Structural Dynamics Of The Actin Binding Cleft Of Dictyostelium Myosin II Analyzed By Stopped Flow Time-Resolved FRET

Abstract

Using site-directed fluorescence labeling, transient time resolved FRET ([TR]2FRET) and time resolved fluorescence anisotropy, we have measured actin- and nucleotide-induced structural changes within the actin binding cleft of Dictyostelium myosin II. In a recent report using time-resolved pulsed EPR spectroscopy, distances were measured between paramagnetic probes attached to the upper and lower 50 kDa subdomains of Dictyostelium myosin II (Klein et al., PNAS, 105:12867-72). These results support the hypothesis that the actin-binding cleft closes partially upon actin binding, but also suggested that both open and closed conformations are simultaneously present, with nucleotides and actin controlling the open-closed equilibrium. Due to technical constraints, those EPR distance measurements were limited to frozen samples. In the present study, time-resolved fluorescence is used to probe the cleft in solution under more physiologically relevant conditions, including the transient phase of the ATPase reaction. Fluorescent probes were attached to engineered Cys residues in the upper and lower 50 kDa subdomains and used to measure the distance across the cleft. Single probes attached to either subdomain were used in combination with fluorescent nucleotides to monitor the coupling between the actin-binding cleft and the active site. Here we use a combination of transient time resolved FRET and transient time resolved fluorescence anisotropy to probe the equilibrium between open and closed cleft conformations. In the key experiments, a complete nanosecond time-resolved fluorescence decay was measured, defining the detailed distance distribution between probes, every 0.1 ms following rapid mixing (stopped flow), thus yielding high-resolution structural information on the sub-millisecond time scale. The results provide new insights into the coupling between the actin-binding cleft, the active site, and actin binding.