A proton nuclear magnetic resonance and molecular modeling study of cardiac troponin C. Calcium dependence and aromatic spectral assignments.
Proton (1H) NMR at 360 MHz has been used to characterize calcium-induced spectral changes in bovine cardiac troponin C in more detail than hitherto reported (Hincke, M. T., Sykes, B. D., and Kay, C. M. (1981) Biochemistry 20, 3286-3294). The observed changes are consistent with two equivalents of calcium occupying high affinity sites, with subsequent binding of a single equivalent to a lower affinity site. Two-dimensional J-correlated and nuclear Overhauser effect NOE-correlated and conventional one-dimensional NOE experiments, combined with a consideration of the titration behavior, have allowed all the aromatic signals, and several prominently shifted alpha-CH and methyl group signals, as well as some methionine methyl signals of the calcium-saturated protein, to be assigned. This exercise was facilitated by the construction of a model of the calcium-bound protein based on crystal structure data of the homologous calmodulin and skeletal troponin C, using mutations, energy minimizations, and molecular dynamics simulations, combined with the ring-current shift and NOE prediction program PARSNIP (Reid, D. G., and Saunders, M. R. (1989) J. Biol. Chem. 264, 2003-2012).
MacLachlan
L K
LK
Department of Physical Organic Chemistry, Smith Kline & French Research Limited, Welwyn, Herts, United Kingdom.
Reid
D G
DG
Carter
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012984:Software; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
D014336:Troponin; D019209:Troponin C; D006859:Hydrogen; D002118:Calcium