Perturbed pKA-values in the denatured states of proteins.
Perturbed pKA-values in the denatured states of proteins.
复制标题
蛋白质变性状态下的 pKA 值受到干扰。
DOI：
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发表时间：
1995
影响因子：
5.6
通讯作者：
A. Fersht
中科院分区：
文献类型：
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作者：
Y. Tan;M. Oliveberg;B. Davis;A. Fersht
在这项研究中，我们表明，在纯水中的变性蛋白质的电离平衡是不一致的“完全展开”的构象是一个扩展的线圈，其中的残基是相互隔离的介入溶剂。研究了酸和盐对大麦胰凝乳蛋白酶抑制剂2（CI 2）稳定性的影响，并通过NMR测定了天然蛋白中所有羧酸残基的pKa值。实验测定的蛋白质稳定性的pH依赖性与使用天然状态下观察到的pKa值计算的pH依赖性的比较表明，变性状态下的pKa值平均比模型化合物低0.3个pH单位。离子强度的增加消除了变性状态下的这些pKA变化。这表明CI 2的变性状态存在静电相互作用。由于之前对芽孢杆菌RNA酶和卵类粘蛋白第三结构域的研究也报告了变性状态的异常滴定行为，因此似乎变性状态下的扰动pKa值是一种普遍现象，表明纯水中的未折叠构象是相当紧凑的物种。此外，我们使用突变方法来确定天然和变性状态下羧酸基团的pKa值。通过该方法获得的天然状态下的pKa值与通过NMR获得的pKa值精确一致。
We show in this study that the ionisation equilibria of denatured proteins in pure water are inconsistent with the "fully-unfolded" conformation being an extended coil where the residues are isolated from one another by the intervening solvent. The effects of acid and salt on the stability of the barley chymotrypsin inhibitor 2 (CI2) were investigated and the pKA-values of all carboxylate residues in the native protein were determined by NMR. A comparison of the experimentally determined pH-dependence of the protein stability and that calculated using observed pKA-values in the native state, reveals that the pKA-values in the denatured state are, on average, 0.3 pH units lower than those of model compounds. An increase in ionic strength eliminates these pKA shifts in the denatured state. This shows that there are electrostatic interactions in the denatured state of CI2. Since previous studies on barnase and the Ovomucoid Third Domain also report anomalous titration behaviours of the denatured states, it appears that perturbed pKA-values in the denatured state is a general phenomenon, indicating that the unfolded conformation in pure water is a fairly compact species. In addition, we used a mutational approach to determine the pKA-values of a carboxylate group in both the native and denatured states. The pKA-value in the native state obtained by this method is in precise agreement with that obtained by NMR.