Institute of Structural Biology, Biomedical Center, University of Bonn, Bonn, Germany.
Department of Life Sciences, Imperial College London, London, UK.
Methods Mol Biol. 2020;2168:313-333. doi: 10.1007/978-1-0716-0724-4_15.
Every membrane protein is involved in close interactions with the lipid environment of cellular membranes. The annular lipids, that are in direct contact with the polypeptide, can in principle be seen as an integral part of its structure, akin to the first hydration shell of soluble proteins. It is therefore desirable to investigate the structure of membrane proteins and especially their conformational flexibility under conditions that are as close as possible to their native state. This can be achieved by reconstituting the protein into proteoliposomes, nanodiscs, or bicelles. In recent years, PELDOR/DEER spectroscopy has proved to be a very useful method to study the structure and function of membrane proteins in such artificial membrane environments. The technique complements both X-ray crystallography and cryo-EM and can be used in combination with virtually any artificial membrane environment and under certain circumstances even in native membranes. Of the above-mentioned membrane mimics, bicelles are currently the least often used for PELDOR studies, although they offer some advantages, especially their ease of use. Here, we provide a step-by-step protocol for studying a bicelle reconstituted membrane protein with PELDOR/DEER spectroscopy.
每种膜蛋白都与细胞膜的脂质环境密切相互作用。与多肽直接接触的环形脂质，原则上可以被视为其结构的一个组成部分，类似于可溶性蛋白质的第一水合壳。因此，理想的情况是在尽可能接近其天然状态的条件下研究膜蛋白的结构，尤其是它们的构象灵活性。这可以通过将蛋白质重构成脂蛋白体、纳米盘或双体来实现。近年来，PELDOR/DEER 光谱学已被证明是一种非常有用的方法，可用于研究此类人工膜环境中的膜蛋白的结构和功能。该技术补充了 X 射线晶体学和冷冻电镜，并可与几乎任何人工膜环境结合使用，在某些情况下甚至可在天然膜中使用。在上述膜模拟物中，双体目前在 PELDOR 研究中使用得最少，尽管它们具有一些优势，尤其是易于使用。在这里，我们提供了一个使用 PELDOR/DEER 光谱学研究双体重组膜蛋白的分步协议。
