Strategy for Stabilization of CutA1 Proteins Due to Ion-Ion Interactions at Temperatures of over 100 °C
Strategy for Stabilization of CutA1 Proteins Due to Ion-Ion Interactions at Temperatures of over 100 °C
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DOI：
10.1021/acs.biochem.8b00103
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发表时间：
2018-05-08
期刊：
影响因子：
2.9
通讯作者：
Yutani, Katsuhide
中科院分区：
文献类型：
--
作者：
Matsuura, Yoshinori;Takehira, Michiyo;Yutani, Katsuhide
为了阐明带电残基在100℃以上温度下对蛋白质稳定性的贡献，我们从大肠杆菌中构建了许多CutA1蛋白的突变体（EcCutA1）。目的是观察是否可以获得与变性温度接近150℃的超嗜热性嗜热球菌中的CutA1相同的稳定性。变性温度（T-d）为113.2℃的EcCutA1 （Ec0VV）疏水突变体被用作突变模板。由单个带电残基取代的Ec0VV突变体的最高T-d为118.4℃，通过单个突变体的组合构建了多个离子突变体，发现其热稳定性有所提高。多突变体的最高稳定性是由9个带电荷残基取代的突变体，其Td为142.2℃。为了评估突变蛋白的离子-离子相互作用能量，我们使用了300 K时分子动力学模拟获得的结构系综。离子突变体的T-d随离子突变蛋白的离子-离子相互作用计算能量的增加而线性增加，甚至达到接近140℃的温度，这表明离子-离子相互作用累积有助于蛋白质在高温下的稳定。
In order to elucidate the contribution of charged residues to protein stabilization at temperatures of over 100 degrees C, we constructed many mutants of the CutA1 protein (EcCutA1) from Escherichia coli. The goal was to see if one can achieve the same stability as for a CutA1 from hyperthermophile Pyrococcus horikoshii that has the denaturation temperature near 150 degrees C. The hydrophobic mutant of EcCutA1 (Ec0VV) with denaturation temperature (T-d) of 113.2 degrees C was used as a template for mutations. The highest T-d of Ec0VV mutants substituted by a single charged residue was 118.4 degrees C. Multiple ion mutants were also constructed by combination of single mutants and found to have an increased thermostability. The highest stability of multiple mutants was a mutant substituted by nine charged residues that had a Td of 142.2 degrees C. To evaluate the energy of ion-ion interactions of mutant proteins, we used the structural ensemble obtained by a molecular dynamics simulation at 300 K. The T-d of ionic mutants linearly increases with the increments of the computed energy of ion-ion interactions for ionic mutant proteins even up to the temperatures near 140 degrees C, suggesting that ion-ion interactions cumulatively contribute to the stabilization of a protein at high temperatures.