Muscular contraction is initiated by calcium binding to troponin C in skeletal and cardiac muscles. This binding triggers a series of molecular events which ultimately lead to the cyclic interaction between myosin and actin as ATP is hydrolyzed. This cyclic interaction results in development of tension. The long-term goal of our research program is to understand how the calcium signal is transmitted from its site at troponin C to several other proteins. This signal transmission is fundamental to the overall regulatory mechanism. The approaches taken in this proposal are based on a combination of energetic analysis of multiple-ligand interactions involving calcium and the various proteins that comprise the muscle thin filament and structural perturbations on these proteins induced by calcium binding to troponin C. Proteins that are specifically altered in their sequence and synthetic oligopeptides will be used to assist in delineating alternative routes for information transfer. The methodologies chosen for these studies include steady state fluorimetry, time-resolved millisecond, nanosecond, and picosecond fluorescence spectroscopy, stopped-flow kinetic technique, and other physical methods. The role of tryptophanyl and tyrosyl residues in signal transfer will be studied through analysis of their emission decay and anisotropy decay. The rapid molecular motions of these residues in troponin C, calmodulin and possibly other proteins will be compared with simulation results based on molecular dynamics. Moleculare distances will be determine by fluorescence resonance energy transfer and will serve to report on global structural perturbations that occur when actomysosin ATPase is activated. The relevance of these changes in calcium regulation will be investigated by rapid kinetics techniques. Finally, a comparison will be made between cardiac regulatory proteins and skeletal regulatory proteins in terms of the energetics of multiple-ligand binding and the nature and extent of structural perturbations that can be correlated with functional properties. The proposed studies are expected to advance our knowledge in signal transmission in muscle and contribute toward our understanding of the role of multiple-ligand binding in biological regulation. The proposed work will also enhance our knowledge of the role of very rapid molecular motions in biological functions.
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