Hydrogen-1 nuclear magnetic resonance investigation of bovine cardiac troponin C. Comparison of tyrosyl assignments and calcium-induced structural changes to those of two homologous proteins, rabbit skeletal troponin C and bovine brain calmodulin

Abstract

The effect of Ca2+ binding on the 270 MHz 1H NMR spectrum of bovine cardiac troponin C (cTnC) was examined. Assignment of resonances in the aromatic spectral region to tyrosine residues 10, 111 and 150 was made for apo-cTnC and Ca-bound cTnC on the basis of decoupling experiments, pH titrations, temperature-induced changes, and gadolinium broadening experiments. The sequence homology which these tyrosine residues display with residues in 2 previously studied proteins, rabbit skeletal troponin C (sTnC) and bovine brain calmodulin was also used in the assignments. High-affinity Ca binding (up to 2 mol/cTnC) causes large alterations in the environments of tyrosines-10 and -150, indicating that the N terminus is probably buried in the protein interior. The evidence suggests that the environment of tyrosine-150 in Ca-saturated cTnC must closely resemble that of tyrosine-138 in calmodulin, in that it experiences the hydrophobic core of the protein. There is no similarity between these environments in the apoproteins. Dramatic alterations in phenylalanine resonances are seen during the binding of the 3rd mol of Ca, corresponding to filling the sole low affinity site. Comparison of the spectral features of cTnC with those previously reported for sTnC and calmodulin reveals many structural similarities which stem from their high degree of primary sequence homology.