The long term goal of this research is to determine the molecular mechanism of muscular contraction. In this proposal we hope to provide direct evidence for the "rotating myosin head" hypothesis of Huxley (1969), the widely accepted working model for the contractile mechanism. Our approach is to correlate the structural changes of the myosin head (orientation and mobility) with force production (mechanical cycle) and the utilization of ATP (biochemical cycle). In order to delineate physical changes taking place during different steps of the contractile cycle, steady- state equilibria will be perturbed mechanically and biochemically including "trapping" the cycle at certain steps. The orientation and mobility of muscle proteins will be measured using electron paramagnetic resonance spectroscopy (EPR) following the labeling of these proteins with paramagnetic probes. EPR is uniquely suited for this purpose because it is site-specific and it is sensitive to static orientation and rotational mobility of proteins. We have just developed a number of computational methods to extract the myosin head orientation (rather than the spin probe orientation) from EPR spectra. This new technology will give a detailed (3 degree resolution) picture of myosin head reorientation during transitions between the different states in the contractile cycle. We will be able to distinguish between pivoting and spinning motions of the myosin head.
