Conformational changes of DEAD-box helicases monitored by single molecule fluorescence resonance energy transfer.

DEAD-box proteins catalyze the ATP-dependent unwinding of RNA duplexes. The common unit of these enzymes is a helicase core of two flexibly linked RecA domains. ATP binding and phosphate release control opening and closing of the cleft in the helicase core. This movement coordinates RNA-binding and ATPase activity and is thus central to the function of DEAD-box helicases. In most DEAD box proteins, the helicase core is flanked by ancillary N-and C-terminal domains. Here, we describe single molecule fluorescence resonance energy transfer (smFRET) approaches to directly monitor conformational changes associated with opening and closing of the helicase core. We further outline smFRET strategies to determine the orientation of flanking N- and C-terminal domains of DEAD-box helicases and to assess the effects of regulatory proteins on DEAD-box helicase conformation.