The long term goal of this proposal is to elucidate the quantitative relationships between fluctuations of free Ca2+ and cyclic nucleotides in heart muscle. The regulatory protein calmodulin (CaM) plays a key role in this system since it mediates Ca2+ stimulation of the Ca2+ sensitive phosphodiesterase and several other enzymes important for regulation of cardiac function. In this study, particular emphasis is placed on the energetics and chemistry for interactions between CaM and CaM regulated enzymes. Thermodynamic calculations for interactions between Ca2+, CaM, and CaM regulated enzymes predict that the Ca2+ concentration dependence for activation of each enzyme will vary in a manner tht can be quantitatively predicted from the free energy of coupling for binding of Ca2+ and the enzyme to CaM. It is also hypothesized that Ca2+ activation of CaM regulated enzymes will exhibit positive cooperativity to an extent which varies with the intrinsic energy coupling of the system, the concentration of the enzyme, and substrates.
Specific aims of this proposal include quantitation of Ca2+ activation and Ca2+ binding to the CaM sensitive phosphodiesterase-CaM system and the myosin light chain kinase-CaM system and an examination of the effect of phosphorylation on energy coupling and Ca2+ sensitivity of the systems. Furthermore, we propose to quantitate binding of CaM to the CaM binding domain of myosin light chain kinase and related peptides in which the primary sequence has been systematically varied. These experiments will allow us to systematically define the chemistry for CaM-enzyme interactions and evaluate the importance of specific amino acid residues for the stability of CaM-enzyme complexes.
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