The means by which calmodulin regulates a large variety of enzymes involved in a diverse collection of cellular processes continues to be the subject of intense investigation. The mediation by calmodulin of cellular responses to changes in calcium concentration is an intriguing example of the coupling of extracellular messengers to fundamental biological responses. Processes influenced by calmodulin-dependent calcium regulation include muscle contraction, nerve conduction, endo- and exocytosis, cell motility, cytoplasmic transport, cell proliferation and fertilization. Its primary function is the modulation of the activity of many enzymes in response to changes in calcium concentrations. Over two dozen enzymes have been found to be regulated by calmodulin. Calcium dependent regulation occurs through a tight binding interaction of calmodulin with specific domains of the regulated enzymes. An understanding of the molecular basis of the transduction of changes in calcium levels to changes in target enzyme activity is only now beginning to emerge. Quite naturally, the central role of calmodulin in a number of vital cellular responses and processes makes it a target for rational drug design in the context of several muscle, digestive, neurological and oncologic disorders and diseases. Calmodulin is found in all eucaryotic organisms, including the yeasts, and in all tissues. It is a small (148 amino acid residues), acidic, highly conserved protein capable of binding four calcium ions with dissociation constants in the micromolar range. The central goal of this proposal is to contribute to the understanding of the interactions responsible for the calcium dependent, high affinity interaction of calmodulin with target enzymes. The basic approach is to examine structural and dynamic consequences of the interaction of calmodulin with peptides corresponding to the calmodulin-binding domains of regulated enzymes by application of high resolution multidimensional and multinuclear NMR spectroscopy. Specifically, studies are proposed to continue the highly successful experiments designed to efficiently determine the structures of the bound peptides by using isotopically enriched peptides in combination with heteronuclear spectroscopy and characterize the stability of their secondary structure by hydrogen exchange and NMR relaxation methods; refine a current model for the structure of the complex of calmodulin and a peptide corresponding to the smooth muscle myosin light chain kinase calmodulin-binding domain (smMLCKp); undertake the resonance assignment assignment and structural characterization of the high affinity complexes of calmodulin with the binding domain of neuromodulin which forms in both the presence and absence of calcium; undertake the resonance assignment assignment and structural characterization of the high affinity complexes of calmodulin with the binding domains of phosphorylase b, one of which is likely to violate the amphiphilic helix model. These studies should augment, unify and clarify a wide body of information regarding the underlying principles and subtleties associated with calcium-dependent regulation of a enzymes by calmodulin.
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