J. Biol. Chem., Vol. 265, Issue 17, 9754-9763, Jun, 1990
A proton nuclear magnetic resonance and molecular modeling study of cardiac troponin C. Calcium dependence and aromatic spectral assignments
LK MacLachlan, DG Reid and N Carter
Department of Physical Organic Chemistry, Smith Kline & French Research Limited, Welwyn, Herts, United Kingdom.
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).
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