THE FOLDING OF AN ENZYME .2. SUBSTRUCTURE OF BARNASE AND THE CONTRIBUTION OF DIFFERENT INTERACTIONS TO PROTEIN STABILITY
THE FOLDING OF AN ENZYME .2. SUBSTRUCTURE OF BARNASE AND THE CONTRIBUTION OF DIFFERENT INTERACTIONS TO PROTEIN STABILITY
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DOI：
10.1016/0022-2836(92)90562-x
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发表时间：
1992-04-05
影响因子：
5.6
通讯作者：
FERSHT, AR
中科院分区：
文献类型：
--
作者：
SERRANO, L;KELLIS, JT;FERSHT, AR
从解剖角度描述了Barnase的亚结构及其堆积方式。计算了埋藏在结构单元形成和堆积上的疏水残基的表面积。已经测量了突变的稳定性变化，在本研究中构建了41个突变，其战略定位在蛋白质上。其目的是提供：(1)关于残基突变稳定能变化幅度的信息，这些突变对维持结构是重要的；(2)用于后续研究的折叠途径的探针。大多数突变会删除侧链的功能部分或进行等位元改变。相互作用的能量学是可变的，并与背景有关。然而，从这项关于稳定蛋白质的相互作用类别的研究中，可以得出以下一般性结论。(1)通常情况下，截断埋藏的疏水侧链对稳定性的影响最大。对于完全掩埋的残基，这平均为每个亚甲基1.5kcal−1，标准偏差为±0.6kcal−1。截断部分暴露的亮氨酸、异亮氨酸或缬氨酸残基的溶剂可及区域(甘氨酸-X-甘氨酸三肽总溶剂可及区域的30%至50%，即那些与表面堆积的区域)对稳定性的影响较小，但相对恒定。每亚甲基0.81kcal−1，统计标准偏差为±0.18kcal−1。(2)对于大量的疏水突变体，其疏水表面积与自由能变化之间的相关性很差。自由能变化与所删除疏水基团半径为6？半径内的亚甲基数目之间的相关性最好。(3)将苏氨酸的羟基埋入用于γ-甲基的Valine的口袋中的成本为2.5kcal摩尔−1，在预期失去两个氢键的范围内。在以前的研究中发现：(1)表面离子对或盐桥的破坏只会使稳定性降低很小的量(0.3千卡−1)，但对埋藏对的破坏却是大的(>；3千卡分子−1)；(2)去除未带电基团的氢键伙伴会损失0.5~2千卡分子−1(氢键的能量加上相关的范德华相互作用)；以及(3)去除带电基团的伙伴与破坏盐桥具有相似的效果。
Barnase is described anatomically in terms of its substructures and their mode of packing. The surface area of hydrophobic residues buried on formation and packing of the structural elements has been calculated. Changes in stability have been measured for 64 mutations, 41 constructed in this study, strategically located over the protein. The purpose is to provide: (1) information on the magnitudes of changes in stabilization energy for mutations of residues that are important in maintaining the structure; and (2) probes for the folding pathway to be used in subsequent studies. The majority of mutations delete functional moieties of side-chains or make isosteric changes. The energetics of the interactions are variable and context-dependent. The following general conclusions may be drawn, however, from this study about the classes of interactions that stabilize the protein. (1) Truncation of buried hydrophobic side-chains has, in general, the greatest effect on stability. For fully buried residues, this averages at 1.5 kcal mol−1per methylene group with a standard deviation of ±0.6 kcal mol−1. Truncation of partly exposed leucine, isoleucine or valine residues that are in the range of 50 to 80 Å2of solvent-accessible area (30 to 50 % of the total solvent-accessible area on a Gly-X-Gly tripeptide, i.e. those packed against the surface) has a smaller, but relatively constant effect on stability, at 0.81 kcal mol−1per methylene group with a statistical standard deviation of±0.18 kcal mol−1. (2) There is a very poor correlation between hydrophobic surface area buried and the free energy change for an extensive data set of hydrophobic mutants. The best correlation is found to be between the free energy change and the number of methylene groups within a 6 Å radius of the hydrophobic groups deleted. (3) Burial of the hydroxyl group of threonine in a pocket that is intended for a γ-methyl group of valine costs 2.5 kcal mol−1, in the range expected for the loss of two hydrogen bonds. In extension of previous studies, it is found that: (1) disruption of surface ion pairs or salt bridges lowers stability by only small amounts (0.3 to 1 kcal mol−1) but disruption of buried pairs, by larger amounts (>3 kcal mol−1); (2) removal of the hydrogen bonding partner of an uncharged group loses 0.5 to 2 kcal mol−1(the energy of the hydrogen bond plus associated van der Waals' interactions); and (3) removal of the partner of a charged group has similar effects to the disruption of salt bridges.