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

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, KD(II)≈20 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, cTnI128–147, 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.