Our goal is to understand structure/function relationships within the cardiac TnC-TnI complex which lead to a reduction in Ca2+ affinity from either cardiac hypoxia or ischemia, as a consequence of a reduction in pH, or an increase in cAMP dependent phosphorylation of cTnI. It has recently been demonstrated that both acidosis and cTnI phosphorylation results in decreased Ca2+ sensitivity of the cardiac contractile apparatus, leading to impairment of heart function. Specifically, our research objectives are (1) to provide structural insight into pH dependent cTnC-cTnI interactions involved in reduction of Ca2+ affinity for the regulatory site by acidosis and (2) to describe cTnI phosphorylation induced conformational changes ina the cTnIC complex, resulting in a reduced sensitivity of cardiac myofibrils to Ca2+. Working structural models obtained from these studies will provide a molecular basis for development of strategies to artificially modify athe muscle regulatory mechanisms. To identify structural regions responsible for pH dependent cTnIC interactions, we will utilize NMR methodology to define, at the atomic level, pH induced changes occurring in the cTnIC complex. Due to the large molecular weight of the cTnIC complex, about 40 kDa, we will determine pH induced effects on a heterodimeric structure consisting of the Ca2+ saturated N-terminal domain of cTnC, cTnC (1-86), and the C-terminal domain of cTnI, cTnI (138-211). The N-terminal domain of cTnC contains the regulatory Ca2+ binding site and the C-terminal domain of cTnI contains the well known inhibitory peptide and the Ca2+ specific binding site. Spatial relationships between cTn1 and cTnC will be explored using relaxation data from selectively spin labeled proteins generated by protein engineering methods. Cardiac TnIC complexes will be prepared with isotopically labeled cTnC or cTnI. Isotope-edited proton NMR will be used to selectively visualize isotopically labeled amino acids, simplifying the spectrum and decreasing spectral overlap in this large complex. Paramagnetic effects from covalently attached spin labels to isotopically labeled amino acids will provide distance restraints necessary for mapping phosphorylation dependent conformational change in cTnIC and refinement of our current cTnIC model.
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