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首页> 外文期刊>The protein journal >Additive effect of single amino acid replacements on the kinetic stability of β-glucosidase B
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Additive effect of single amino acid replacements on the kinetic stability of β-glucosidase B

机译：单个氨基酸替代物对β-葡萄糖苷酶B动力学稳定性的加和作用

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摘要

Previously, we applied in vitro evolution to generate the thermoresistant triple mutant H62R/N223Y/M319I of β-glucosidase B (BglB) from Paenibacillus polymyxa. In order to dissect the energetic contributions to protein stabilization achieved by these mutations, we measured the kinetic constants of the heat denaturation of wild type BglB, the triple mutant and the three single mutants (H62R, N223Y, M319I) by circular dichroism at various temperatures. Our results show that all four mutants delayed the denaturation process. Based on the Transition State theory, the increase of the activation barrier for the thermal denaturation of the triple mutant (ΔΔG N→TS ) is equivalent to that produced by the sum of the contributions from the three single mutants, whose C β s are located at least 18 ? apart. This analysis provides a formal demonstration of the generally accepted idea that protein thermal stability can be increased through sequential addition of individual mutations. Each of the mutations described here contribute in part to the overall effect, which in this case affects the unfolding barrier.
机译：以前，我们应用了体外进化来从多粘芽孢杆菌产生β-葡萄糖苷酶B（BglB）的耐热三联突变体H62R / N223Y / M319I。为了剖析这些突变对蛋白质稳定化的能量贡献，我们在不同温度下通过圆二色性测量了野生型BglB，三重突变体和三个单一突变体（H62R，N223Y，M319I）的热变性的动力学常数。 。我们的结果表明，所有四个突变体均延迟了变性过程。根据过渡状态理论，三重突变体（ΔΔGN→TS）的热变性的激活势垒的增加等同于三个单突变体的贡献之和，这三个突变体的Cβs位于至少18岁？分开。该分析提供了公认的想法的正式证明，即可以通过顺序添加单个突变来增加蛋白质的热稳定性。此处描述的每个突变均部分影响整体效果，在这种情况下会影响展开障碍。

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