Loladze V V, Ibarra-Molero B, Sanchez-Ruiz J M, Makhatadze G I
Department of Biochemistry and Molecular Biology, Pennsylvania State College of Medicine, Hershey 17033-0850, USA.
Biochemistry. 1999 Dec 14;38(50):16419-23. doi: 10.1021/bi992271w.
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.
通过对天然蛋白质表面带电荷残基的分布进行适当重新设计来提高蛋白质稳定性的一个简单理论模型得到了实验验证。以泛素分子作为模型系统，我们预测了该蛋白质表面可能导致其稳定性增加的氨基酸取代。通过使用远紫外圆二色光谱监测尿素诱导的去折叠来测量单点取代泛素变体的稳定性，从而实现了对这一预测的实验验证。我们表明，所产生的泛素变体确实比野生型蛋白质更稳定，这与理论预测在定性上是一致的。作为阳性对照，也考虑了对泛素分子表面使氨基酸取代不稳定的理论预测。这些预测也使用相应设计的泛素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，这再次与理论预测一致。这些观察结果为合理设计更稳定的蛋白质提供了指导方针，并提示了嗜热生物蛋白质结构稳定性的一种可能机制。
