Molecular mechanisms of pH-driven conformational transitions of proteins:: Insights from continuum electrostatics calculations of acid unfolding
Molecular mechanisms of pH-driven conformational transitions of proteins:: Insights from continuum electrostatics calculations of acid unfolding
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DOI：
10.1002/prot.20797
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发表时间：
2006-04-01
影响因子：
2.9
通讯作者：
García-Moreno, B
中科院分区：
文献类型：
--
作者：
Fitch, CA;Whitten, ST;García-Moreno, B
葡萄球菌核酸酶（SNase）的酸展开是非常合作的（Whitten and Garcia-Moreno, chemistry; 2000;39:14 - 29）。在pH 3.2 ~ 3.9范围内，相对于天然(N)态，有多达7个氢离子（H+）优先以酸未折叠态结合。为了研究酸展开的机理，采用多种连续介质静电方法进行了基于结构的pK(a)计算。计算成功地再现了pH为5 ~ 9时N态的H+结合特性，但系统地高估了酸展开时H+结合的数量。所有羧基残基在N态下的pK(a)值都比实际值低。通过使用较高的蛋白质介电常数、分子动力学放宽结构或其他人先前实施的其他经验修改来最大化测量值和计算值之间的一致性，酸展开时观察到的H+摄取与计算的H+摄取之间的差异并没有得到改善。这表明主链的构象波动是羧基pK(a)值的重要决定因素。由于以前在酸展开区以上的酸性条件下没有观察到SNase的整体或亚整体构象变化，因此这些波动一定是局部的。SNase的酸展开似乎不涉及通过积累分子内排斥相互作用而破坏N态，也不涉及键离子配对羧基残基的质子化。它更符合许多H+结合基团的适度贡献，在H+结合与整体结构转变之间的耦合中，局部构象波动起着重要作用。
The acid unfolding of staphylocoecal nuclease (SNase) is very cooperative (Whitten and Garcia-Moreno, Biochemistry 2000;39:1429214304). As many as seven hydrogen ions (H+) are bound preferentially by the acid-unfolded state relative to the native (N) state in the pH range 3.2-3.9. To investigate the mechanism of acid unfolding, structure-based pK(a) calculations were performed with a variety of continuum electrostatic methods. The calculations reproduced successfully the H+ binding properties of the N state between pH 5 and 9, but they systematically overestimated the number of H+ bound upon acid unfolding. The calculated pK(a) values of all carboxylic residues in the N state were more depressed than they should be. The discrepancy between the observed and the calculated H+ uptake upon acid unfolding was not improved by using high protein dielectric constants, structures relaxed with molecular dynamics, or other empirical modifications implemented previously by others to maximize agreement between measured and calculated pK(a) values. This suggests an important role for conformational fluctuations of the backbone as important determinants of pK(a) values of carboxylic groups. Because no global or subglobal conformational changes have been observed previously for SNase under acidic conditions above the acid-unfolding region, these fluctuations must be local. The acid unfolding of SNase does not seem to involve the disruption of the N state by accruement of intramolecular repulsive interactions, nor the protonation of key ion paired carboxylic residues. It is more consistent with modest contributions from many H+ binding groups, with an important role for local conformational fluctuations in the coupling between H+ binding and the global structural transition.