SIMILAR HYDROPHOBIC REPLACEMENTS OF LEU99 AND PHE153 WITHIN THE CORE OF T4-LYSOZYME HAVE DIFFERENT STRUCTURAL AND THERMODYNAMIC CONSEQUENCES
SIMILAR HYDROPHOBIC REPLACEMENTS OF LEU99 AND PHE153 WITHIN THE CORE OF T4-LYSOZYME HAVE DIFFERENT STRUCTURAL AND THERMODYNAMIC CONSEQUENCES
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DOI：
10.1006/jmbi.1993.1077
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发表时间：
1993-02-05
影响因子：
5.6
通讯作者：
MATTHEWS, BW
中科院分区：
文献类型：
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作者：
ERIKSSON, AE;BAASE, WA;MATTHEWS, BW
噬菌体T4溶菌酶核心内的两个大体积氨基酸各自依次被一系列疏水氨基酸取代。在一组实验中，Leu 99被Phe、Met、Ile、瓦尔和Ala取代。在第二个系列中，Phe 153被Leu、Met、Ile、瓦尔和Ala取代。还构建了Leu99和Phe153均被丙氨酸取代的双突变体。已经确定了与每个取代相关的蛋白质稳定性的变化和每个变体的晶体结构。在位置99处的替换的情况下，蛋白质以相对刚性的方式表现，并且响应于替换而变化非常小。相比之下，该蛋白质更灵活，并且响应于Phe153的取代而调整得更多。在这两种情况下，突变蛋白相对于野生型的稳定性（ΔΔG）与基于溶剂转移测量的参与取代的氨基酸的疏水强度差异（ΔΔGtr）之间存在大致线性依赖性。然而，ΔΔGis的变化远大于ΔΔGtr。对于Phe153替代物，差异约为1.9倍，而对于Leu99系列，差异约为2.6倍。突变体如Leu99→Ala，其蛋白质基本上保持刚性，倾向于产生更大的空腔，因此产生更大的不稳定能量。突变体如Phe153→Ala，其蛋白质结构倾向于松弛，导致更小的空腔，因此不太稳定。突变体L99I和L99V的稳定性低于预期，这是由于Leu99被不同形状的残基取代而引入应变，从而考虑到转移自由能和空腔形成。突变体F153L比参考野生型更稳定，即使Leu的转移自由能小于Phe。稳定性的增加显然是由于存在于野生型溶菌酶中的Phe153侧链中的扭转应变，但在突变体结构中得到缓解。
Two bulky amino acids within the core of phage T4 lysozyme have each been replaced in turn with a series of hydrophobic amino acids. In one set of experiments, Leu99 was replaced with Phe, Met, Ile, Val and Ala. In the second series, Phe153 was replaced with Leu, Met, Ile, Val and Ala. The double mutant in which both Leu99 and Phe153 were replaced with alanine was also constructed. The change in stability of the protein associated with each substitution and the crystal structure of each variant have been determined. In the case of replacement at position 99 the protein behaves in a relatively rigid manner, and changes very little in response to substitutions. In contrast, the protein is more flexible and adjusts much more in response to substitutions of Phe153. In both cases there is a roughly linear dependence between the stability of the mutant protein relative to wild-type (ΔΔG) and the difference in the hydrophobic strength of the amino acids involved in the substitution based on solvent transfer measurements (ΔΔGtr). The change in ΔΔGis, however, much greater than ΔΔGtr. For the Phe153 replacements the discrepancy is about 1·9-fold, while for the Leu99 series it is about 2·6-fold. Mutants such as Leu99→Ala, for which the protein remains essentially rigid, tend to create larger cavities and so incur a larger energy of destabilization. Mutants such as Phe153→Ala, for which the protein structure tends to relax, results in smaller cavities and so are less destabilized. Mutants L99I and L99V are less stable than expected from considerations of transfer free energy and cavity formation due to introduced strain caused by the replacement of Leu99 with a residue of different shape. Mutant F153L is more stable than the reference wild-type, even though the transfer free energy of Leu is less than that of Phe. The increase in stability is apparently due to torsional strain in the side-chain of Phe153 that is present in wild-type lysozyme, but is relieved in the mutant structure.