Baldwin E, Xu J, Hajiseyedjavadi O, Baase W A, Matthews B W
Institute of Molecular Biology, University of Oregon, Eugene, 97403, USA.
J Mol Biol. 1996 Jun 14;259(3):542-59. doi: 10.1006/jmbi.1996.0338.
Previous analysis of randomly generated multiple mutations within the core of bacteriophage T4 lysozyme suggested that the "large-to-small" substitution Leu121 to Ala (L121A) and the spatially adjacent "small-to-large" substitution Ala129 to Met (A129M) might be mutually compensating. To test this hypothesis, the individual variants L121A and A129M were generated, as well as the double "size-switch" mutant L121A/A129M. To make the interchange symmetrical, the combination of L121A with A129L to give L121A/A129L was also constructed. The single mutations were all destabilizing. Somewhat surprisingly, the small-to-large substitutions, which increase hydrophobic stabilization but can also introduce strain, were less deleterious than the large-to-small replacements. Both Ala129 --> Leu and Ala129 --> Met offset the destabilization of L121A by about 50%. Also, in contrast to typical Leu --> Ala core substitutions, which destabilize by 2 to 5 kcal/mol, Leu121 --> Ala slightly stabilized A129L and A129M. Crystal structure analysis showed that a combination of side-chain and backbone adjustments partially accommodated changes in side-chain volume, but only to a limited degree. For example, the cavity that was created by the Leu121 to Ala replacement actually became larger in L121A/A129L. The results demonstrate that the destabilization associated with a change in volume of one core residue can be specifically compensated by an offsetting volume change in an adjacent residue. It appears, however, that complete compensation is unlikely because it is difficult to reconstitute an equivalent set of interactions. The relatively slow evolution of core relative to surface residues appears, therefore, to be due to two factors. First, a mutation in a single core residue that results in a substantial change in size will normally lead to a significant loss in stability. Such mutations will presumably be selected against. Second, if a change in bulk does occur in a buried residue, it cannot normally be fully compensated by a mutation of an adjacent residue. Thus, the most probable response will tend to be reversion to the parent protein.
先前对噬菌体T4溶菌酶核心区域随机产生的多个突变的分析表明，“大到小”的取代，即Leu121突变为Ala（L121A）以及空间上相邻的“小到大”取代，即Ala129突变为Met（A129M），可能会相互补偿。为了验证这一假设，分别构建了单个变体L121A和A129M，以及双“大小切换”突变体L121A/A129M。为了使互换对称，还构建了L121A与A129L组合而成的L121A/A129L。所有单个突变都会使结构不稳定。有点令人惊讶的是，“小到大”的取代虽然增加了疏水稳定性，但也可能引入张力，其有害性比“大到小”的取代要小。Ala129突变为Leu和Ala129突变为Met都能使L121A的稳定性下降抵消约50%。此外，与典型的Leu突变为Ala导致核心结构稳定性下降2至5千卡/摩尔不同，Leu121突变为Ala会使A129L和A129M略有稳定。晶体结构分析表明，侧链和主链调整的组合部分适应了侧链体积的变化，但程度有限。例如，由Leu121突变为Ala所产生的空腔在L121A/A129L中实际上变得更大。结果表明，一个核心残基体积变化所导致的稳定性下降可以通过相邻残基的抵消性体积变化得到特异性补偿。然而，似乎不太可能实现完全补偿，因为难以重建一组等效的相互作用。因此，核心残基相对于表面残基进化相对缓慢似乎是由两个因素导致的。首先，单个核心残基的突变如果导致大小发生显著变化，通常会导致稳定性大幅下降。这样的突变可能会被淘汰。其次，如果一个埋藏残基的体积发生变化，通常无法通过相邻残基的突变得到完全补偿。因此，最可能的反应往往是回复到亲本蛋白。
