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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 |
|---|---|
| Personen und Körperschaften: | , , , , |
| In: | Protein Science, 7, 1998, 11, S. 2451-2459 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
Wiley
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| Schlagwörter: |
| author_facet |
Hazard, Andrea L. Kohout, Susy C. Stricker, Nicole L. Putkey, John A. Falke, Joseph J. Hazard, Andrea L. Kohout, Susy C. Stricker, Nicole L. Putkey, John A. Falke, Joseph J. |
|---|---|
| author |
Hazard, Andrea L. Kohout, Susy C. Stricker, Nicole L. Putkey, John A. Falke, Joseph J. |
| spellingShingle |
Hazard, Andrea L. Kohout, Susy C. Stricker, Nicole L. Putkey, John A. Falke, Joseph J. Protein Science The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements Molecular Biology Biochemistry |
| author_sort |
hazard, andrea l. |
| spelling |
Hazard, Andrea L. Kohout, Susy C. Stricker, Nicole L. Putkey, John A. Falke, Joseph J. 0961-8368 1469-896X Wiley Molecular Biology Biochemistry http://dx.doi.org/10.1002/pro.5560071123 <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> The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements Protein Science |
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Wiley, 1998 |
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Wiley, 1998 |
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1998 |
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Wiley |
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ai |
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ai |
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Protein Science |
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49 |
| title |
The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements |
| title_unstemmed |
The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements |
| title_full |
The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements |
| title_fullStr |
The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements |
| title_full_unstemmed |
The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements |
| title_short |
The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements |
| title_sort |
the kinetic cycle of cardiac troponin c: calcium binding and dissociation at site ii trigger slow conformational rearrangements |
| topic |
Molecular Biology Biochemistry |
| url |
http://dx.doi.org/10.1002/pro.5560071123 |
| publishDate |
1998 |
| physical |
2451-2459 |
| description |
<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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| author | Hazard, Andrea L., Kohout, Susy C., Stricker, Nicole L., Putkey, John A., Falke, Joseph J. |
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| description | <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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| imprint | Wiley, 1998 |
| imprint_str_mv | Wiley, 1998 |
| institution | DE-Brt1, DE-Zwi2, DE-D161, DE-Zi4, DE-Gla1, DE-15, DE-Pl11, DE-Rs1, DE-14, DE-105, DE-Ch1, DE-L229, DE-D275, DE-Bn3 |
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| spelling | Hazard, Andrea L. Kohout, Susy C. Stricker, Nicole L. Putkey, John A. Falke, Joseph J. 0961-8368 1469-896X Wiley Molecular Biology Biochemistry http://dx.doi.org/10.1002/pro.5560071123 <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> The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements Protein Science |
| spellingShingle | Hazard, Andrea L., Kohout, Susy C., Stricker, Nicole L., Putkey, John A., Falke, Joseph J., Protein Science, The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements, Molecular Biology, Biochemistry |
| title | The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements |
| title_full | The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements |
| title_fullStr | The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements |
| title_full_unstemmed | The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements |
| title_short | The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements |
| title_sort | the kinetic cycle of cardiac troponin c: calcium binding and dissociation at site ii trigger slow conformational rearrangements |
| title_unstemmed | The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements |
| topic | Molecular Biology, Biochemistry |
| url | http://dx.doi.org/10.1002/pro.5560071123 |