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+ s ignaling protein which triggers heart muscle contraction. Previous stu dies have shown that IAANS covalently coupled to Cys84 of wild-type cT nC 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 IAAN S labeling. Site II Ca2+ affinities for cTnC-wt, cTnC-C35S, cTnC-wt-IA ANS(2), and cTnC-C35S-IAANS were similar (K-D = 2-5 mu M at 25 degrees C; K-D = 2-8 mu M at 4 degrees C), indicating that neither the IAANS label nor the C35S mutation strongly perturbs site II Ca2+ affinity. T o directly determine the rate of Ca2+ dissociation from site II, the C a2+-loaded protein was rapidly mixed with a spectroscopically sensitiv e chelator in a stopped flow spectrometer. The resulting site II Ca2off-rates were k(off) = 700-800 s(-1) (4 degrees C) for both cTnC-wt a nd cTnC-C35S, yielding calculated macroscopic site II Ca2+ on-rates of k(on) = k(off)/K-D = 2-4 x 10(8) M-1 s(-1) (4 degrees C). As observed for Ca2+ affinities, neither the C35S mutation nor IAANS labeling sig nificantly altered the Ca2+ on- and off-rates. Using IAANS fluorescenc e as a monitor of the protein conformational state, the intramolecular conformational changes (Delta) induced by Ca2+ binding and release at site II were found to be significantly slower than the Ca2+ on- and o ff-rates. The conformational rate constants measured for cTnC-wt-LAANS (2) and cTnC-C35S-IAANS were k(Delta on) = 120-210 s(-1) and k(off) = 90-260 s(-1) (4 degrees C). Both conformational events were slowed in cTnC-wt-IAANS(2) relative to cTnC-C35S IAANS, presumably due to the bu lky IAANS probe coupled to Cys35. Together, the results provide a near ly complete kinetic description of the Ca2+ activation cycle of isolat ed cTnC, revealing rapid Ca2+ binding and release at site II accompani ed by slow conformational steps that are likely to be retained by the full troponin complex during heart muscle contraction and relaxation.