Determination of rate constants for conformational changes of RNA helicases by single-molecule FRET TIRF microscopy.

Abstract

RNA helicases couple nucleotide-driven conformational changes to the unwinding of RNA duplexes. Interaction partners can regulate helicase activity by altering the rate constants of these conformational changes. Single-molecule FRET experiments on donor/acceptor-labeled, immobilized molecules are ideally suited to monitor conformational changes in real time and to extract rate constants for these processes. This article provides guidance on how to design, perform, and analyze single-molecule FRET experiments by TIRF microscopy. It covers the theoretical background of FRET and single-molecule TIRF microscopy, the considerations to prepare proteins of interest for donor/acceptor labeling and surface immobilization, and the principles and procedures of data analysis, including image analysis and the determination of FRET time traces, the extraction of rate constants from FRET time traces, and the general conclusions that can be drawn from these data. A case study, using the DEAD-box protein eIF4A as an example, highlights how single-molecule FRET studies have been instrumental in understanding the role of conformational changes for duplex unwinding and for the regulation of helicase activities. Selected examples illustrate which conclusions can be drawn from the kinetic data obtained, highlight possible pitfalls in data analysis and interpretation, and outline how kinetic models can be related to functionally relevant states.