title: The kinetic cycle of cardiac troponin C: calcium binding and dissociation at site II trigger slow conformational rearrangements.
abstract: 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 (KD = 2-5 microM at 25 degrees C; KD = 2-8 microM at 4 degrees 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 degrees C) for both cTnC-wt and cTnC-C35S, yielding calculated macroscopic site II Ca2+ on-rates of k(on) = k(off)/KD = 2-4 x 10(8) M(-1) s(-1) (4 degrees 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 (delta) 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(delta on) = 120-210 s(-1) and k(delta off) = 90-260 s(-1) (4 degrees 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
authors.forename: A L
authors.initials: AL
authors.lastname: Hazard
authors.forename: S C
authors.initials: SC
authors.lastname: Kohout
authors.forename: N L
authors.initials: NL
authors.lastname: Stricker
authors.forename: J A
authors.initials: JA
authors.lastname: Putkey
authors.forename: J J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D007700:Kinetics; D009206:Myocardium / Q000737:chemistry*; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D019209:Troponin C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D011994:Recombinant Proteins; D019209:Troponin C; C011488:IAANS; D002118:Calcium