The goal of this research is to relate physiological, structural and biochemical hypotheses of muscle contraction by measurement of protein motions and biochemical and biochemical rate constants of the actomyosin ATPase in isolated individual myosin molecules and in physiological active skeletal muscle preparations. The mechanical events of the cross-bridge cycle will be correlated with the elementary biochemical processes of the actomyosin ATPase and with structural changes of the contractile proteins. At pre-determined times following sound mechanical and chemical perturbations, the cross-bridge cycle will be arrested by the ultra-rapid freezing for high resolution electron microscopic structural analysis. Structural changes of the myosin head will be monitored using novel bifunctional fluorescent probes having pre-defined orientation relative to the protein crystal coordinates. These reporter groups will enable quantitative determination of relative domain motions when myosin heads decorate actin filaments within muscle fibers. Protein rotational motions and relative domain motions will be measured using a novel time-resolved fluorescence polarization microscope that will resolve the orientation of single molecules in real time. Genetic manipulations will be utilized to investigate the protein structural requirements and the structure-function relationships of muscle-specialized for oscillatory contraction. Results from the project should significantly advance knowledge of the contractile process and thus lead to a greater understanding of both normal and pathological states of skeletal and heart muscle and other types of cell motility.
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