Myosin structural regions that determine muscle shorten*

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): The kinetic and structural mechanisms by which myosin converts the chemical energy of ATP hydrolysis to force and motion are far from being understood. Generally accepted myosin cross-bridge theories have Pi release associated with the force producing power stroke, but steps associated with ADP release rate are thought to be rate limiting for unloaded shortening velocity. However, recent evidence from myosin molecular studies suggests that ADP release is not the only step of the cycle that can influence unloaded myosin shortening velocity. Further, kinetic studies of Drosophila myosins and flight muscle fibers suggest that fast Drosophila myosins may be limited by steps associated with Pi release rather than ADP release rate. Therefore, the KINETIC HYPOTHESIS to be tested is that the unloaded velocity of very fast myosins is limited by steps associated with Pi release while slow myosin velocities are limited primarily by ADP release rate. SPECIFIC AIMS: (1) Test our kinetic hypothesis at the molecular level by varying ATP, Pi and ADP levels in the in vitro sliding filament assay. We will contrast the effect on velocity for two very fast adult Drosophila isoforms compared with two slow embryonic isoforms. (2) Test if Pi release limits unloaded velocity at the fiber level by varying ATP, Pi and ADP levels in the bathing solution of skinned TDT (jump) muscle transgenically expressing the four myosin isoforms. (3) Determine which structural region sets shortening velocity of myosin isoforms. Our STRUCTURAL HYPOTHESIS is that the myosin converter region is primarily responsible for setting differences in unloaded velocity. We will test this hypothesis by performing the same molecular and fiber experiments as described in Aims 1 and 2 on two myosin chimeras previously made by exchanging converter regions between a very fast and a very slow myosin isoform. Significance: Depending on the validity of our kinetic hypothesis, we will either be determining which region influences ADP release or which region influences Pi release. The latter would be highly significant as regions of myosin that set Pi release rate have not been identified. In either case, information on the function of the converter region will be highly informative as the converter is a hotspot for mutations that lead to familial hypertrophic cardiomyopathy (FHC). FHC is an inherited genetic disease that is a major cause of sudden death among young adults.