Cardiac muscle contraction is regulated by a Ca2+-dependent modulation of protein interactions within the thin filament. A major challenge in cardiac muscle research is to understand this regulation in terms of the structures and structural dynamics of thin filament proteins. This research proposal continues investigations of the mechanism of muscle contraction emphasizing two structural events that underlie its regulation. The first involves the Ca2+ cardiac troponin (cTn) mediated structural perturbation in cardiac troponin I (cTnl) that disrupts the cTnl-actin complex, and the second involves functional movements of cardiac tropomyosin (cTm) during thin filament activation. We hypothesize that the structural changes in cTnl which disrupt the cTnl-actin complex are coupled to functional movements of cTm. These structural and dynamic studies take on special significance in the context of hypertrophic cardiomyopathy (HCM), where single point mutations in cTm lead to heart failure by unknown mechanisms. We will explore some of these mechanisms by testing the hypothesis that certain HCM mutations perturb functional conformational transitions of cTm on the thin filament. The project has four specific aims: (1), establish structural relationships between 33 different probe loci on cTnl with respect to a fixed locus on actin +/- Ca2+-and myosin; (2), establish structural relationships between 10 different probe loci on the C-terminus of cTm with respect to a fixed locus on actin +/- Ca2+-and myosin. These structural relationships are determined from high precision measurements of fluorescence resonance energy transfer (FRET) efficiency, orientation factor KE2, fluorescence polarization (FP) on single, Ca2+-regulated filaments and by fitting these data into current models of the thin filament; (3), characterize slow (ms-ms) structural transitions within cTm during Ca2+-activation of thin filaments; (4), characterize the effects of specific HCM mutations on the structural dynamics of cTm during Ca2+-activation of thin filaments. A particularly novel aspect of the proposed research is that the structural relationships among multiple loci on cTnl and cTm in relaxed and Ca2+-activated thin filaments will be established in the context of human cardiac muscle contraction. Overall, implementation of the proposed work will advance our understanding of the molecular basis of muscle contraction and provide new insights into the mechanisms through which specific mutations in cTm lead to heart failure. ? ?
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