Protein Science, Vol 7, Issue 11 2451-2459, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements

A. L. HAZARD, S. C. KOHOUT, N. L. STRICKER, J. A. PUTKEY and J. J. FALKE
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215

The goal of this study is to characterize the kinetic mechanism of Ca(2+) activation and inactivation of cardiac troponin C (cTnC), the Ca(2+) signaling protein which triggers heart muscle contraction. Previous studies have shown that IAANS covalently coupled to Cys84 of wild-type cTnC is sensitive to conformational change caused by Ca(2+) binding to the regulatory site II; the present study also utilizes the C35S mutant, in which Cys84 is the lone cysteine, to ensure the specificity of IAANS labeling. Site II Ca(2+) affinities for cTnC-wt, cTnC-C35S, cTnC-wt-IAANS(2), and cTnC-C35S-IAANS were similar (K(D) = 2-5 {mu}M at 25{deg}C; K(D) = 2-8 {mu}M at 4{deg}C), indicating that neither the IAANS label nor the C35S mutation strongly perturbs site II Ca(2+) affinity. To directly determine the rate of Ca(2+) dissociation from site II, the Ca(2+)-loaded protein was rapidly mixed with a spectroscopically sensitive chelator in a stopped flow spectrometer. The resulting site II Ca(2+) off-rates were k(off) = 700-800 s(-1) (4{deg}C) for both cTnC-wt and cTnC-C35S, yielding calculated macroscopic site II Ca(2+) on-rates of k(on) = k(off)/K(D) = 2-4 X 10(8) M(-1) s(-1) (4{deg}C). As observed for Ca(2+) affinities, neither the C35S mutation nor IAANS labeling significantly altered the Ca(2+) on- and off-rates. Using IAANS fluorescence as a monitor of the protein conformational state, the intramolecular conformational changes ({Delta}) induced by Ca(2+) binding and release at site II were found to be significantly slower than the Ca(2+) on- and off-rates. The conformational rate constants measured for cTnC-wt-IAANS(2) and cTnC-C35S-IAANS were k({Delta}on) = 120-210 s(-1) and k({Delta}off) = 90-260 s(-1) (4{deg}C) . Both conformational events were slowed in cTnC-wt-IAANS(2) relative to cTnC-C35S-IAANS, presumably due to the bulky IAANS probe coupled to Cys35. Together, the results provide a nearly complete kinetic description of the Ca(2+) activation cycle of isolated cTnC, revealing rapid Ca(2+) binding and release at site II accompanied by slow conformational steps that are likely to be retained by the full troponin complex during heart muscle contraction and relaxation.
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