Kinetic studies of calcium and cardiac troponin I peptide binding to human cardiac troponin C using NMR spectroscopy
Kinetic studies of calcium and cardiac troponin I peptide binding to human cardiac troponin C using NMR spectroscopy
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DOI：
10.1007/s00249-002-0227-1
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发表时间：
2002-07-01
影响因子：
2
通讯作者：
Sykes, BD
中科院分区：
文献类型：
--
作者：
Li, MX;Saude, EJ;Sykes, BD
用2D {H-1，N-15} HSQC NMR研究了Ca~（2+）和人心肌肌钙蛋白I（cTnI）肽与人心肌肌钙蛋白C（cTnC）的结合。分析光谱强度、化学位移和线形变化，以获得30 ℃下的解离常数（K-D）和解离速率常数（k（off））。结果表明，位点III和IV的Ca~（2+）亲和力比位点II高100倍（K（D（III，IV））约为0.2 μ M，K（D（II））约为20 μ M），但在位点III和IV饱和之前，位点II已被部分占据。前两个当量的Ca 2+的添加使90%的位点III和IV以及20%的位点II饱和。这表明所有三个位点的Ca 2+占据可能有助于肌肉收缩中的Ca 2+依赖性调节。我们已经确定了在30 ℃下位点II Ca 2+解离的k（off）为5000 s（-1）。如此快速的解离速率以前从未测量过。将三种cTnI肽cTnI（34 - 71）、cTnI（128 - 147）和cTnI（147 - 163）滴定至Ca2+饱和cTnC。在每种情况下，结合以1：1的化学计量发生。对于cTnI（34 - 71），测定的K-D和k（off）值分别为1 μ M和5 s（-1）;对于cTnI（128 - 147），测定的K-D和k（off）值分别为78 +/-10 μ M和5000 s（-1）;对于cTnI（147 - 163），测定的K-D和k（off）值分别为150 +/-10 μ M和5000 s（-1）。因此，Ca2+从位点II和cTnI 128 - 147和cTnI（147 - 163）从cTnC的解离足够快以参与心肌的收缩/舒张周期，而cTnI（34 - 71）从cTnC的解离可能太慢而不能参与该过程。
Ca2+ and human cardiac troponin I (cTnI) peptide binding to human cardiac troponin C (cTnC) have been investigated with the use of 2D {H-1, N-15} 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 degreesC. The results show that sites III and IV exhibit 100-fold higher Ca2+ affinity than site II (K(D(III,IV))approximate to0.2 muM, K(D(II))approximate to20 muM), 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 degreesC. 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 muM and 5 s(-1) for cTnI(34-71), 78 +/- 10 muM and 5000 s(-1) for cTnI(128-147), and 150 +/- 10 muM and 5000 s(-1) for cTnI(147-163), respectively. Thus, the dissociation of Ca2+ from site II and cTnI128-147 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.