The goal of this project is to determine three-dimensional structures of several muscle proteins, with special emphasis on those involved in the regulation of muscle contraction, and to relate the resulting structures to physiological events. Tropomyosin and troponin are key components of thin-filament based regulation in skeletal muscle and tropomyosin is also found as a component of actin filaments in a range of smooth muscle and non-muscle cells. We are proposing to complete the structural analysis of tropomyosin to the limit of resolution of the crystals, which is about 3.5 angstroms at present. We will construct an atomic model for the protein, and refine it using restraining methods and/or molecular dynamics. The resulting structure will be related to its role in muscle regulation. We will also determine low resolution structures of complexes of tropomyosin and troponin and fragments of actin in the Bailey crystals at low and high calcium concentrations, to reveal gross changes in the regulatory complex during activation. We are proposing to solve the structure of smooth muscle regulatory complexes, including the tropomyosin-caldesmon complex at low (17 angstrom) resolution to reveal the attachment site of caldesmon on tropomyosin. We will also bind caldesmon fragments containing the putative tropomyosin binding regions of caldesmon in crystals and determine their binding sites on tropomyosin. The results will be compared to those of troponin-containing tropomyosin crystals. We will also determine the locations of the AMP and ATP sites in adenylate kinase, and resulting conformational changes that result on substrate binding, by completing the structure of this enzyme to 2.2 angstroms resolution. The significance of this work is two-fold. The enzyme is a key player in the """"""""energy charge"""""""" of the cell, catalyzing the reaction MgATP + AMP <--> MgADP + ADP. Furthermore, adenylate kinase is a close """"""""relative"""""""" of one domain of the cystic fibrosis gene product, and although not implicated in cystic fibrosis, the study of adenylate kinase may reveal aspects of the function of the cft protein.
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