The broad, long-term objective is to elucidate the molecular details of the regulation of striated muscle contraction by determining how trooping and Ca2+ and myosin, regulate the important tropomyosin-actin interaction. Health Relatedness: The role of each component and their interactions are required to understand the malfunctions associated with certain muscle diseases, e.g., familial hypertrophic cardiomyopathy (FHC) resulting from mutations in Tm, TnT, Tnl and myosin. Tm, in its interaction with actin, plays a key role in determining the equilibria between the 3 states of the muscle thin filament, Blocked, Closed and Open (contraction takes place in the Open state). By interacting with actin and Tm, troponin (assisted by Ca2+) and myosin heads change the equilibrium between the states, thereby turning contraction on and off.
Specific Aims : (a) to determine the relationship between the flexibility of Tm on actin and the number of actin subunits activated (the cooperative unit size); to determine the altered regulatory properties of the FHC Tm mutants; (b) to determine the location of the Tnl and TnT components of the Tn complex on the actinTm filament and the changes that take place in shifting between activity states; (c) to determine the changing interactions of the thin filament components in the organized sarcomere. Methods: In addition to characterization of the 3 states in solution with ATPase, binding and kinetic studies, extensive use will be made of high resolution distance measurements between components with time-resolved fluorescence-detected energy transfer (FRET) in solution and in regions of the sarcomere of components exchanged into single myofibrils. These measurements between specific labeled amino acid residues will provide information about the dynamics of the changes between the components involved in the regulatory process.
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