A Conformational Change in F-Actin When Myosin Binds: Fluorescence Resonance Energy Transfer Detects an Increase in the Radial Coordinate of Cys-374

Interactions of myosin with actin filaments probably induce conformational changes in actin which are crucial for its function. Fluorescence resonance energy transfer spectroscopy can determine changes in distance (range 10-100 A) between two probes and therefore can sense conformational changes in proteins. We have investigated changes in the radial coordinates of fluorescent probes bound to Cys-374 of F-actin when either of the isozymes (S1A1 and S1A2) of myosin subfragment 1 (S-1) bind. Using 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide as donor and acceptor probes, respectively, we calculated a radius of 13-14 A. This distance increased by approximately 4.5 A upon addition of S-1. No differences were detected between the effects of S1A1 and S1A2. This increase is reversed by MgATP. The average position of the probes on Cys-374 is closer to the filament axis than expected from the current models of F-actin. S-1 increases the radial position of Cys-374 either by direct interaction or via an allosteric conformational change associated with its binding.