Department of Biochemistry and Molecular Biology, The University of Kansas School of Medicine, Kansas City, KS, United States.
Methods Enzymol. 2021;653:21-48. doi: 10.1016/bs.mie.2020.12.002. Epub 2021 Jan 18.
Transient Receptor Potential (TRP) channels play numerous important physiological roles in humans. Notably, they are involved in temperature sensing and regulation, in the proper functioning of immune and cardiac systems, in skin, hair, and bone physiology and in many types of cancer. Because of their physiological significance there has been much interest in elucidating their molecular mechanisms of action. Recent improvements in eukaryotic protein expression and purification techniques and in cryo-electron microscopy (cryo-EM) have greatly facilitated TRP channel studies. The TRP Vanilloid 2 (TRPV2) channel has emerged as particularly amenable to structural studies and its structure has been solved by both X-ray crystallography and by cryo-EM. Here, we provide an overview of demands posed by X-ray crystallography and cryo-EM on protein sample preparation and outline a step-by-step protocol for preparing the TRPV2 protein for structure determination by both of these techniques.
瞬时受体电位 (TRP) 通道在人类中发挥着许多重要的生理作用。值得注意的是，它们参与温度感应和调节、免疫和心脏系统的正常功能、皮肤、头发和骨骼生理学以及许多类型的癌症。由于它们的生理意义，人们一直热衷于阐明它们的分子作用机制。真核蛋白表达和纯化技术以及冷冻电镜 (cryo-EM) 的最新进展极大地促进了 TRP 通道的研究。TRP 香草素 2 (TRPV2) 通道特别适合于结构研究，其结构已通过 X 射线晶体学和 cryo-EM 解决。在这里，我们概述了 X 射线晶体学和 cryo-EM 对蛋白质样品制备的要求，并概述了使用这两种技术确定 TRPV2 蛋白结构的分步方案。
