University of Muenster, Institute for Physical Chemistry, Muenster, Germany.
Methods Enzymol. 2022;673:251-310. doi: 10.1016/bs.mie.2022.03.043. Epub 2022 Apr 22.
RNA helicases are a diverse group of enzymes that catalyze the unwinding of RNA duplex regions in an ATP-dependent reaction. Both the helicase itself and its RNA substrate undergo conformational changes during the reaction, which are amenable to Förster resonance energy transfer (FRET) studies. Single-molecule FRET studies in solution by confocal microscopy and on surfaces by total internal reflection microscopy provide information on different conformers present, their fractional populations in equilibrium, and the rate constants of their inter-conversion. Collectively, the information gained can be integrated into a kinetic and thermodynamic framework that quantitatively describes the conformational dynamics of the helicase studied. FRET experiments also provide distance information to map and model the structures of individual conformational states. The integrated model provides a comprehensive description of the structure and dynamics of the helicase, which can be linked to its biological function. Single-molecule FRET studies have tremendous potential to define the relationship between structure, function and dynamics of RNA helicases and to understand the mechanistic basis for their broad range of biological functions. The focus of this chapter is on providing guidance in the design of single-molecule FRET experiments and on the interpretation of the data obtained. Selected examples illustrate important considerations when analyzing single-molecule experiments, as well as their limitations and possible pitfalls.
RNA 解旋酶是一大类酶，能够在 ATP 依赖的反应中催化 RNA 双链区域的解旋。在反应过程中，解旋酶本身及其 RNA 底物都会发生构象变化，这使得Förster 共振能量转移（FRET）研究成为可能。通过共焦显微镜在溶液中以及通过全内反射显微镜在表面上进行的单分子 FRET 研究，可以提供有关存在的不同构象体、它们在平衡中的分数种群以及它们相互转化的速率常数的信息。综合这些信息可以整合到一个定量描述所研究解旋酶构象动力学的动力学和热力学框架中。FRET 实验还提供距离信息，以绘制和模拟单个构象态的结构。整合模型提供了对解旋酶结构和动力学的全面描述，可以将其与生物学功能联系起来。单分子 FRET 研究具有极大的潜力，可以定义 RNA 解旋酶的结构、功能和动力学之间的关系，并深入了解其广泛的生物学功能的机制基础。本章的重点是提供设计单分子 FRET 实验的指导，并解释获得的数据。选择的示例说明了在分析单分子实验时需要考虑的重要因素，以及它们的局限性和可能的陷阱。
