Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Biochemistry. 2013 Apr 23;52(16):2839-47. doi: 10.1021/bi400076f. Epub 2013 Apr 12.
A goal of protein engineering technology is developing methods to increase protein stability. However, rational design of stable proteins is difficult because the stabilization mechanism of proteins is not fully understood. In this study, we examined the structural dependence of protein stabilization by introducing single amino acid substitution into ribonuclease H1 from the psychotropic bacterium Shewanella oneidensis MR-1 (So-RNase H1), which was our model protein. We performed saturation mutagenesis at various sites. Mutations that stabilized So-RNase H1 were screened using an RNase H-dependent temperature-sensitive Escherchia coli strain. Stabilizing mutations were identified by the suppressor mutagenesis method. This method yielded 39 stabilized mutants from 513 mutations at 27 positions. This suggested that more than 90% of mutations caused destabilization even in a psychotropic protein. However, 17 positions had stabilizing mutations, indicating that the stabilization factors were dispersed over many positions. Interestingly, the identified mutations were distributed mainly at exposed or nonconserved sites. These results provide a novel strategy for protein stabilization.
蛋白质工程技术的目标之一是开发提高蛋白质稳定性的方法。然而，由于对蛋白质稳定机制的了解还不完全，合理设计稳定的蛋白质是困难的。在这项研究中，我们通过在来自精神细菌希瓦氏菌（Shewanella oneidensis MR-1）的核糖核酸酶 H1（So-RNase H1）中引入单个氨基酸取代，研究了蛋白质稳定的结构依赖性，这是我们的模型蛋白。我们在各种位置进行饱和突变。使用依赖于核糖核酸酶 H 的温度敏感大肠杆菌菌株筛选稳定 So-RNase H1 的突变。通过抑制突变方法鉴定稳定突变。这种方法从 27 个位置的 513 个突变中产生了 39 个稳定的突变体。这表明即使在精神蛋白中，超过 90%的突变导致不稳定。然而，有 17 个位置有稳定的突变，表明稳定因子分散在许多位置。有趣的是，鉴定出的突变主要分布在暴露或非保守的位置。这些结果为蛋白质稳定提供了一种新策略。
