Uncovering pH-dependent transient states of proteins with buried ionizable residues.
Uncovering pH-dependent transient states of proteins with buried ionizable residues.
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DOI：
10.1021/ja5012564
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发表时间：
2014-06-18
影响因子：
15
通讯作者：
Brooks CL 3rd
中科院分区：
文献类型：
--
作者：
Goh GB;Laricheva EN;Brooks CL 3rd
pH 在调节生物活性中的作用是普遍存在的，并且理解 pH 介导的活性传统上依赖于分析接近生理 pH 值的高密度基态的静态生物分子结构。然而，最近的进展表明瞬态填充态的重要性与日俱增，其表征极具挑战性，但随着弛豫色散核磁共振波谱等技术的发展变得合理。为了用原子级细节解开这些瞬态的 pH 依赖性，我们将最近开发的显式溶剂常数 pH 分子动力学 (CPHMDMSλD) 框架应用于一系列埋藏可电离残基的葡萄球菌核酸酶 (SNase) 突变体，并探讨了它们在不同 pH 环境中的动力学。我们的主要发现之一是所有 SNase 突变体中都存在开放态，这些突变体含有 pKa 高度变化的“埋藏”残基，在质子化位点周围观察到局部溶剂化。计算出的 pKa 与实验 pKa 非常吻合，平均无符号误差低至 1.3 pKa 单位，相关系数 R2 = 0.78。在各自的 pH 范围内对开放态和封闭态进行采样（预计它们将占主导地位）对于重现实验 pKa 是必要的，并且在测量的 pKa 变化的最极端示例中，可以解释为开放态结构在生理 pH 下是瞬态的，只占 1-2% 的一小部分。这表明埋藏的可电离残基可以引发构象波动，这种波动可以在生理 pH 下观察到瞬态结构。此外，开放态和封闭态的耦合关系及其在概括宏观实验可观测值中的作用表明，埋藏残基的结构分析可能受益于观察结构对，而不是观察单个静态基态构象的传统方法。
The role of pH in regulating biological activity is ubiquitous, and understanding pH-mediated activity has traditionally relied on analyzing static biomolecular structures of highly populated ground states solved near physiological pH. However, recent advances have shown the increasing importance of transiently populated states, the characterization of which is extremely challenging but made plausible with the development of techniques such as relaxation dispersion NMR spectroscopy. To unlock the pH dependence of these transient states with atomistic-level details, we applied the recently developed explicit solvent constant pH molecular dynamics (CPHMDMSλD) framework to a series of staphylococcal nuclease (SNase) mutants with buried ionizable residues and probed their dynamics in different pH environments. Among our key findings is the existence of open states in all SNase mutants containing “buried” residues with highly shifted pKa’s, where local solvation around the protonation site was observed. The calculated pKa demonstrated good agreement with experimental pKa’s, with a low average unsigned error of 1.3 pKa units and correlation coefficient R2 = 0.78. Sampling both open and closed states in their respective pH range, where they are expected to be dominant, was necessary to reproduce experimental pKa’s, and in the most extreme examples of pKa shifts measured, it can be interpreted that the open-state structures are transient at physiological pH, contributing a small population of 1–2%. This suggests that buried ionizable residues can trigger conformational fluctuations that may be observed as transient-state structures at physiological pH. Furthermore, the coupled relationship of both open and closed states and their role in recapitulating macroscopic experimental observables suggest that structural analysis of buried residues may benefit from looking at structural pairs, as opposed to the conventional approach of looking at a single static ground-state conformation.
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