Muscle contraction results from cyclic interactions between actin and myosin. The magnitude of developed force is a function of the number actin-myosin contacts that are formed. This interaction in vertebrate striated muscle is regulated by the binding of activator calcium to the NH/2-domain of troponin C (TnC), and this binding results in a series of changes in both the structures of the proteins associated with the thin filament and the energetics of interactions among those proteins. The details of the immediate molecular events resulting from activator calcium binding are still poorly understood. Modeling studies suggest that activator calcium induces substantial reorientations of several helices located in the NH/2-terminal domain of TnC, resulting in an """"""""open"""""""" conformation. This conformation in turn provides specific sites for a strong interaction of TnC with troponin I (TnI). This strong interaction is believed to be responsible for triggering the actin-myosin interaction, thus activating actomyosin ATPase and contractile events. TnI from cardiac muscle has an activity which is related to phosphorylation of the protein in the NH/2-terminal segment and which is not found in skeletal muscle TnI. While the sites of phosphorylation are known, little is known about how this segment may modulate Ca2+ signaling in thin-filament regulation. This application addresses certain aspects of the calcium regulatory mechanism in cardiac muscle. The proposed work has four major goals. (1) The first is identification of pairs of specific residues or helices on TnC that move relative to each other resulting from activator calcium binding. Equilibrium experiments will be done using fluorescence resonance energy transfer (FRET) to establish such calcium-induced conformational changes, followed by transient kinetic experiments to time- resolve the conformational transitions. TnC mutants from cardiac muscle and cardiac-skeletal chimeras will be used for these experiments. (2) The kinetic mechanism of reversible binding of activator calcium to cardiac TnC and the cardiac regulatory system will be studied using transient kinetic methods. It is expected that these kinetic results additionally can be used to track movements of helices in the NH/2-domain. (3) Cardiac TnI has an extension at the NH/2-terminus which is absent in skeletal TnI. We will investigate the relationship of phosphorylation of this extension with its overall conformation. Full-length recombinant mutants and truncated mutants of TnI will be used in FRET, hydrodynamic, and binding studies. (4) The last goal will investigate the relationships of amino acid sequences of the calcium-binding loops and flanking helices in the NH/2-domain of TnC with binding affinity and biological activities. Cardiac muscle TnC mutants with specific alterations in amino acid sequences will be designed and used for this goal. The proposed studies are expected to advance our knowledge of the molecular determinants that are important in thin filament regulation of cardiac muscle. We also expect that the anticipated results may provide a basis to understand the extent to which the calcium triggering mechanism plays a role in abnormal cardiac functions.
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