Engineering a thermostable protein via optimization of charge-charge interactions on the protein surface
Engineering a thermostable protein via optimization of charge-charge interactions on the protein surface
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DOI：
10.1021/bi992271w
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发表时间：
1999-12-14
期刊：
影响因子：
2.9
通讯作者：
Makhatadze, GI
中科院分区：
文献类型：
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作者：
Loladze, VV;Ibarra-Molero, B;Makhatadze, GI
实验测试了通过充分重新设计天然蛋白质表面上的带电残基分布来增加蛋白质稳定性的简单理论模型。使用泛素分子作为模型系统，我们预测了这种蛋白质的表面上可能的氨基酸取代，这将导致其稳定性的增加。实验验证这一预测实现了通过测量的稳定性的单网站取代的泛素变体使用尿素诱导的展开监测远紫外CD光谱。我们表明，产生的变体的泛素确实比野生型蛋白质更稳定，在定性与理论预测。作为阳性对照，还考虑了泛蛋白分子表面不稳定氨基酸取代的理论预测。这些预测也用相应设计的泛蛋白变体进行了实验测试。我们发现这些变体比野生型蛋白质更不稳定，再次与理论预测一致。这些观察结果提供了更稳定的蛋白质的合理设计的指导方针，并建议从嗜热生物的蛋白质的结构稳定性的可能机制。
A Simple theoretical model for increasing the protein stability by adequately redesigning the distribution of charged residues on the surface of the native protein was tested experimentally. Using the molecule of ubiquitin as a model system, we predicted possible amino acid substitutions on the surface of this protein which would lead to an increase in its stability. Experimental validation for this prediction was achieved by measuring the stabilities of single-site-substituted ubiquitin variants using urea-induced unfolding monitored by far-UV CD spectroscopy. We show that the generated variants of ubiquitin are indeed more stable than the wild-type protein, in qualitative agreement with the theoretical prediction. As a positive control, theoretical predictions for destabilizing amino acid substitutions on the surface of the ubiquitin molecule were considered as well. These predictions were also tested experimentally using correspondingly designed variants of ubiquitin. We found that these variants are less stable than the wild-type protein, again in agreement with the theoretical prediction. These observations provide guidelines for rational design of more stable proteins and suggest a possible mechanism of structural stability of proteins from thermophilic organisms.