Ca2+ ions play a major role in the regulation of intra-cellular events in all eukaryotes and the protein calmodulin mediates much of this regulation. Free CA2+ ions bind to calmodulin producing conformational changes in the protein. As a result, calmodulin is able to bind to various target proteins and modify their activity. Calmodulin has four structurally related Ca2+-binding sites but the individual roles of these sites are not well understood. The studies proposed are directed at an analysis of the functions of the individual Ca2+-binding sites of the protein. Using site-directed mutagenesis and bacterial expression, mutant versions of the calmodulin of Drosophila melanogaster in each of which one of the Ca2+-binding sites have been successfully modified so as to prevent Ca2+ binding. It is now planned to analyze the effects of each of these mutations on the regulatory functions of the protein. The Ca2+-induced conformational changes produced in each mutant will be analyzed by a series of techniques including fluorescence enhancement for reporter molecules, tryptic digestion studies, 1H and 113 CD NMR and X-ray crystallography. The capacity of each mutant to bind and activate a series of target enzymes will also be examined. The correlation of data from these two types of experiments should provide much insight into the roles of the individual Ca2+ binding events.
