Increase in concentration of Calcium (Ca2+), arguably the most utilized second messenger in cellular signaling, activates the ubiquitous Ca2+sensor calmodulin which then proceeds to bind and activate numerous enzymes including Ca2+/calmodulin-dependent protein kinase II (CaMKIl). CaMKIl functions as an interpreter of information conveyed by the amplitude, duration and frequency of intracellular calcium transients. The readout of the calcium signal is mediated by a complex series of conformational changes that occur in the confines of the unique dodecameric CaMKIl oligomer. Activation persists well after return of calcium concentration to basal levels through autophosphorylation. The conformational memory of activation is essential for the enzyme's physiological function and is integral for learning and memory. The underlying structural and dynamic basis of activation and of conformational memory is poorly defined. Although an X-ray structure of a monomeric catalytic domain has recently been solved, some of its features seem incompatible with biochemical data. The long term goal of this research is to bridge the structural gap by mapping conformational changes in well defined catalytic intermediates of CaMKIl. The specific aims will test the hypothesis that inactive CaMKIl is autoinhibited by extensive catalytic-regulatory interaction and that Ca2+/CaM binding and autophosphorylation disrupt autoinhibition by disengaging regulatory and catalytic domains. We will utilize site directed spin labeling and electron paramagnetic resonance (SDSL-EPR) to derive local environmental constraints and global distance restraints. The feasibility of this approach is established in our preliminary results which challenge many features of the monomer crystal structure. The SDSL-EPR studies will be extended to the holoenzyme to gain novel insight into the basis of cooperative calmodulin binding and self regulation by autophosphorylation. CaMKIl plays a critical role in learning and memory and may also serve as an autoregulated structural scaffold to assemble protein complexes at key intracellular locations, such as neuronal postsynaptic densities or on cardiac T-tubule membranes. It is implicated in a number of diseases including Parkinson's disease and Schizophrenia making it an attractive target for drug development. Determining CaMKIl mechanisms is of fundamental biochemical importance and may well facilitate development of new compounds with better therapeutic action and minimal side effects.