Laser photochemically induced dynamic nuclear polarization proton nuclear magnetic resonance studies on three homologous calcium binding proteins: cardiac troponin-C, skeletal troponin-C, and calmodulin

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Abstract
Laser photo-CIDNP [photochemically induced dynamic nuclear polarization] 1H NMR experiments were performed with rabbit skeletal troponin C (sTn-C), bovine cardiac troponin C (cTn-C) and bovine brain calmodulin to study the exposure of histidine and tyrosine residues. In cTn-C, tyrosine residues, 5, 111 and 150 were exposed in the apoprotein, becoming buried as Ca2+ was bound. A similar phenomenon was observed for tyrosine residues 10 and 109 of sTn-C. In calmodulin, only tyrosine-99 was accessible in the apoprotein. The lack of exposure of tyrosine-138 observed with this technique correlates with the buried nature of this residue implied by other criteria. In 6 M urea each apoprotein was unfolded from the standpoint of the tyrosine environments. A large tyrosyl CIDNP effect was obtained for each protein which decreased as Ca2+ was bound, with a stoichiometry of 1 metal ion per protein. This was correlated for cTn-C with the appearance of native resonances representing tyrosine residues 111 and 150 in Ca2+-saturated cTn-C, also with a stoichiometry of one. Analysis of the NMR findings, in the light of other spectroscopic and model building studies on these systems, suggests that the sole high-affinity Ca2+ binding site of cTn-C and sTn-C remaining in 6 M urea is site IV.