Puthenveetil Robbins, Christenson Eric T, Vinogradova Olga
Section on Structural and Chemical Biology of Membrane Proteins, Neurosciences and Cellular and Structural Biology Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 35A Convent Dr., Bethesda, MD 20892, USA.
OriGene Technologies, Inc., Rockville, MD 20850, USA.
Membranes (Basel). 2022 Feb 16;12(2):227. doi: 10.3390/membranes12020227.
A plethora of membrane proteins are found along the cell surface and on the convoluted labyrinth of membranes surrounding organelles. Since the advent of various structural biology techniques, a sub-population of these proteins has become accessible to investigation at near-atomic resolutions. The predominant bona fide methods for structure solution, X-ray crystallography and cryo-EM, provide high resolution in three-dimensional space at the cost of neglecting protein motions through time. Though structures provide various rigid snapshots, only an amorphous mechanistic understanding can be inferred from interpolations between these different static states. In this review, we discuss various techniques that have been utilized in observing dynamic conformational intermediaries that remain elusive from rigid structures. More specifically we discuss the application of structural techniques such as NMR, cryo-EM and X-ray crystallography in studying protein dynamics along with complementation by conformational trapping by specific binders such as antibodies. We finally showcase the strength of various biophysical techniques including FRET, EPR and computational approaches using a multitude of succinct examples from GPCRs, transporters and ion channels.
大量膜蛋白存在于细胞表面以及围绕细胞器的复杂膜迷宫中。自从各种结构生物学技术出现以来，这些蛋白中的一部分已能够在接近原子分辨率的水平上进行研究。用于解析结构的主要可靠方法，即X射线晶体学和冷冻电镜，在三维空间中提供了高分辨率，但代价是忽略了蛋白质随时间的运动。尽管结构提供了各种静态快照，但只能从这些不同静态状态之间的插值推断出模糊的机制理解。在本综述中，我们讨论了用于观察从刚性结构中难以捉摸的动态构象中间体的各种技术。更具体地说，我们讨论了诸如核磁共振（NMR）、冷冻电镜和X射线晶体学等结构技术在研究蛋白质动力学方面的应用，以及通过抗体等特定结合剂的构象捕获进行补充。我们最后通过来自G蛋白偶联受体（GPCR）、转运蛋白和离子通道的大量简洁示例展示了包括荧光共振能量转移（FRET）、电子顺磁共振（EPR）和计算方法在内的各种生物物理技术的优势。
