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The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements
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| Zeitschriftentitel: | Protein Science |
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| Personen und Körperschaften: | , , , , |
| In: | Protein Science, 7, 1998, 11, S. 2451-2459 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
Wiley
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| Schlagwörter: |
| Zusammenfassung: | <jats:title>Abstract</jats:title><jats:p>The goal of this study is to characterize the kinetic mechanism of Ca<jats:sup>2+</jats:sup> activation and inactivation of cardiac troponin C (cTnC), the Ca<jats:sup>2+</jats:sup> 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<jats:sup>2+</jats:sup> 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<jats:sup>2+</jats:sup> affinities for cTnC‐wt, cTnC‐C35S, cTnC‐wt‐IAANS<jats:sub>2</jats:sub>, and cTnC‐C35S‐IAANS were similar (<jats:italic>K</jats:italic><jats:sub><jats:italic>D</jats:italic></jats:sub> = 2–5 μM at 25°C; <jats:italic>K<jats:sub>D</jats:sub> =</jats:italic> 2–8 μM at 4°C), indicating that neither the IAANS label nor the C35S mutation strongly perturbs site II Ca<jats:sup>2+</jats:sup> affinity. To directly determine the rate of Ca<jats:sup>2+</jats:sup> dissociation from site II, the Ca<jats:sup>2+</jats:sup>‐loaded protein was rapidly mixed with a spectroscopically sensitive chelator in a stopped flow spectrometer. The resulting site II Ca<jats:sup>2+</jats:sup> off‐rates were <jats:italic>k<jats:sub>on</jats:sub> =</jats:italic> 700–800 s<jats:sup>−1</jats:sup> (4°C) for both cTnC‐wt and cTnC‐C35S, yielding calculated macroscopic site II Ca<jats:sup>2+</jats:sup> on‐rates of <jats:italic>k<jats:sub>on</jats:sub> = k<jats:sub>off</jats:sub>/K<jats:sub>D</jats:sub> =</jats:italic> 2–4 × 10<jats:sup>8</jats:sup> M<jats:sup>−1</jats:sup> s<jats:sup>−1</jats:sup> (4°C). As observed for Ca<jats:sup>2+</jats:sup> affinities, neither the C35S mutation nor IAANS labeling significantly altered the Ca<jats:sup>2+</jats:sup> on‐ and off‐rates. Using IAANS fluorescence as a monitor of the protein conformational state, the intramolecular conformational changes (Δ) induced by Ca<jats:sup>2+</jats:sup> binding and release at site II were found to be significantly slower than the Ca<jats:sup>2+</jats:sup> on‐ and off‐rates. The conformational rate constants measured for cTnC‐wt‐IAANS<jats:sub>2</jats:sub> and cTnC‐C35S‐IAANS were <jats:italic>k<jats:sub>Δon</jats:sub> =</jats:italic> 120–210 s<jats:sup>−1</jats:sup> and <jats:italic>k<jats:sub>Δoff</jats:sub> =</jats:italic> 90–260 s<jats:sup>−1</jats:sup> (4°C). Both conformational events were slowed in cTnC‐wt‐IAANS<jats:sub>2</jats:sub> 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<jats:sup>2+</jats:sup> activation cycle of isolated cTnC, revealing rapid Ca<jats:sup>2+</jats:sup> 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.</jats:p> |
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| Umfang: | 2451-2459 |
| ISSN: |
0961-8368
1469-896X |
| DOI: | 10.1002/pro.5560071123 |