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

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 (K_D) and off-rate (k_off) constants at 30 °C. The results show that sites III and IV exhibit 100-fold higher Ca²⁺ affinity than site II (K_D(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 k_off 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, cTnI_34-71, cTnI_128-1477, and cTnI_147-163, were titrated to Ca²⁺-saturated cTnC. In each case, the binding occurs with a 1:1 stoichiometry. The determined K_D and k_off values are 1 μM and 5 s^-1 for cTnI_34-71, 78 ± 10 μM and 5000 s^-1 for cTnI_128-147, and 150 ± 10 μM and 5000 s^-1 for cTnI_147-163, respectively. Thus, the dissociation of Ca²⁺ from site II and cTnI_128-147 and cTnI_147-163 from cTnC are rapid enough to be involved in the contraction/relaxation cycle of cardiac muscle, while that of cTnI_{34_71} from cTnC may be too slow for this process.