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.
Laser photo-CIDNP 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 of the apoproteins were observed to be 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 one 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 our 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.
D000818:Animals; D001665:Binding Sites; D001921:Brain; D002118:Calcium / Q000032:analysis; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002417:Cattle; D007834:Lasers; D009682:Magnetic Resonance Spectroscopy; D009124:Muscle Proteins; D009132:Muscles / Q000032:analysis*; D009206:Myocardium / Q000032:analysis*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013329:Structure-Activity Relationship; D014336:Troponin; D019209:Troponin C; D014508:Urea
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D009124:Muscle Proteins; D014336:Troponin; D019209:Troponin C; D014508:Urea; D002118:Calcium