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

The goal of this study is to characterize the kinetic mechanism of Ca²⁺ activation and inactivation of cardiac troponin C (cTnC), the Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ affinities for cTnC-wt, cTnC-C35S, cTnC-wt-IAANS₂, and cTnC-C35S- IAANS were similar (K(D) = 2-5 μM at 25 °C; K(D) = 2-8 μM at 4 °C), indicating that neither the IAANS label nor the C35S mutation strongly perturbs site II Ca²⁺ affinity. To directly determine the rate of Ca²⁺ dissociation from site II, the Ca²⁺-loaded protein was rapidly mixed with a spectroscopically sensitive chelator in a stopped flow spectrometer. The resulting site II Ca²⁺ off-rates were k(off) = 700-800 s^-1 (4°C) for both cTnC-wt and cTnC-C35S, yielding calculated macroscopic site II Ca²⁺ on-rates of k(on) = k(off)/K(D) = 2-4 x 10⁸ M^-1 s^-1 (4°C). As observed for Ca²⁺ affinities, neither the C35S mutation nor IAANS labeling significantly altered the Ca²⁺ on- and off-rates. Using IAANS fluorescence as a monitor of the protein conformational state, the intramolecular conformational changes (Δ) induced by Ca²⁺ binding and release at site II were found to be significantly slower than the Ca²⁺ on- and off-rates. The conformational rate constants measured for cTnC-wt-IAANS₂ and cTnC-C35S- IAANS were k(Δon) = 120-210 s^-1 and k(Δoff) = 90-260 s^-1 (4°C) . Both conformational events were slowed in cTnC-wt-IAANS₂ 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 Ca²⁺ activation cycle of isolated cTnC, revealing rapid Ca²⁺ 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.