Remarkable improvement in the heat stability of CutA1 from Escherichia coli by rational protein design
Remarkable improvement in the heat stability of CutA1 from Escherichia coli by rational protein design
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DOI：
10.1093/jb/mvq079
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发表时间：
2010-10-01
影响因子：
2.7
通讯作者：
Yutani, Katsuhide
中科院分区：
文献类型：
--
作者：
Matsuura, Yoshinori;Ota, Motonori;Yutani, Katsuhide
为了提高大肠杆菌CutA1蛋白（EcCutA1）的热稳定性，使其在变性温度（T-d）为150℃时具有与同源焦球菌CutA1蛋白相当的稳定性，我们使用突变蛋白稳定性谱（SPMP）来检测EcCutA1的构象与其天然序列之间的结构-序列（3D-1D）相容性[J]。摩尔。杂志。[j].环境科学学报，1998,23(5):733-738。我们在EcCutA1中发现了7个在二面角和疏水性方面不相容的残基。这些残基被适当的氨基酸取代，并通过DSC和变性剂变性来评估突变蛋白的稳定性变化。在7个位点中的5个位点引入的突变提高了EcCutA1的稳定性。单突变体（S11A）和三突变体（S11V/E61V/Q73V）的T-d值比野生型蛋白（89.9℃）分别提高了16.5和26.6℃。这些分析表明：(1)尽管野生型蛋白的稳定性相当高，但EcCutA1的稳定性通过轻微的取代得到了显著提高；(2)稳定性的显著提高可以基于新解决的天然结构进行定量解释；(3)SPMP是检测取代提高蛋白质稳定性的有力工具。
To enhance the heat stability of the CutA1 protein from Escherichia coli (EcCutA1) so that it has comparable stability to CutA1 from Pyrococcus horikoshii with a denaturation temperature (T-d) of 150 degrees C, we used the Stability Profile of Mutant Protein (SPMP) to examine the structure-sequence (3D-1D) compatibility between the conformation of EcCutA1 and its native sequence [J. Mol. Biol., 248, 733-738, (1995)]. We identified seven residues in EcCutA1 that were incompatible in terms of dihedral angles and hydrophobicity. These residues were replaced with appropriate amino acids, and the mutant proteins were evaluated for changes in stability by DSC and denaturant denaturation. The mutations that were introduced at five out of the seven positions improved the stability of EcCutA1. The T-d values of single (S11A) and triple (S11V/E61V/Q73V) mutants improved by 16.5 and 26.6 degrees C, respectively, compared to that of the wild-type protein (89.9 degrees C). These analyses showed that (1) the stability of EcCutA1 is remarkably improved by slight substitutions, even though the stability of the wild-type protein is considerably high, (2) remarkable improvements in the stability can be quantitatively explained based on the newly solved native structure, and (3) SPMP is a powerful tool to examine substitutions that improve protein stability.