Pocanschi Cosmin L, Dahmane Tassadite, Gohon Yann, Rappaport Fabrice, Apell Hans-Jürgen, Kleinschmidt Jörg H, Popot Jean-Luc
Fachbereich Biologie, Fach M 694, Universität Konstanz, D-78457 Konstanz, Germany.
Biochemistry. 2006 Nov 28;45(47):13954-61. doi: 10.1021/bi0616706.
Among the major obstacles to pharmacological and structural studies of integral membrane proteins (MPs) are their natural scarcity and the difficulty in overproducing them in their native form. MPs can be overexpressed in the non-native state as inclusion bodies, but inducing them to achieve their functional three-dimensional structure has proven to be a major challenge. We describe here the use of an amphipathic polymer, amphipol A8-35, as a novel environment that allows both beta-barrel and alpha-helical MPs to fold to their native state, in the absence of detergents or lipids. Amphipols, which are extremely mild surfactants, appear to favor the formation of native intramolecular protein-protein interactions over intermolecular or protein-surfactant ones. The feasibility of the approach is demonstrated using as models OmpA and FomA, two outer membrane proteins from the eubacteria Escherichia coli and Fusobacterium nucleatum, respectively, and bacteriorhodopsin, a light-driven proton pump from the plasma membrane of the archaebacterium Halobacterium salinarium.
完整膜蛋白（MPs）进行药理学和结构研究的主要障碍包括其天然稀缺性以及以天然形式过量表达的困难。MPs可以以包涵体的形式在非天然状态下过度表达，但诱导它们形成功能性三维结构已被证明是一项重大挑战。我们在此描述了使用两亲聚合物A8-35作为一种新环境，在不存在去污剂或脂质的情况下，使β桶状和α螺旋状MPs都能折叠成天然状态。两亲分子是极其温和的表面活性剂，似乎更有利于形成天然的分子内蛋白质-蛋白质相互作用，而不是分子间或蛋白质-表面活性剂相互作用。分别以来自真细菌大肠杆菌和具核梭杆菌的两种外膜蛋白OmpA和FomA，以及来自古细菌盐生盐杆菌质膜的光驱动质子泵细菌视紫红质作为模型，证明了该方法的可行性。
