Horng Jia-Cherng, Cho Jae-Hyun, Raleigh Daniel P
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794-3400, USA.
J Mol Biol. 2005 Jan 7;345(1):163-73. doi: 10.1016/j.jmb.2004.10.023.
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 (logK) and folding rates (logk(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.
通过对一组组氨酸突变为谷氨酰胺的点突变体的折叠动力学和稳定性进行pH依赖性研究，来表征核糖体蛋白L9的C末端结构域（CTL9）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，在天然状态下被埋藏，而第三个，H144，则更暴露。使用Tanford-Wyman连锁关系计算的pH依赖性稳定性与测量值的比较表明，在变性状态集合中，三个组氨酸残基的表观pK(a)值没有受到显著扰动。动力学测量表明，H134的突变对折叠过程的影响比H106和H144的突变更大。H134的Phi值明显大于其他组氨酸残基的Phi值，在pH 5.45和pH 8.0时，其他组氨酸残基的Phi值接近零。H134在pH 8.0时的Phi值更高，为0.55，而在pH 5.45时为0.39。在pH 5.45时，H134在未折叠状态下被质子化，但在天然状态下被去质子化，而在pH 8.0时，它在两种状态下均被去质子化。对于所有突变体，在pH 5-9范围内，稳定性（logK）和折叠速率（logk(f)）之间存在极好的线性关系。从这些图中，可以为每个突变体计算DeltaQ( not equal)/DeltaQ的比值。DeltaQ( not equal)是结合到过渡态和未折叠态的质子数之差，而DeltaQ代表折叠态和变性态之间的差异。线性图表明，由DeltaQ( not equal)/DeltaQ判断的过渡态集合的相对位置与pH无关。连锁分析与Phi值分析一致，表明H134是pH依赖性相互作用发展的最关键贡献者，包括过渡态集合中的去溶剂化效应。
