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

39 Scopus citations

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 1 2002
Externally publishedYes

Keywords

  • Calcium binding
  • Kinetics
  • NMR
  • Troponin C

ASJC Scopus subject areas

  • Biophysics

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