Coordination of Actin-Activated Phosphate Release and the Myosin Power-Stroke Detected by Transient Time-Resolved FRET

Abstract

We have used transient kinetics and nanosecond time-resolved FRET to resolve a structural transition in the myosin relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with force-transducing rotation of the myosin light chain domain. Previous research in the absence of actin used optical and EPR spectroscopy to demonstrate that the relay helix of Dictyostelium Discoideum myosin II exists in dynamic equilibrium between a bent pre-power-stroke state, stabilized by ATP, and a straight post-power-stroke state that predominates when ADP or no nucleotide is bound. Kinetics studies showed that ATP binding induces a recovery stroke in which this equilibrium is shifted toward the bent state. In the present study, we ask whether actin binding to the myosin.ADP.Pi complex reverses this transition and if so, how actin-activated helix straightening is coordinated with biochemical transitions of the myosin ATPase cycle. We labeled a Cys-lite Dictyostelium myosin motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We measured time-resolved FRET following stopped-flow mixing of myosin (in the presence of ATP) with actin. These experiments allowed us to measure the structural kinetics of the relay helix during the weak-to-strong actin-binding transition. The results clearly show that actin binding straightens the relay helix in the presence of ATP. The helix straightens prior to ADP dissociation and full formation of the strong binding actomyosin interface. Measurements of actin-activated phosphate release demonstrate that dissociation of the hydrolyzed gamma-phosphate is closely coupled to the structural state of the relay helix during the power-stroke.