The long term objective of the proposed research is to elucidate the structural and chemical events in myosin, which lead to the contraction of muscle. This objective will be pursued by undertaking three inter-related research projects aimed at the clarification of (1) the structure and interaction of myosin heads with actin at the molecular level; (2) the function and structural changes in myosin filaments in intact myofibrils and muscle fibers; (3) the assembly of myosin molecules into functional filament units, and the properties of such filaments. (1) The specific aims of the proposed studies on the myosin head are: i) to clarify the role of specific sites on myosin and actin in actomyosin binding; ii) to derive new structural information and define structure-function relationships on the myosin head. These studies will involve diverse biochemical and biophysical methods including site specific chemical modifications, chemical cross-linkings, selective proteolysis, immunochemical techniques, electron microscopy, and fluorescence techniques. (2) The specific aims of the studies on the function and structural changes in myosin filaments are: i) to determine the binding of myosin to actin in the presence of ATP by using synthetic filament systems, myofibrils, and muscle fibers; ii) to clarify the effect of nucleotide analogue (AMPPNP) on the conformation of myosin in myofibrils and muscle fibers; iii) to explore contraction and stress-induced changes in myosin. This work will utilize proteolytic methods, chemical cross-linkings, and chemical modification reactions to test different regions on myosin, and to measure its binding to actin. (3) The specific aims of the work on the assembly of myosin into filaments are: i) to characterize the nucleation step in filament assembly; ii) to clarify the factors involved in the regulation of filament nucleation and growth. These studies will employ hydrodynamic, light scattering, electron microscopy, and spectroscopic techniques. The research proposed in this application will bring an improved understanding of contractile processes in muscle and non-muscle cells.
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