Analysis of the pH-dependent folding and stability of histidine point mutants allows characterization of the denatured state and transition state for protein folding
Analysis of the pH-dependent folding and stability of histidine point mutants allows characterization of the denatured state and transition state for protein folding
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DOI：
10.1016/j.jmb.2004.10.023
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发表时间：
2005-01-07
影响因子：
5.6
通讯作者：
Raleigh, DP
中科院分区：
文献类型：
--
作者：
Horng, JC;Cho, JH;Raleigh, DP
对一组His到Gln点突变的折叠动力学和稳定性的pH依赖性研究被用来表征核糖体蛋白L9(CTL9)C末端结构域的变性状态和过渡态集合。CTL9含有三个组氨酸残基，其中两个H106和H134以天然状态埋藏，第三个H144暴露更多。用Tanford-Wyman键关系计算的pH依赖的稳定性与实测值的比较表明，在变性状态下，三个组氨酸残基的表观pK(A)值没有明显的扰动。动力学测量表明，H134突变对折叠过程的影响大于H106和H144突变。H134的phi值明显大于其他组氨酸残基的phi值，后者在pH 5.45和pH 8.0时都接近于零。PH 8.0时，H134的pH值为0.55，高于pH 5.45时的0.39。在pH 5.45时，H134在未折叠状态下质子化，而在自然状态下去质子化，而在pH 8.0时，两者都去质子化。在pH 5-9的范围内，所有突变体的稳定性(LogK)和折叠率(logk(F))之间都有很好的线性关系。根据这些曲线图，可以计算出每个突变体的DeltaQ(不等于)/DeltaQ的比率。DeltaQ(不等于)是结合到过渡态和未折叠状态的质子数之差，而DeltaQ表示折叠状态和变性状态之间的差异。线性图表明，由DeltaQ(不等于)/DeltaQ判断的过渡态系综的相对位置与pH无关。连锁分析与PHI值分析是一致的，表明H134是包括脱溶在内的pH依赖相互作用发展的最关键贡献者。过渡态系综中的效应。(C)2004爱思唯尔有限公司。保留所有权利。
pH-dependent studies of the folding kinetics and stability of a set of His to Gln point mutants were used to characterize the denatured state and transition state ensembles for the C-terminal domain of the ribosomal protein L9 (CTL9). CTL9 contains three histidine residues, two of which, H106 and H134, are buried in the native state, while the third, H144, is more exposed. Comparison of the pH-dependent stability calculated using the Tanford-Wyman linkage relationship to the measured values demonstrates that the apparent pK(a) values of the three histidine residues are not significantly perturbed in the denatured state ensemble. Kinetic measurements show that mutation of H134 has a larger effect on the folding process than does mutation of H106 and H144. The Phi-value for H134 is significantly larger than the Phi-values for the other histidine residues, which are near zero at both pH 5.45 and pH 8.0. The Phi-value for H134 is higher, 0.55, at pH 8.0 than at pH 5.45, 0.39. At pH 5.45, H134 is protonated in the unfolded state but deprotonated in the native state, while at pH 8.0 it is deprotonated in both. There is an excellent linear relationship between stability (log K) and folding rates (log k(f)) over the range of pH 5-9 for all mutants. From these plots, the ratio of DeltaQ(not equal)/DeltaQ can be calculated for each mutant. DeltaQ(not equal) is the difference in the number of protons bound to the transition state and to the unfolded state, while DeltaQ represents the difference between folded and denatured state. The linear plots indicate that the relative position of the transition state ensemble as judged by DeltaQ(not equal)/DeltaQ is independent of pH. The linkage analysis is consistent with the Phi-value analysis, showing that H134 is the most critical contributor to the development of pH-dependent interactions, including desolvation. effects in the transition state ensemble. (C) 2004 Elsevier Ltd. All rights reserved.