CHEMISTRY AND FUNCTION OF DICTYOSTELIUM CALMODULIN

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Calmodulin is a calcium-binding protein found in all eukaryotic cells; it appears to be responsible for mediating the regulatory effects of calcium on numerous biological processes. The protein has been highly conserved, so its structure and functions can be conveniently examined in lower eukaryotes as well as vertebrate cells. This project would characterize calmodulin from the eukaryotic microorganism Dictyostelium discoideum and explore its roles, particularly in the regulation of motility. Earlier work has revealed that this protein, although functionally similar to vertebrate calmodulins, has several amino acid differences, including the absence of trimethylated lysine residue. Determination of the primary structure will be completed, and the consequences of the amino acid differences will be explored. Interaction with Ca++, troponin I, and vertebrate enzymes will be examined in this regard. Monoclonal antibodies will be raised against Dictyostelium calmodulin; the site (or sites) toward which these antibodies are directed will be determined, and mutants producing calmodulin altered at this site will be sought. Proteins that interact with Dictyostelium calmodulin will be identified and characterized, with emphasis on proteins that may be involved in regulating the structural states or interaction of myosin and actin. The ability of calmodulin to modulate myosin force production through a light chain kinase or other means will be tested directly. Antibodies against Dictyostelium calmodulin, myosin, and selected calmodulin-binding proteins will be used to visualize the localization of these proteins in mitotic and chemotaxing cells; for some of these proteins, monoclonal antibodies will be raised to facilitate purification and to map functional domains. The gene(s) encoding Dictyostelium calmodulin will be isolated and sequenced, as an initial step toward a long range goal of analyzing the function of the protein by genetically modifying it.