重新设计高亲和度非特异性核酶,并改变了序列偏好
Yi-Ting Wang1, Jon D Wright, Lyudmila G Doudeva
1Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.
Journal of the American Chemical Society
|November 26, 2009
概括
研究人员设计了一种细菌毒素.
科学领域:
- 生物化学 生物化学
- 结构生物学 结构生物学
- 计算生物学 计算生物学
背景情况:
- 了解蛋白质-DNA相互作用是分子生物学和生物技术的关键.
- 素E7 (nColE7) 的核酶域是研究DNA结合的模型系统.
- 野生类型的nColE7更喜欢在胺和腺之后分裂DNA.
研究的目的:
- 为了产生具有增强DNA结合亲和力和改变序列特异性的重新设计的核酶.
- 研究改善蛋白质-DNA相互作用的结构和能量基础.
主要方法:
- 系统的计算查,以预测高亲和度的nColE7突变.
- 针对DNA结合的局部定向突变发生,蛋白质净化和光动力学测试.
- 用DNA足迹测试来确定序列切割偏好.
- 进行X射线晶体学和自由能量分解分析,以获得结构性见解.
主要成果:
- 在五种工程化nColE7突变中,四种突变呈现出3至5倍更高的DNA结合亲和力.
- 三种突变 (D493N,D493Q,D493R) 显示出对在关氨酸残留物上分裂DNA的偏好增加.
- 结构分析显示,D493的替代解决了不利的静电排斥,改善了蛋白质-DNA相互作用.
结论:
- 计算选对于设计具有改进DNA结合的核酶是有效的.
- 突变可以改变DNA结合亲和力和序列分裂特异性.
- 这种方法为设计定制的DNA结合蛋白提供了一个强大的策略.
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