Biochemistry and kinetics of the myosin structural change responsible for force generation in active, spin-labeled muscle fibers

This thesis seeks to more completely describe at the molecular level the structural change that is responsible for generation of muscular force. The goal is to explain how this structural change is coupled to the generation of force and the biochemistry of the muscle protein myosin. Much evidence suggests that the light-chain (LC) domain of myosin rotates as a lever arm to cause actin and myosin filaments to slide past each other, leading to force generation and muscle contraction. In this thesis, biochemical and spectroscopic methods are used to describe the motion of the lever arm in active muscle fibers in both the steady-state and transient time domains. Specifically, an EPR (electron paramagnetic resonance) active site-directed spectroscopic probe has been incorporated into the regulatory light chain of myosin to provide direct information about the orientation of the LC domain in relaxed and contracting muscle. Computer modeling was then used to estimate the behavior of this spectroscopic probe when attached to the regulatory light chain of myosin. To study the coupling between myosin biochemistry and myosin structure, spectroscopic changes upon perturbation of the myosin ATPase cycle with products of the hydrolysis reaction and ATP analogs were observed. Transient changes in the orientation of the LC domain upon relaxation and contraction were also monitored. As this spectroscopic data was being acquired, the force generated by these fibers was simultaneously measured, allowing direct correlation between myosin structural changes and muscle force generation. By combining the information obtained from these studies, as well as information about the known molecular structure and biochemical kinetics of myosin, molecular models of muscle contraction have been evaluated.