在人工金属β-乳酸酶的设计和定向演变中,脚手架灵活性/刚性的重要性
Woon Ju Song1,2, Jaeseung Yu2, F Akif Tezcan1
1Department of Chemistry and Biochemistry, University of California, San Diego , La Jolla, California 92093-0356, United States.
Journal of the American Chemical Society
|October 11, 2017
概括
研究人员设计了一个超分子蛋白质支架, 定向进化改善了催化活性,突出了蛋白质灵活性和酶设计稳定性的平衡.
科学领域:
- 生物化学
- 蛋白质工程
- 酶催化
背景情况:
- 超分子蛋白质支架为设计新型催化活动提供了多功能平台.
- 之前的工作证明了金属模板脚手架的设计 (Zn4:C96RIDC14) 与可调节的协调点.
研究的目的:
- 在一个新的超分子蛋白质支架上研究催化酶活性的设计和演变 (Zn8:AB54).
- 描述工程脚手架的结构和功能特性,并评估其可变性.
- 探索蛋白质灵活性和稳定性在酶设计和进化中的作用.
主要方法:
- 使用细胞染色体cb562构建超分子支架的金属模板策略.
- 使用体内选择来提高催化效率的定向进化.
- 工程蛋白变体的结构和功能表征,包括动力分析 (迈凯利斯-门动力学) 和β-乳糖酶活性评估.
- 通过消除二硫化物连接来改变局部刚性的蛋白质结构的修改.
主要成果:
- 一种新的变体,Zn8:AB54,是用不和协调部位进行的,在没有优化的情况下表现出迈凯利斯 - 门动力学.
- Zn8:AB54的定向演化显示出有限的催化改善,这归因于局部蛋白质刚性和缺乏可演化的循环.
- 通过去除二硫化物链接来缓解局部刚性,导致结构转化和增强β- 乳糖酶活性.
结论:
- 超分子支架显示了工程催化酶活性的潜力.
- 蛋白质的灵活性和稳定性是影响酶设计和定向进化的关键因素.
- 调节局部蛋白质刚性可以显著影响酶功能和可进化性.
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