To understand the functionally significan states of a regulatory protein complex, one must directly measure its thermodynamic and kinetic driving forces in conjunction with its ligand induce conformational responses. The major goal of this proposal is to elucidate molecular mechanisms of cooperative structural transitions in the regulatory calcium binding protein calmodulin (CaM) by probing the linkage between calciu binding and conformational change and determining the distinct roles of each o the two homologous domains. Cooperative binding of 4 calcium ions to CaM cause large conformational changes that control its activation of enzymes and structural proteins. CaM has roles in neurotransmission, muscle contraction, fertility and other fundamental physiological processes. Because CaM is essential for eukaryotes, it is difficult to isolate functional mutants that might reveal its molecular logic. In Paramecium, C. Kung found two classes of defective swimmers that were traced to mutations of CaM; these segregated by domain. Mutations in the N domain of Paramecium CaM (PCaM) affected the calciu dependent sodium current while mutations in the C domain affected the Ca dependent potassium current. Three hypotheses are that (1) mutations in N domain primarily affect interdomain interactions rather than calcium affinity of sites I and II, (2) mutations in C domain primarily affect calcium affinity of sites III & IV, and (3) recognition and binding of target proteins by PCaM depend on both calcium affinity of each domain and domain domain interactions. The Research Design will test these by (a) determining the molecular defects that lead to dysfunctional calcium activation of both classes of mutant PCaMs and (b) studying the interactions between mutant PCaMs and selected targets (enzymes, inhibitory peptides & antagonists). Calcium induced conformational switching and energetics of calcium binding will be determined using quantitative proteolytic footprinting, NMR, fluorescence, CD, differential scanning calorimetry, and hydrodynamic methods (analytical ultracentrifugation, chromatography). This analysis of PCaM mutants will contribute to understandin pathways of domain interactions and the distinct roles these domains play in target activation. This may lead to a better understanding of how synchronized changes in calcium levels modulate diverse physiological processes in eukaryotes.
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