Dynamic modulation of the regulatory domain of myosin heads by pH, ionic strength, and RLC phosphorylation in synthetic myosin filaments

The position of the myosin head with respect to the filament backbone is th ought to be a function of pH, ionic strength (mu) and the extent of regulat ory light chain (RLC) phosphorylation [Harrington (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5066-5070]. The object of this study is to examine the dyna mics of the proximal part of the myosin head (regulatory domain) which acco mpany the changes in head disposition. The essential light chain was labele d at Cys177 with the indanedione spin-label followed by the exchange of the labeled proteins into myosin. The mobility of the labeled domain was inves tigated with saturation transfer electron paramagnetic resonance in reconst ituted, synthetic myosin filaments. We have found that the release of the h eads from the myosin filament surface by reduction of electrostatic charge is accompanied by a 2-fold increase in the mobility of the regulatory domai n. Phosphorylation of the RLC by myosin light chain kinase resulted in a sm aller 1.5-fold increase of motion, establishing that the head disordering o bserved by electron microscopy [Levine et al. (1996) Biophys. J. 71, 898-90 7] is due to increased mobility of the heads. This result indirectly suppor ts the hypothesis that the RLC phosphorylation effect on potentiation of fo rce arises from a release of heads from the filament surface and a shift of the heads toward actin.