The overall aims of this research are to understand the molecular mechanism by which muscle proteins convert chemical energy into mechanical work, and to obtain a precise correlation between the physiological, biochemical and structural events of muscular contraction. Novel methods will be applied to striated muscle fibers to probe the relations between biochemical reactions of the contractile proteins, the elementary mechanical steps of the cross-bridge cycle and the corresponding structural motions. Rapid changes in the chemical concentrations of pertinent biochemical species, such as ATP, will be made by laser pulse photolysis of photolabile """"""""caged"""""""" precursors. A newly developed method to secure the fiber ends will be used to improve the uniformity and reproducibility of the contractions. The orientation of fluorescent molecules covalently bound to the myosin heads will be monitored at high time resolution to determine the rates and identity of specific structural changes of the protein molecules. This method combined with laser photolysis, new chromophoric probes, and additional labelling sites made available by recombinant technology will provide orientation and mobility information from several regions of the myosin head. The experiments will be carried out on single muscle fibers of rabbit psoas and frog semitendinosus muscles. Results from this project should significantly advance knowledge of the contractile process and thus bring a greater understanding of both normal and pathological states of striated muscle and other types of cell motility.
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