title: Fluorescence methods in the investigation of the DEAD-box helicase mechanism.
abstract: DEAD-box proteins catalyze the ATP-dependent unwinding of RNA duplexes and accompany RNA molecules throughout their cellular life. Conformational changes in the helicase core of DEAD-box proteins are intimately linked to duplex unwinding. In the absence of ligands, the two RecA domains of the helicase core are separated. ATP and RNA binding induces a closure of the cleft between the RecA domains that is coupled to the distortion of bound RNA, leading to duplex destabilization and dissociation of one RNA strand. Reopening of the helicase core occurs after ATP hydrolysis and is coupled to phosphate release and dissociation of the second RNA strand.Fluorescence spectroscopy provides an array of approaches to study intermolecular interactions, local structural rearrangements, or large conformational changes of biomolecules. The fluorescence intensity of a fluorophore reports on its environment, and fluorescence anisotropy reflects the size of the molecular entity the fluorophore is part of. Fluorescence intensity and anisotropy are therefore sensitive probes to report on binding and dissociation events. Fluorescence resonance energy transfer (FRET) reports on the distance between two fluorophores and thus on conformational changes. Single-molecule FRET experiments reveal the distribution of conformational states and the kinetics of their interconversion. This chapter summarizes fluorescence approaches for monitoring individual aspects of DEAD-box protein activity, from nucleotide and RNA binding and RNA unwinding to protein and RNA conformational changes in the catalytic cycle, and illustrates exemplarily how fluorescence-based methods have contributed to understanding the mechanism of DEAD-box helicase-catalyzed RNA unwinding.
authors.affiliation: Institute for Physical Chemistry, University of Muenster, Corrensstrasse 30, 48149, Muenster, Germany, aandr_01@uni-muenster.de.
authors.forename: Alexandra Z
authors.initials: AZ
authors.lastname: Andreou
authors.forename: Dagmar
authors.initials: D
authors.lastname: Klostermeier
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D015336:Molecular Probe Techniques; D009690:Nucleic Acid Conformation; D061848:Optical Imaging / Q000379:methods*; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D012313:RNA; D000255:Adenosine Triphosphate; D053487:DEAD-box RNA Helicases