Myosin heavy chain (MyHC) is the major contractile protein required for skeletal muscle function. This protein is represented by a multigene family in the genome of all vertebrates. As muscles mature and age, different genes encoding distinct MyHC isoforms are expressed. The long term goal of this project is to determine the significance of MyHC isoform diversity to skeletal muscle function during development and aging. To achieve this goal, the functional role of conserved and divergent regions in the myosin rod domain of the fast family of MyNC isoforms will be determined. The myosin rod encodes the structural information for the assembly of myosin molecules into thick filaments, the functional form of myosin in skeletal muscle. The precise mechanism(s) of skeletal muscle myosin thick filament assembly has not been established, nor has the mechanism(s) by which myosin molecules within filaments are replaced by newly synthesized myosin isoforms. In this project we will produce skeletal muscle myosin rods, myosin rod fragments, and myosin rod fusion proteins in bacterial expression systems and use site directed mutagenesis to alter conserved and variable regions of the myosin rod sequence. Recombinant myosin proteins will be used to determine the role of various rod regions and of specific amino acid residues in [1] regulating dimerization specificity of myosin rod a helical coiled coils during biosynthesis, [2] determining the unique solubility characteristics of myosin proteins, [3] the aggregation of myosin rod fragments into ordered paracrystalline structures, [4] the formation of bipolar myosin filaments, and [5] the etiology of human myopathies resulting from myosin rod mutations. The data gained from this project will provide new insights into the structural role of different myosin isoforms that are found in embryonic, neonatal, and adult muscles as well as how changes in the sequences of the myosin rod contribute to MyHC isoform specific properties that are associated with muscle aging. The significance of this research to human health is underscored by recent studies showing that point mutations in the rod domain of myosin genes are associated with human myopathies in cardiac and skeletal muscles.
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