The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements
✍ Scribed by Andrea L. Hazard; Susy C. Kohout; Nicole L. Stricker; John A. Putkey; Joseph J. Falke
- Book ID
- 105356571
- Publisher
- Cold Spring Harbor Laboratory Press
- Year
- 1998
- Tongue
- English
- Weight
- 983 KB
- Volume
- 7
- Category
- Article
- ISSN
- 0961-8368
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
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 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 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~on~ = 700–800 s^−1^ (4°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 × 10^8^ M^−1^ s^−1^ (4°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 (Δ) 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~Δon~ = 120–210 s^−1^ and k~Δoff~ = 90–260 s^−1^ (4°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.