Biochemical regulation is of fundamental importance to healthy function, and breakdown of regulatory mechanisms generally leads to pathological conditions and to uncontrolled proliferation or cell death. Divalent calcium is a major regulator of intracellular processes and the objective of this proposal is to investigate the molecular basis for this regulation. To date, studies have focused on the structures of the calcium-binding proteins calmodulin and troponin C, as well as target proteins whose activities they regulate: myosin light chain kinase and phosphorylase kinase. In addition, studies have been done on the cAMP- dependent Protein Kinase and the Protein Kinase Inhibitor protein. In the next funding period we propose to pursue structural studies of: * the calmodulin/myosin light chain kinase interaction using truncated and substituted model peptides as well as myosin light chain kinase truncation mutants; * the calmodulin/phosphorylase kinase interaction using model peptides as well as intact catalytic subunit of phosphorylase kinase; * troponin C complexed with its target protein troponin I and with various peptides. Small-angle X-ray scattering and neutron scattering with contrast variation will be used to characterize the overall shapes and interactions of the components in these systems in different states of activation and/or association. Circular Dichroism and Fourier Transform Infrared spectroscopy will be used to evaluate secondary structure content, and to monitor changes in secondary structure that accompany changes in functional state and/or association. The long term goal is to understand the molecular mechanisms in calcium regulation of biochemical processes.
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