The long term objective of the proposed research is to elucidate the structural and chemical basis of muscle contraction. This goal will be pursued by working on four related projects aimed at clarifying: (I and II) the interactions of myosin and actin in the weakly and strongly bound states (+/-ATP); (III) regulation of contraction; and (IV) dynamic transitions in myosin heads (S1). (I) The specific aims of the first project are: (i) to complete the mapping of clusters of charged residues on actin involved in the formation of weakly bound actomyosin complexes; (ii) to test the hypothesis on the key role of the N-terminus of actin in the transition between weakly and strongly bound actomyosin states; (iii) to test ionic actomyosin interactions under physiological conditions; and (iv) to alter the N- terminus of actin for labeling with spectroscopic probes. (II) The specific aims of this project are: (i) to prepare action mutants for mapping the sites of strong myosin binding; (ii) to test models of acto-S1 structure by using actin and myosin mutants; and (iii) to determine the functional role of specific actomyosin interaction sites. (III) The aims of the regulation project are: (i) to test model predicted shifts in the arrangement of tropomyosin on actin; and (ii) to clarify the role of troponin I in the regulation of actomyosin function. (IV) The aims of the last project are: (i) to characterize the recently crystallized motor domain of Dictyostelium myosin head (SldC); and (ii) to clarify the role of specific sites on S1 in myosin function and in the structural changes which determine the function. The research proposed in these projects will combine mutagenic, immunochemical, peptide and chemical preparation of materials for probing specific protein sites. The binding and functional interactions of myosin and actin mutants will be evaluated in the in vitro motility assays, force measurements, binding and enzymatic activity measurements, and a large array of biochemical and biophysical experiments. The research proposed in this application will lead to a better understanding of the fundamental biological processes of force and motion generation and the molecular basis of familial cardiac myopathies caused by mutations in beta cardiac myosin.
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