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 ( 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.

The cardiac-specific N-terminal region of troponin I positions the regulatory domain of troponin C

“…Wild type-or C35S,C84S-cTnC was expressed in Escherichia coli using a pET3a-derived expression vector (45) and purified according to previously published protocols (46). In brief, purification involved three chromatographic steps: anion exchange, hydrophobic, and gel filtration chromatography.…”

Elucidation of Isoform-dependent pH Sensitivity of Troponin I by NMR Spectroscopy

“…The engineering of the vector and the expression of 15 N-and 13 C, 15 N-labeled proteins in Escherichia coli were as described previously (27). GL Biochem Ltd. (Shanghai, China) synthesized cTnI(147-163) (acetyl-RISADAMMQALLGARAK-amide) and Alberta Peptide Institute (API) synthesized sTnI(115-131) (acetyl-RMS-ADAMLKALLGSKHK-amide).…”

Structure and Dynamics of the Acidosis-Resistant A162H Mutant of the Switch Region of Troponin I Bound to the Regulatory Domain of Troponin C

“…We performed a pH titration of cChimera A162H monitored by 1 H, 15 N-HSQC NMR experiments at each titration point to investigate the pK a of H162 in this system. The pK a of a specific residue can be determined by plotting the chemical shift change as a function of the pH.…”

“…For structure determination, the sample contained 0.8−1 mM 15 N-cNTnC or 15 N, 13 C-cNTnC and a 4:1 molar excess of cTnI A162H to ensure saturation of cNTnC, and the pH was set to 6.1. For backbone dynamics experiments, the sample contained 0.8 mM 15 N-cChimera A162H and the pH was set to 6.4 or 7.4 accordingly.…”