【作者】 葛猛；

【导师】 潘宪明；

【作者基本信息】 清华大学 ， 生物学， 2009， 博士

【摘要】 比较极端嗜热蛋白与同源嗜温蛋白,有显著性差别的是极端嗜热蛋白含有较多的盐桥及带电残基,其他参数的差异仅仅有统计倾向性。然而,大量有关电荷相互作用是否维持蛋白稳定的实验结果却相互矛盾。本工作以极端嗜热蛋白Ssh10b为模型,主要研究盐桥以及带电残基与蛋白热稳定性的内在联系。理论分析提出:盐桥对温度变化的极度不敏感使得它成为蛋白在高温条件下维持稳定的重要因素。这一理论很好的解释了盐桥与蛋白热稳定性的内在关系,但是,至今没有实验证据。本研究通过双突变循环测定了Ssh10b分子中两个最为保守的盐桥在不同温度下的耦合自由能。结果显示,尽管Ssh10b的稳定自由能随着温度升高而显著下降,但这两个盐桥对蛋白稳定能的直接贡献却几乎不随温度改变,证明了盐桥是高温条件下维持蛋白稳定的重要因素。通过比较本研究的实验结果和一些已经发表的数据,我们发现总体上那些由两个在一级序列上离得较远的残基所形成的盐桥对蛋白稳定能的直接贡献明显要大于那些由两个在一级序列上离得很近的残基所形成的盐桥,这一发现对蛋白质工程颇有意义。本研究中,我们还通过理论分析提出:在中性pH区域,带电残基可解离基团的pKa漂移所引起的蛋白天然态构象熵增加可以促进蛋白稳定。酸碱滴定实验显示,相对于变性态,天然态Ssh10b中有许多可解离基团的pKa发生了漂移。理论计算结果显示一些碱性残基可解离基团的pKa发生了向下漂移,这使得在中性pH区域,天然态时构成蛋白的酸碱平衡分布远比在变性态中更为混乱,由此增加了天然态蛋白的构象熵,从而极大地提高了Ssh10b稳定性。此外,为了研究脯氨酸残基对蛋白稳定的贡献,我们表达纯化了一系列有关脯氨酸的Ssh10b突变体,进行了相关的去折叠实验;提出了一个包含脯氨酸异构化平衡的去折叠模型,根据模型,脯氨酸残基对蛋白稳定能的贡献依赖于其天然态及变性态异构化平衡分布;实验结果与模型预测完全吻合。实验结果还显示,天然态时脯氨酸的异构化导致的构象熵增加可以促进Ssh10b的稳定,从而间接为可解离基团pKa漂移可以促进蛋白稳定这一观点提供了实验证据。更多还原

【Abstract】 A statistical research showed that comparing the structural parameters between hyperthermophilic proteins and mesophilic ones, the only generally observed rule is an increase in the number of salt bridges and charged amino acid residues with increasing growth temperature. However, estimates of the energy contribution of electrostatic interaction to protein stability have led to conflicting conclusions. In this work, we took the hyperthermophilic protein Ssh10b as model, made some researches mainly focused on the relationships between enhanced protein stability and salt bridges/charged residues.Theoretical research showed that salt bridges are extremely resilient to temperature increases and thus are specially suited to promoting protein stability at high temperatures. However, so far there are no reports of experiments to measure the stability contribution of salt bridges at high temperatures and to provide evidence for these theoretical predictions. In this work, a double mutant cycle (DMC) approach was employed to estimate the effect of temperature on the contribution of two highly conserved salt bridges to protein stability in protein Ssh10b. The stability free energy of Ssh10b decrease greatly with increasing temperature, while the direct contribution of these two salt bridges to protein stability remain almost constant, providing evidence supporting the theoretical prediction that salt bridges are extremely resilient to temperature increases and thus are specially suited to improving protein stability at high temperatures.Moreover, comparing our results with published DMC data for the contribution of salt bridges to stability in other proteins, we found that the direct contribution to protein stability of a salt bridge formed by two charged residues far apart in the primary sequence is higher than that of those formed between two very close ones. Implications of this finding are useful for engineering proteins with enhanced thermostability.In this work, we also proposed through theoretical analysis that the increase of conformational entropy in the native state due to pKa shifts of ionizable residues could enhance protein stability. Acid/base titration demonstrated that relative to the unfolded state, the pKa values of many ionizable residues are shifted in the native Ssh10b. Theoretical prediction suggested that the pKa values of some alkali residues are downshifted in the native state, which should result in that at neutral pH region, the native Ssh10b is existed as a conformation ensemble with these ionizable residues in more disordered acid/base equilibrium. Theoretical calculation indicated that the increased conformational entropy in native state induced by pKa shifts at neutral pH region could stabilize the protein greatly.In addition, in order to study the stability contribution of the proline residues, in this work, we expressed and purified a series of Ssh10b mutants about proline, made correlative unfolding experiments in detail. We proposed a new unfolding mechanism including proline isomerization. According to this model, the contribution of a proline residue to protein stability is associated with the thermodynamic equilibrium between the cis- and trans- isomers in both unfolded and folded states, agreeing well with unfolding experimental results. The results also demonstrated that the increased conformational entropy due to two native forms induced by proline isomerization contributes to stabilize the folded protein, providing experimental evidence indirectly for the viewpoint that the increase of conformational entropy in the native state due to pKa shifts of ionizable residues could enhance protein stability.更多还原