Type II Calmodulin-Dependent Protein Kinase

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With the goal of elucidating molecular mechanisms by which calmodulin-activated enzymes aid transformation of CA2+-signals into biochemical responses, this project addresses relationships between structure, function, and regulation of the type II calmodulin-dependent protein kinase. The major target is a recombinant, monomeric form of the kinase which contains catalytic and regulatory domains but lacks the association domain. Site-specific mutagenesis will be used to produce additional forms of the kinase and calmodulin. How Mg2+, calmodulin, substrates, and autophosphorylation regulate domain folding, interactions, and topographical arrangements will be a major focus. Chemical crosslinking and active site-directed reagents will be used to probe sequences of events in kinase activation. Fluorescent probes will be attached to specific sites of calmodulin and the kinase for fluorescence and energy transfer studies; suitable metal ions and nucleotide analogs provide additional fluoroprobes. Changes in circular dichroism and in the susceptibility of the protein to proteolytic cleavage will be correlated with corresponding changes in kinase activity and calmodulin- dependence. The X-ray structure of the truncated kinase will be determined in a collaborative project. Current working models of the structure and regulation of this prominent calmodulin-dependent enzyme will be refined by computer modeling.