Institute for Physical Chemistry, University of Muenster, Muenster, Germany.
Methods Mol Biol. 2021;2209:119-132. doi: 10.1007/978-1-0716-0935-4_8.
Förster resonance energy transfer (FRET) is a versatile tool to study the conformational dynamics of proteins. Here, we describe the use of confocal and total internal reflection fluorescence (TIRF) microscopy to follow the conformational cycling of DEAD-box helicases on the single molecule level, using the eukaryotic translation initiation factor eIF4A as an illustrative example. Confocal microscopy enables the study of donor-acceptor-labeled molecules in solution, revealing the population of different conformational states present. With TIRF microscopy, surface-immobilized molecules can be imaged as a function of time, revealing sequences of conformational states and the kinetics of conformational changes.
Förster 共振能量转移（FRET）是研究蛋白质构象动力学的一种通用工具。在这里，我们描述了使用共聚焦和全内反射荧光（TIRF）显微镜在单细胞水平上跟踪 DEAD 盒解旋酶构象循环的方法，以真核翻译起始因子 eIF4A 为例。共聚焦显微镜能够研究溶液中供体-受体标记分子，揭示存在的不同构象状态的群体。使用 TIRF 显微镜，可以随时间对表面固定化分子进行成像，揭示构象状态序列和构象变化的动力学。
