The ultimate goal of the proposed research is to understand structure/function relationships that allow myosin to act as a molecular motor, transducing chemical energy into mechanical work during muscle contraction. Efforts in this proposal are focused on elucidating dynamic structural changes at the actin-binding interface of myosin, and correlating these dynamic structural changes with biochemical and mechanical states of the contractile cycle. Fluorescent probes will be introduced at selected sites within the acto-myosin interface using both chemical and photoreactive labelling techniques. The upper 50 kD subdomain of myosin will be photolabelled with methyl coumarin near the 50/20 kD junction and Lys-553 of the lower 50 kD subdomain of myosin will be lableled with fluorescein at Lys-553. A combination of state-of-the-art spectroscopic, biochemical, and mechanical methods will be used to rigorously test current molecular models of muscle contraction. Specifically, the applicant intends to identify the roles of the upper and lower 50 kD subdomains of myosin in the formation of weakly and strongly bound complexes with actin, examine the role of the cleft splitting the 50 kD domain of myosin in mediating the acto- myosin interaction, determine the fraction of actin-attached myosin molecules during isometric contraction and its relationship to muscle fiber stiffness, determine whether or not all strongly bound myosin cross-bridges generate force, and examine how the acto-myosin duty cycle varies with mechanical load.
