The broad, long-term objective is to elucidate the molecular basis of the regulation of muscle contraction by defining the role of each muscle component: actin, tropomyosin (Tm), the troponin components, TnI, TnT, TnC, and myosin. Health Relatedness: This information will help in the understanding of muscle diseases e.g., hypertrophic cardiomyopathy resulting from mutations in Tm, TnT, TnI and myosin and stunned myocardium resulting from coronary artery disease. 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/Open (contraction takes place in the Open state). By interacting with actinTm, the troponin complex and myosin heads change the equilibrium between the states, thereby turning contraction on and off.
The specific aims are to test the hypotheses that: (a) Ca2+ and myosin induce changes in position and conformation of Tm on actin which results in a shift of the equilibrium toward the Open biochemical state, thereby allowing contraction; (b) in the absence of Ca2+, TnI, interacts with Tm in addition to actin, to stabilize the thin filament in the Blocked-state; (c) TnT: (i) inhibits actinTm-S1 ATPase by altering Tm conformation, (ii) increases the cooperativity between actinTm units, (iii) interacts differently with Tm in each of the 3 biochemical states; (d) Tm movement to the thin filament On-activity state (Open-state) occurs during myosin binding after Ca2+-induced TnI dissociation from actin; (e) the two heads of HMM act cooperatively to shift actinTm from the Closed to the Open-state; (f) mutants of Tm found in patients with FHC alter the equilibria between the 3 biochemical states of the thin filament thereby affecting contraction. Methods: In addition to characterization of the 3 states in solution with ATPase, myosin titrations and stopped-flow techniques, extensive use will be made of high resolution distance measurements between components with time-resolved fluorescence energy transfer.
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