Premium
The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements
Author(s) -
Hazard Andrea L.,
Kohout Susy C.,
Stricker Nicole L.,
Putkey John A.,
Falke Joseph J.
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560071123
Subject(s) - chemistry , dissociation (chemistry) , biophysics , kinetic energy , troponin c , calcium , binding site , troponin , stereochemistry , crystallography , biochemistry , cardiology , biology , medicine , physics , organic chemistry , myocardial infarction , quantum mechanics
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.