Zhang H, Skinner M M, Sandberg W S, Wang A H, Terwilliger T C
Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign 61801, USA.
J Mol Biol. 1996 May 31;259(1):148-59. doi: 10.1006/jmbi.1996.0309.
The basis for the context dependence of the effects of core mutations on protein stability was investigated by comparing the structures of three gene V protein mutants with that of the wild-type protein. We previously examined a "swapped" mutant in which core residues Val35 and Ile47 were simply reversed so that the mutant had no hydrophobicity change from the native protein. The swapped mutant was destabilized by 3 kcal/mol per gene V protein dimer relative to the wild-type protein, demonstrating that factors other than hydrophobicity must make substantial contributions to the effects of mutations on the stability of the protein. Here we have determined the structure of this swapped mutant (V35I/I47V) as well as those of the two constituent mutants (V35I and I47V). We find that the structures of the mutant proteins are very similar to that of the wild-type protein except for the necessary addition or deletion of methylene groups and for slight positional shifts of atoms around each mutated residue. The structure of the double mutant is a composite of the structures of the two single mutants. In the mutant structures, the V35I mutation fills a cavity that exists in the wild-type protein and the I47V mutation creates a new cavity. The structures of the mutants indicate further that the reason the V35I and I47V mutations do not have opposite effects on stability is that the cavity in the wild-type protein filled by the V35I mutation is not optimally shaped for accommodating the additional methylene group of the isoleucine. These results support the concepts that the details of core packing have substantial influence on the effects of core mutations on protein stability and that these packing effects are major determinants of the context dependence of core mutation effects on stability.
通过比较三种基因V蛋白突变体与野生型蛋白的结构，研究了核心突变对蛋白质稳定性影响的上下文依赖性基础。我们之前研究过一个“交换”突变体，其中核心残基Val35和Ile47简单地互换，因此该突变体与天然蛋白相比没有疏水性变化。相对于野生型蛋白，每个基因V蛋白二聚体的交换突变体的稳定性降低了3千卡/摩尔，这表明除疏水性外的其他因素必须对突变对蛋白质稳定性的影响做出重大贡献（起到重要作用）。在这里，我们确定了这个交换突变体（V35I/I47V）以及两个组成突变体（V35I和I47V）的结构。我们发现，突变蛋白的结构与野生型蛋白非常相似，只是需要添加或删除亚甲基，以及每个突变残基周围的原子有轻微的位置偏移。双突变体的结构是两个单突变体结构的组合。在突变体结构中，V35I突变填充了野生型蛋白中存在的一个腔，而I47V突变产生了一个新的腔。突变体的结构进一步表明，V35I和I47V突变对稳定性没有相反影响的原因是，被V35I突变填充的野生型蛋白中的腔的形状并非最适合容纳异亮氨酸的额外亚甲基。这些结果支持了以下概念：核心堆积的细节对核心突变对蛋白质稳定性的影响有重大影响，并且这些堆积效应是核心突变对稳定性影响的上下文依赖性的主要决定因素。
