The long term objective of the proposed research is to determine how the structure of Ca++ binding proteins dictate function. The primary system to be utilized is calmodulin dependent regulation of smooth muscle myosin light chain kinase (MLCK) that results in the phosphorylation of myosin light chain (LC) required for myosin movement. Calmodulin, MLCK and LC have been expressed in bacteria. Site specific mutagenesis will be employed together with direct binding and enzyme activity assays to determine the interaction of the three proteins in molecularly precise turns. Portions of calmodulin, troponin C, S100 beta and parvalbumin cDNA will be used to create hybrid proteins in bacteria. The isolated proteins will be evaluated for binding to and activation of MLCK as well as activation of calmodulin kinase II, calcineurin and cGMP phosphodiesterase. Both MLCK and calmodulin kinase 11 contain a substrate inhibitory region contiguous to the calmodulin binding site. Portions of these regulatory areas will be substituted between the two enzymes to examine the functional consequences. We will attempt to define the interaction of the pseudosubstrate region of MLCK with the constitutive enzyme to allow the eventual development of pharmacological agents to specifically inhibit the vascular smooth muscle form of MLCK. Finally, gene substitution by homologous recombination will be utilized to define functions of calmodulin in a genetically fascile fungal system and second site suppressors will be employed to identify calmodulin targets in this organism. Together these studies should contribute to a better understanding of how calmodulin serves as a multipurpose regulator of cellular homeostasis.
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