We plan to continue current physico-chemical studies on the molecular mechanism of force-generation in vertebrate skeletal muscle. In these studies, we hope to test the helix-coil model proposed for force-generation in muscle and compare the results with those expected according to the classical rotating cross- bridge mechanism. An infrared, iodine photodissociation laser will be used to heat fibers (approximately 5 degrees C) under 1 mu sec and the coupling between transient melting in (the S-2 region of) rigor cross-bridges and the development of tension will be investigated in detail. Experiments will be carried out under ionic conditions where the S-2 elements of rigor bridges are stabilized by association with the thick filament backbone and where they are released from the backbone at various temperatures and sarcomere lengths. The kinetic constants and amplitudes of the force transients will be compared to those of activated fibers under a variety of environmental conditions in these two states in an attempt to demonstrate the presence or absence of common features which could distinguish between the two mechanisms. We also plan to continue our investigations of cross-linking of the rod segments within the thick filament core of glycerinated rigor fibers in an attempt to decouple force generation in the S-2 and S-1 regions of the cross-bridges. The effect of polyclonal antibodies specific to the S-2 region of myosin on contractile force in glycerinated fibers is also under study. These experiments, like the cross-linking studies, are designed to determine whether modulation of S-2 binding of a cycling bridge to the thick filament surface has a direct effect on force-generation. The long range goal of these studies is to understand the process of muscle contraction at a fundamental level. An understanding of this process has wide-ranging medical implications.
