Fast Pressure Jumps Can Perturb Calcium and Magnesium Binding to Troponin C F29W
Fast Pressure Jumps Can Perturb Calcium and Magnesium Binding to Troponin C F29W
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DOI：
10.1021/bi801150w
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发表时间：
2008-11-18
期刊：
影响因子：
2.9
通讯作者：
Geeves, Michael A.
中科院分区：
文献类型：
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作者：
Pearson, David S.;Swartz, Darl R.;Geeves, Michael A.
我们采用快速压力跃变和停流荧光法研究了钙和镁与F29 W鸡骨骼肌钙蛋白C的结合。在存在和不存在镁的情况下，增加的压力扰乱了钙与N-末端位点的结合，并提供了钙结合后体积变化的估计值（约12 mL/mol）。我们观察到一个双相响应的压力变化，其特征在于快速和缓慢的倒数弛豫时间的顺序1000/s和100/s。在pCa 8-5.4和肌钙蛋白C浓度为8-28 μ M之间，慢弛豫时间不变，表明蛋白质异构化是限速的。仅在非常窄的pCa范围（5.6-5.4）内检测到快速事件。我们设计了一个基于Monod-Wyman-Changeux合作机制的模型，其中钙结合到调节位点和闭合到开放蛋白质异构化步骤的体积变化分别为-9和+6 mL/mol。在不存在镁的情况下，我们发现可以检测到与C-末端位点结合的钙，尽管它们的位置远离钙敏感的色氨酸，体积变化为+25 mL/mol。我们使用这种新的观察来测量竞争性镁结合到C-末端位点，并推导出在200-300 μ M范围内的亲和力（和+35 mL/mol的体积变化）。这种亲和力比基于直接竞争结合模型所建议的平衡荧光数据更紧密一个数量级。因此，镁间接调节与N-末端位点的结合，这可能在体内起到微调机制的作用。
We have used rapid pressure jump and stopped-flow fluorometry to investigate calcium and magnesium binding to F29W chicken skeletal troponin C. Increased pressure perturbed calcium binding to the N-terminal sites in the presence and absence of magnesium and provided an estimate for the volume change upon calcium binding (-12 mL/mol). We observed a biphasic response to a pressure change which was characterized by fast and slow reciprocal relaxation times of the order 1000/s and 100/s. Between pCa 8-5.4 and at troponin C concentrations of 8-28 mu M, the slow relaxation times were invariant, indicating that a protein isomerization was rate-limiting. The fast event was only detected over a very narrow pCa range (5.6-5.4). We have devised a model based on a Monod-Wyman-Changeux cooperative mechanism with volume changes of -9 and +6 mL/mol for the calcium binding to the regulatory sites and closed to open protein isomerization steps, respectively. In the absence of magnesium, we discovered that calcium binding to the C-terminal sites could be detected, despite their position distal to the calcium-sensitive tryptophan, with a volume change of +25 mL/mol. We used this novel observation to measure competitive magnesium binding to the C-terminal sites and deduced an affinity in the range 200-300 mu M (and a volume change of +35 mL/mol). This affinity is an order of magnitude tighter than equilibrium fluorescence data suggest based on a model of direct competitive binding. Magnesium thus indirectly modulates binding to the N-terminal sites, which may act as a fine-tuning mechanism in vivo.