Calcium is one of the most important regulators of biological systems; its intracellular levels are increased upon stimulation by extracellular agents, such as hormones, drugs, an neurotransmitters. The increase in the concentration of Ca2+ triggers a series of biochemical events that ultimately alter many processes, including those responsible for muscle contraction and the transmission of nerve impulses. The molecular mechanisms by which Ca2+ elicits these responses are poorly understood, though many appear to be mediated by calmodulin, a modulator protein of a large number of regulatory enzymes. To fully understand the Ca2+-triggered responses at the molecular level, we investigate structure-function relationships and mechanisms of regulation of calmodulin-regulated enzymes in vitro. The focus of this particular application is on structural features of the catalytic sites of two prominent enzymes from brain, the type II calmodulin- dependent protein kinase and calcineurin, the calmodulin-regulated phosphoprotein phosphatase. A novel approach to photoaffinity labeling is proposed to identify amino acid residues lining the substrate-binding pockets and to locate these sites on the protein sequences of the target enzymes. Although photoaffinity labeling has been widely used to identify ligand-binding subunits of enzymes and hormone receptors, a low efficiency of photolabeling usually rendered this technique unsuitable for the identification of amino acid residues lining the respective binding sites. According to results of photomechanistic studies this problem can be overcome if one uses photomechanistically competent probes and a frozen matrix. This strategy will be applied to the proposed structure-function work. Substrate analogs will be synthesized which are photochemically competent an form tight complexes with the target enzymes; matrix conditions will be used to modify the target sites. The specificity of photolabeling will be determined by accepted criteria. Following proteolytic cleavage of the modified enzyme, the labeled peptides will be isolated and identified. With the results of the proposed study we expect to locate the target sites on the respective protein sequences and hope to gain a better understanding of enzyme-ligand recognition for calcineurin and the type II calmodulin-dependent protein kinase. In addition, results of the proposed approach to photoaffinity labeling in a frozen matrix may significantly aid future applications of photoaffinity labeling to structure-function analyses of peptide-binding sites of biological importance.
