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.