高度敏感，即使在 6 周后也是如此。
