Reconstitution of Membrane Proteins into Platforms Suitable for Biophysical and Structural Analyses.
Reconstitution of Membrane Proteins into Platforms Suitable for Biophysical and Structural Analyses.
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DOI：
10.1007/978-1-0716-0373-4_14
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发表时间：
2020
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由于其在水溶液中的溶解度低，整体膜蛋白一直是生物物理研究的一个具有挑战性的目标。然而，它们对细胞间的化学和电子信号的重要性使它们成为对细胞和生理过程调节感兴趣的研究人员的迷人目标。由于膜蛋白分流了细胞膜施加的屏障，它们也可以作为药物的入口点，增加了药物研究和开发的兴趣。近年来，由于单颗粒冷冻电镜、x射线晶体学和核磁共振获得了高分辨率的结构信息，对膜蛋白功能的详细了解大大增加。为了进一步推进我们对膜蛋白的机制理解并促进药物开发，在特定条件下生成更多关于这些蛋白质的生物物理和功能数据至关重要。为此，已经开发了不同的技术来稳定天然环境中的整体膜蛋白，这些环境允许进行结构和生物物理研究——双孔、脂质小束和脂质纳米盘。在本章中，我们提供了根据这三种技术重组膜蛋白的详细方案。我们还概述了每种技术的一些可能的应用，并讨论了它们的优点和可能的注意事项。
Integral membrane proteins historically have been challenging targets for biophysical research due to their low solubility in aqueous solution. Their importance for chemical and electrical signaling between cells, however, makes them fascinating targets for investigators interested in the regulation of cellular and physiological processes. Since membrane proteins shunt the barrier imposed by the cell membrane, they also serve as entry points for drugs, adding pharmaceutical research and development to the interests. In recent years, detailed understanding of membrane protein function has significantly increased due to high-resolution structural information obtained from single-particle cryoEM, X-ray crystallography, and NMR. In order to further advance our mechanistic understanding on membrane proteins as well as foster drug development, it is crucial to generate more biophysical and functional data on these proteins under defined conditions. To that end, different techniques have been developed to stabilize integral membrane proteins in native-like environments that allow both structural and biophysical investigations – amphipols, lipid bicelles, and lipid nanodiscs. In this chapter, we provide detailed protocols for the reconstitution of membrane proteins according to these three techniques. We also outline some of the possible applications of each technique and discuss their advantages and possible caveats.