Wang Shizhen, Brettmann Joshua B, Nichols Colin G
Center for the Investigation of Membrane Excitability Diseases and Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
Methods Mol Biol. 2018;1684:163-180. doi: 10.1007/978-1-4939-7362-0_13.
Single-molecule FRET (smFRET) can visualize conformational dynamics of individual ion channels in lipid bilayers of defined composition. Dynamic and distance measurements from smFRET, combined with single channel recordings, can provide previously unattainable direct mechanistic insights into ion channel function and modulation. smFRET measurements require site-specific fluorophore labeling between two distinct sites, which is a major challenge for multimeric ion channels. This chapter aims to provide a step-by-step protocol: (1) to design concatemeric constructs with only two cysteine residues within a homotetrameric channel; (2) to express, purify, label, and reconstitute channel proteins; (3) to perform smFRET imaging on channel proteins in liposomes with an objective-based Total Internal Reflection (TIRF) microscope; and finally (4) to analyze the FRET distributions and dynamics that reflect the dynamic conformational transitions of ion channels in membranes.
单分子荧光共振能量转移（smFRET）能够可视化特定组成脂质双层中单个离子通道的构象动力学。结合单通道记录，smFRET进行的动力学和距离测量可为离子通道功能及调节提供前所未有的直接机制性见解。smFRET测量需要在两个不同位点之间进行位点特异性荧光团标记，这对于多聚体离子通道而言是一项重大挑战。本章旨在提供一个逐步的方案：（1）设计在同四聚体通道内仅含两个半胱氨酸残基的串联构建体；（2）表达、纯化、标记并重组通道蛋白；（3）使用基于物镜的全内反射（TIRF）显微镜对脂质体中的通道蛋白进行smFRET成像；最后（4）分析反映膜中离子通道动态构象转变的FRET分布和动力学。
