Renault Marie, Saurel Olivier, Demange Pascal, Reat Valérie, Milon Alain
UPS, Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UMR 5089, Toulouse, France.
Methods Mol Biol. 2010;654:321-39. doi: 10.1007/978-1-60761-762-4_17.
Structure determination of integral membrane proteins is one of the most important challenges of structural biology. Over the last 7 years, solution-state NMR spectroscopy has become an increasingly useful approach for 3D structure determination and dynamical analysis of membrane proteins solubilized in detergent micelles. We describe herein an ensemble of methods applied in this context, including isotopic labelling, in vitro refolding procedure, and state-of-the-art NMR experiments required for the structure determination of high molecular weight molecular complexes. Furthermore, the basic principles of spectrum interpretation and 3D structure calculation are reported. This approach is illustrated with the structural study of the transmembrane domain of the outer membrane protein A from Klebsiella pneumoniae (KpOmpA).
整合膜蛋白的结构测定是结构生物学面临的最重要挑战之一。在过去7年中，溶液态核磁共振光谱已成为一种越来越有用的方法，用于确定溶解在去污剂胶束中的膜蛋白的三维结构并进行动力学分析。我们在此描述了在此背景下应用的一系列方法，包括同位素标记、体外重折叠程序以及确定高分子量分子复合物结构所需的最新核磁共振实验。此外，还报告了光谱解释和三维结构计算的基本原理。通过对肺炎克雷伯菌外膜蛋白A（KpOmpA）跨膜结构域的结构研究来说明这种方法。
