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 degrees C. The results show that sites III and IV exhibit 100-fold higher Ca2+ affinity than site II ( K(D(III,IV)) approximately 0.2 microM, K(D(II)) approximately 20 microM), 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 degrees C. Such a rapid off-rate had not been previously measured. Three cTnI peptides, cTnI(34-71), cTnI(128-147), 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 microM and 5 s(-1) for cTnI(34-71), 78+/-10 microM and 5000 s(-1) for cTnI(128-147), and 150+/-10 microM 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.
CIHR Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Monica X
MX
Li
Erik J
EJ
Saude
Xu
X
Wang
Joyce R
JR
Pearlstone
Lawrence B
LB
Smillie
Brian D
BD
Sykes
D002118:Calcium / Q000737:chemistry*; D006801:Humans; D009206:Myocardium / Q000737:chemistry*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019209:Troponin C / Q000737:chemistry*; D019210:Troponin I / Q000737:chemistry*
D009587:Nitrogen Isotopes; D011522:Protons; D019209:Troponin C; D019210:Troponin I; D002118:Calcium