The major contractile protein required for skeletal muscle function is the myosin heavy chain (MyHC). This protein is encoded by a large and diverse multigene family whose members are differentially expressed in developing muscle. The long term goal of the research described in this application is to determine the significance of myosin isoform diversity in skeletal muscle development. To achieve this goal the functional role of conserved and divergent domains in chicken fast MyHC isoforms will be determined. In the first objective the functional properties associated with a highly conserved 28 aa sequence within the filament-forming light meromyosin (LMM) domain will be studied. The entire LMM of a chicken MyHC has been cloned and produced in a bacterial expression system. Site-directed mutagenesis will be used to determine the role of the conserved sequence in myosin aggregation. Fusion proteins containing a globular domain at the N-terminus of the myosin rod will be produced to distinguish the effects of LMM mutations on the parallel and anti-parallel interactions involved in bipolar filament formation.
In aim 2, divergent regions in the LMM domain responsible for the observation that only identical MyHC isoforms form a stable a-helical coiled-coil will be identified using bacterial expression vectors encoding chimeric LMM proteins comprised of neonatal and adult sequences. The ability of these chimeric LMMs to form a-helical coiled-coil dimers with neonatal and adult LMMs will be determined. Subsequent studies will identify the minimum neonatal LMM sequence necessary to inhibit the formation of a dimer with adult LMM. In the final aim we test the hypothesis that sequence diversity in the hinge domain of myosin rods affects myosin-based motility. Cloned MyHC and light chain subunits are being co-expressed to produce recombinant myosin active in an -in vitro motility assay. The effect of mutations to the hinge domain of MyHC will be determined. The data gained from our studies will increase our understanding of the mechanism of filament formation and which aa changes in chicken isoforms inhibit the formation of heterodimeric myosins. In addition, the production of a complete and active recombinant myosin will provide a tool for determining how sequence diversity among isoforms affects myosin function.
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