Kinetic studies of calcium and cardiac troponin I peptide binding to human cardiac troponin C using NMR spectroscopy

Monica X. Li, Erik J. Saude, Xu Wang, Joyce R. Pearlstone, Lawrence B. Smillie, Brian D. Sykes

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Ca2+ and human cardiac troponin I (cTnI) peptide binding to human cardiac troponin C (cTnC) have been investigated with the use of 2D {1H,15N} HSQC NMR spectroscopy. The spectral intensity, chemical shift, and line-shape changes were analyzed to obtain the dissociation (KD) and off-rate (koff) constants at 30 °C. The results show that sites III and IV exhibit 100-fold higher Ca2+ affinity than site II (KD(III,IV)≈0.2 μM), but site II is partially occupied before sites III and IV are saturated. The addition of the first two equivalents of Ca2+ saturates 90% of sites III and IV and 20% of site II. This suggests that the Ca2+ occupancy of all three sites may contribute to the Ca2+-dependent regulation in muscle contraction. We have determined a koff of 5000 s-1 for site II Ca2+ dissociation at 30 °C. Such a rapid off-rate had not been previously measured. Three cTnI peptides, cTnI34-71, cTnI128-1477, and cTnI147-163, were titrated to Ca2+-saturated cTnC. In each case, the binding occurs with a 1:1 stoichiometry. The determined KD and koff values are 1 μM and 5 s-1 for cTnI34-71, 78 ± 10 μM and 5000 s-1 for cTnI128-147, and 150 ± 10 μM and 5000 s-1 for cTnI147-163, respectively. Thus, the dissociation of Ca2+ from site II and cTnI128-147 and cTnI147-163 from cTnC are rapid enough to be involved in the contraction/relaxation cycle of cardiac muscle, while that of cTnI34_71 from cTnC may be too slow for this process.

Original languageEnglish (US)
Pages (from-to)245-256
Number of pages12
JournalEuropean Biophysics Journal
Volume31
Issue number4
DOIs
StatePublished - Jul 2002
Externally publishedYes

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Troponin C
Troponin I
Magnetic Resonance Spectroscopy
Calcium
Peptides
Muscle Contraction
Myocardium

Keywords

  • Calcium binding
  • Kinetics
  • NMR
  • Troponin C

ASJC Scopus subject areas

  • Biophysics

Cite this

Kinetic studies of calcium and cardiac troponin I peptide binding to human cardiac troponin C using NMR spectroscopy. / Li, Monica X.; Saude, Erik J.; Wang, Xu; Pearlstone, Joyce R.; Smillie, Lawrence B.; Sykes, Brian D.

In: European Biophysics Journal, Vol. 31, No. 4, 07.2002, p. 245-256.

Research output: Contribution to journalArticle

Li, Monica X. ; Saude, Erik J. ; Wang, Xu ; Pearlstone, Joyce R. ; Smillie, Lawrence B. ; Sykes, Brian D. / Kinetic studies of calcium and cardiac troponin I peptide binding to human cardiac troponin C using NMR spectroscopy. In: European Biophysics Journal. 2002 ; Vol. 31, No. 4. pp. 245-256.
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abstract = "Ca2+ and human cardiac troponin I (cTnI) peptide binding to human cardiac troponin C (cTnC) have been investigated with the use of 2D {1H,15N} HSQC NMR spectroscopy. The spectral intensity, chemical shift, and line-shape changes were analyzed to obtain the dissociation (KD) and off-rate (koff) constants at 30 °C. The results show that sites III and IV exhibit 100-fold higher Ca2+ affinity than site II (KD(III,IV)≈0.2 μM), but site II is partially occupied before sites III and IV are saturated. The addition of the first two equivalents of Ca2+ saturates 90{\%} of sites III and IV and 20{\%} of site II. This suggests that the Ca2+ occupancy of all three sites may contribute to the Ca2+-dependent regulation in muscle contraction. We have determined a koff of 5000 s-1 for site II Ca2+ dissociation at 30 °C. Such a rapid off-rate had not been previously measured. Three cTnI peptides, cTnI34-71, cTnI128-1477, and cTnI147-163, were titrated to Ca2+-saturated cTnC. In each case, the binding occurs with a 1:1 stoichiometry. The determined KD and koff values are 1 μM and 5 s-1 for cTnI34-71, 78 ± 10 μM and 5000 s-1 for cTnI128-147, and 150 ± 10 μM and 5000 s-1 for cTnI147-163, respectively. Thus, the dissociation of Ca2+ from site II and cTnI128-147 and cTnI147-163 from cTnC are rapid enough to be involved in the contraction/relaxation cycle of cardiac muscle, while that of cTnI34_71 from cTnC may be too slow for this process.",
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AU - Li, Monica X.

AU - Saude, Erik J.

AU - Wang, Xu

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AU - Smillie, Lawrence B.

AU - Sykes, Brian D.

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N2 - Ca2+ and human cardiac troponin I (cTnI) peptide binding to human cardiac troponin C (cTnC) have been investigated with the use of 2D {1H,15N} HSQC NMR spectroscopy. The spectral intensity, chemical shift, and line-shape changes were analyzed to obtain the dissociation (KD) and off-rate (koff) constants at 30 °C. The results show that sites III and IV exhibit 100-fold higher Ca2+ affinity than site II (KD(III,IV)≈0.2 μM), but site II is partially occupied before sites III and IV are saturated. The addition of the first two equivalents of Ca2+ saturates 90% of sites III and IV and 20% of site II. This suggests that the Ca2+ occupancy of all three sites may contribute to the Ca2+-dependent regulation in muscle contraction. We have determined a koff of 5000 s-1 for site II Ca2+ dissociation at 30 °C. Such a rapid off-rate had not been previously measured. Three cTnI peptides, cTnI34-71, cTnI128-1477, and cTnI147-163, were titrated to Ca2+-saturated cTnC. In each case, the binding occurs with a 1:1 stoichiometry. The determined KD and koff values are 1 μM and 5 s-1 for cTnI34-71, 78 ± 10 μM and 5000 s-1 for cTnI128-147, and 150 ± 10 μM and 5000 s-1 for cTnI147-163, respectively. Thus, the dissociation of Ca2+ from site II and cTnI128-147 and cTnI147-163 from cTnC are rapid enough to be involved in the contraction/relaxation cycle of cardiac muscle, while that of cTnI34_71 from cTnC may be too slow for this process.

AB - Ca2+ and human cardiac troponin I (cTnI) peptide binding to human cardiac troponin C (cTnC) have been investigated with the use of 2D {1H,15N} HSQC NMR spectroscopy. The spectral intensity, chemical shift, and line-shape changes were analyzed to obtain the dissociation (KD) and off-rate (koff) constants at 30 °C. The results show that sites III and IV exhibit 100-fold higher Ca2+ affinity than site II (KD(III,IV)≈0.2 μM), but site II is partially occupied before sites III and IV are saturated. The addition of the first two equivalents of Ca2+ saturates 90% of sites III and IV and 20% of site II. This suggests that the Ca2+ occupancy of all three sites may contribute to the Ca2+-dependent regulation in muscle contraction. We have determined a koff of 5000 s-1 for site II Ca2+ dissociation at 30 °C. Such a rapid off-rate had not been previously measured. Three cTnI peptides, cTnI34-71, cTnI128-1477, and cTnI147-163, were titrated to Ca2+-saturated cTnC. In each case, the binding occurs with a 1:1 stoichiometry. The determined KD and koff values are 1 μM and 5 s-1 for cTnI34-71, 78 ± 10 μM and 5000 s-1 for cTnI128-147, and 150 ± 10 μM and 5000 s-1 for cTnI147-163, respectively. Thus, the dissociation of Ca2+ from site II and cTnI128-147 and cTnI147-163 from cTnC are rapid enough to be involved in the contraction/relaxation cycle of cardiac muscle, while that of cTnI34_71 from cTnC may be too slow for this process.

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