Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases,Biochemistry

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Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases
Biochemistry ( IF 2.9 ) Pub Date : 2017-12-28 00:00:00 , DOI: 10.1021/acs.biochem.7b01014
Mira D Liu ₁ , Elliot A Warner ₁ , Charlotte E Morrissey ₁ , Caitlyn W Fick ₁ , Taia S Wu ₁ , Marya Y Ornelas ₁ , Gabriela V Ochoa ₁ , Brendan S Zhang , Colin M Rathbun , William B Porterfield , Jennifer A Prescher , Aaron M Leconte ₁

Affiliation

Directed evolution has proven to be an invaluable tool for protein engineering; however, there is still a need for developing new approaches to continue to improve the efficiency and efficacy of these methods. Here, we demonstrate a new method for library design that applies a previously developed bioinformatic method, Statistical Coupling Analysis (SCA). SCA uses homologous enzymes to identify amino acid positions that are mutable and functionally important and engage in synergistic interactions between amino acids. We use SCA to guide a library of the protein luciferase and demonstrate that, in a single round of selection, we can identify luciferase mutants with several valuable properties. Specifically, we identify luciferase mutants that possess both red-shifted emission spectra and improved stability relative to those of the wild-type enzyme. We also identify luciferase mutants that possess a >50-fold change in specificity for modified luciferins. To understand the mutational origin of these improved mutants, we demonstrate the role of mutations at N229, S239, and G246 in altered function. These studies show that SCA can be used to guide library design and rapidly identify synergistic amino acid mutations from a small library.

中文翻译：

用于发现突变荧光素酶的统计耦合分析指导文库设计

定向进化已被证明是蛋白质工程的宝贵工具;然而，仍然需要开发新的方法来继续提高这些方法的效率和效果。在这里，我们展示了一种应用先前开发的生物信息学方法统计耦合分析（SCA）的文库设计新方法。SCA 使用同源酶来识别可变且功能重要的氨基酸位置，并参与氨基酸之间的协同相互作用。我们使用 SCA 来指导蛋白质荧光素酶的文库，并证明在单轮选择中，我们可以鉴定出具有几个有价值的特性的荧光素酶突变体。具体来说，我们鉴定了荧光素酶突变体，这些突变体具有红移发射光谱和相对于野生型酶的稳定性提高。我们还鉴定了对修饰的荧光素特异性发生 >50 倍变化的荧光素酶突变体。为了了解这些改良突变体的突变起源，我们证明了 N229、S239 和 G246 突变在功能改变中的作用。这些研究表明，SCA 可用于指导文库设计，并从小型文库中快速鉴定协同氨基酸突变。

更新日期：2017-12-28

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