Probing RNA Helicase Conformational Changes by Single-Molecule FRET Microscopy.

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.

Citation

Linda Krause, Dagmar Klostermeier. Probing RNA Helicase Conformational Changes by Single-Molecule FRET Microscopy. Methods in molecular biology (Clifton, N.J.). 2021;2209:119-132

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PMID: 33201466

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