In vitro evolutionary thermostabilization of congerin II: a limited reproduction of natural protein evolution by artificial selection pressure.
In vitro evolutionary thermostabilization of congerin II: a limited reproduction of natural protein evolution by artificial selection pressure.
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DOI：
10.1016/j.jmb.2005.01.027
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发表时间：
2005-03
影响因子：
5.6
通讯作者：
C. Shionyu-Mitsuyama;Y. Ito;Ayumu Konno;Y. Miwa;T. Ogawa;K. Muramoto;T. Shirai
中科院分区：
文献类型：
--
作者：
C. Shionyu-Mitsuyama;Y. Ito;Ayumu Konno;Y. Miwa;T. Ogawa;K. Muramoto;T. Shirai
利用体外进化蛋白工程技术提高了海鳗半乳糖凝集素（congerin II）的热稳定性。两轮随机PCR诱变和选择实验将congerin II的热稳定性提高到与其天然热稳定同种型congerin I相当的水平。最耐热的双突变体Y16 S/T88 I和相关的单突变体Y16 S和T88 I的晶体结构分别在2.0 nm、1.8 nm和1.6 nm分辨率下测定。Thr 88 Ile突变排除了两个内部水分子，Tyr 16 Ser突变缓解了相邻构象应力，是对热稳定性的主要贡献。在congerin II突变体中的这些特征与在congerin I中观察到的那些相似。在自然选择的压力下，具有KA/KS比为2.6的congerin基因的自然进化被加速。人工施加于congerin II的热稳定选择压力模拟了congerin I的隐含天然压力。结果表明，人工压力使congerin II部分再现了congerin I的自然进化。
The thermostability of the conger eel galectin, congerin II, was improved by in vitro evolutionary protein engineering. Two rounds of random PCR mutagenesis and selection experiments increased the congerin II thermostability to a level comparative to its naturally thermostable isoform, congerin I. The crystal structures of the most thermostable double mutant, Y16S/T88I, and the related single mutants, Y16S and T88I, were determined at 2.0Å, 1.8Å, and 1.6Å resolution, respectively. The exclusion of two interior water molecules by the Thr88Ile mutation, and the relief of adjacent conformational stress by the Tyr16Ser mutation were the major contributions to the thermostability. These features in the congerin II mutants are similar to those observed in congerin I. The natural evolution of congerin genes, with the KA/KSratio of 2.6, was accelerated under natural selection pressures. The thermostabilizing selection pressure artificially applied to congerin II mimicked the implied natural pressure on congerin I. The results showed that the artificial pressure made congerin II partially reproduce the natural evolution of congerin I.