The long-term objective of this project is to explain force generation and crossbridge kinetics at the molecular level in striated muscle. The primary focus of the proposed work will be to investigate the function of myosin regulatory light chain (MLC-2) in relation to its structure. Molecular structure and function will be correlated using a hybrid of molecular genetics, recombinant DNA technology, and biochemical-biophysical techniques. The primary approach will be to change MLC-2 content or MLC-2 amino acid composition in Drosophila melanogaster indirect flight muscle (IFM), and to correlate these structural alterations with observed changes in contractile function. Structure-function studies of other selected muscle proteins will be initiated using similar approaches. Specifically, in Ca2+-activated skinned IFM fibers, the rate of ATP hydrolysis and the rate at which force responds to length changes will be measured and interpreted within the context of crossbridge theory. ATPase will be measured using a NADH coupled on-line optical assay. The indices of crossbridge function will be compared with those measured in fibers from EMS-induced MLC-2 mutants generated by classical and recombinant DNA techniques. The differences in function between mutant and wild-type fibers will be correlated with observed differences in sarcomeric structure, MLC-2 content, MLC-2 phosphorylation, or protein structure. Using recombinant DNA techniques, a strain of files will also be constructed that is wild type for its expression of the MLC-2 protein in all tubular and supercontractile muscles, but which fail to express the MLC-2 protein in the IFM. The observed differences in contractile function between MLC-2 deficient and wild-type IFM. The observed differences in contractile function between MLC-2 deficient and wild-type IFM will be correlated with the induced structural changes resulting from the complete absence of MLC-2 protein. This mutant strain of flies will serve as the host strain of files for the reintroduction of in vitro mutanized MLC-2 genes by germ line transformation. Structure-function studies on IFM are also planned for other flightless mutants whose genetic lesions map to the IFM actin locus, the myosin heavy chain locus, and the alpha-actinin locus. In addition, functional assays of IFM are planned for four alleles of a putative tropomyosin mutant. In summary, the proposed studies will help define the functional role of specific proteins in contracting muscle and will extend our knowledge of the molecular mechanism of force production and regulation.
