This research, supported by a ROW Career Advancement Award, has the goal of understanding Ca2+-regulated events at the molecular level. Structure-function relationships and mechanisms of regulation of Ca2+/calmodulin-activated enzymes, such as the type II calmodulin-dependent protein kinase (CaM kinase II) are studied. CaM kinase II is found most prominently in the brain, where it is thought to be involved in regulating synaptic activity. Biochemical treatment of CaM kinase II produces a catalytic fragment that can serve as a simplified kinase model for active site-directed structure-function studies. To prepare this monomeric kinase domain in amounts sufficient for biochemical and biophysical analyses, and to add new research capabilities to studies of this enzyme, this research will use recombinant DNA technology. It is the aim of this particular project to subclone a cDNA corresponding to the first 280 amino acids of CaM kinase II into secretion-expression vectors for overexpression in Escherichia coli. The recombinant kinase will be purified and characterized. This work will enhance the possibility of critical structural analysis of an important enzyme.
