The long term objective of the proposed research is to elucidate the structural and chemical basis of muscle contraction. This objective will be pursued by working on three related research projects aimed at the clarification of: (1) the regulation of muscle contraction; (2) function-structure relationships on actin, myosin, and actomyosin interface; and (3) the assembly of myofibrilar structures. (1)The specific aims of the regulation project are: (i) to test the hypothesis that the N-terminal segment of actin is involved in the regulation of contraction in different muscles; (ii) to clarify the interaction of caldesmon with actin; and (iii) to test under physiological conditions the steric block and catalytic mechanisms for regulation of muscle contraction. (2)The goals of the studies on actin, myosin, and actomyosin interface are: (i) to clarify the role of selected sites on actin and myosin in the strong (-ATP) and weak (+ATP) actomyosin binding; and (ii) to determine the role of the SHI peptide, selected sites on actin and myosin, and dynamic motions in actin in the contractile process. The first two projects will involve immunochemical, enzymological, chemical modification, in vitro motility, fluorescence and sedimentation experiments with peptide antibodies, modified and unmodified proteins, actin mutants, myosin and actin filaments, myofibrils, and muscle fibers. (3)The specific aims of the assembly project are: (i) to clarify the role of regulatory factors in the nucleation, growth, and stability of myosin filaments; (ii) to characterize solution properties of titin; and (iii) to examine the formation of myosin filaments in myofibrils by using myosin from skeletal muscle hybridized with light chain-2 from smooth muscle myosin. These studies will employ hydrodynamic, light scattering, electron microscopy, and spectroscopic techniques. The research proposed in this application will result in an improved understanding of contractile processes in muscle and non-muscle cells.
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