Unité Mixte de Recherche 7099, Centre National de Recherche Scientifique and Université Paris 7, Institut de Biologie Physico-Chimique, 13 rue Pierre-et-Marie Curie, F-75005 Paris, France.
Biochemistry. 2012 Feb 21;51(7):1416-30. doi: 10.1021/bi201862v. Epub 2012 Feb 9.
Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.
非离子型两性嵌段聚合物（NAPol）通过两性单体的同尾聚合合成，能够在没有去污剂的情况下保持膜蛋白（MP）的稳定和功能。已经研究了它们的一些生化和生物物理性质及其应用，特别关注它们与经典的基于聚丙烯酸酯的两性嵌段聚合物 A8-35 的互补性。发现盐杆菌中的菌紫质（BR）和莱茵衣藻中的细胞色素 b(6)f 复合物与 NAPol 复合后处于其天然状态且高度稳定。NAPol 捕获的 BR 被证明能够完成其完整的光循环。由于 NAPol 的 pH 不敏感性，可以在 pH 6.8 下记录 NAPol 捕获的来自大肠杆菌的外膜蛋白 X（OmpX）的 NAPol 捕获的溶液核磁共振（NMR）二维（1）H-（15）N 异核单量子相干谱。它们的分辨率与在 pH 8.0 下记录的 OmpX/A8-35 复合物的光谱相似，并可获得溶剂暴露的快速交换酰胺质子的信号。与 A8-35 一样，NAPol 可用于将 MPs 折叠到其天然状态，这里用 BR 和胃饥饿素 G 蛋白偶联受体 GHS-R1a 进行了证明，从而扩展了可折叠条件的范围。在 NAPol 辅助折叠后，GHS-R1a 结合了其四个特异性配体，募集了 arrestin-2，并激活了 G(αq) 蛋白与 GTPγS 的结合。最后，发现 A8-35 和磺化两性嵌段聚合物抑制的 MPs 无细胞合成在 NAPol 存在下非常有效。这些结果为 MP 研究中使用两性嵌段聚合物开辟了广阔的新前景。
