Cardiac and skeletal muscle contraction are regulated by the reversible binding of Ca2+ to the thin filament protein and troponin subunit, TnC. This proposal will investigate the complex allosteric interactions underlying this regulation by assembling thin filaments containing altered forms of three of its constituent proteins: TnC, TnT, and tropomyosin (Tm). The experiments will test specific models for conformational changes within the thin filament, measure long range interactions within the thin filament, probe the importance of specific regions of TnT and Tm for these interactions, and develop a more detailed model of TnT structure. The thin filament will be analyzed as a linear lattice with measurable nearest neighbor interactions along the filament. CBMII, a mutant cardiac TnC that fails to bind Ca2+ at the regulatory, Ca2+ -specific binding site will be used to: (a) measure Ca2+ -sensitive interactions between thin filament-bound troponin molecules and show how these interactions are perturbed by alterations in the important N- and C- termini of Tm; (b) determine the relationship between Ca2+ binding to the thin filament regulatory sites and functional activation of the thin filament; (c) determine the effect of the thick filament protein myosin on the interactions between troponins and the effect of these interactions on myosin binding to actin. Using Tm molecules that are altered at either end, analyze thin filament assembly as a lattice binding problem, and determine the effects of Ca2+ and either end of Tm on the process. Perform structure-function studies of TnT, by determining the functional consequences after deleting specific regions of the molecule, and by investigating the detailed structure of TnT by X-ray crystallography. Study myosin-induced changes in thin filament conformation, assembly, allosteric interactions, and functional activation. By these studies of the molecular processes that directly regulate muscle contraction, obtain insights that aid understanding of normal, adaptive, and pathological cardiac and skeletal muscle function.
