An enzyme-based biosensor for monitoring and engineering protein stability in vivo
An enzyme-based biosensor for monitoring and engineering protein stability in vivo
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用于监测和改造体内蛋白质稳定性的酶生物传感器
DOI：
10.1073/pnas.2101618118
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发表时间：
2021-03
影响因子：
11.1
通讯作者：
Quan Shu
中科院分区：
文献类型：
--
作者：
Ren Chang;Wen Xin;Mencius Jun;Quan Shu
意义 蛋白质稳定性是几种主要人类疾病发病机制的核心，也是广泛研究的关键。蛋白质稳定性工程需要改进的方法。在这里，我们提出了一种高通量筛选策略，通过将蛋白质的稳定性与表达工程细菌酶的细胞的荧光读数联系起来来稳定蛋白质。该策略普遍适用于不同来源、大小和结构特征的蛋白质，包括工业相关酶和疾病相关蛋白质。我们还将这种策略与深度突变扫描相结合，以全面了解突变如何塑造重要表观遗传酶的稳定性景观。我们的策略扩展了当前蛋白质研究的工具箱，还将促进各种蛋白质设计。蛋白质稳定性影响蛋白质的生理功能，也是许多蛋白质工程任务中的一个理想特性，但由于直接监测细胞中蛋白质稳定性的方法的局限性，提高蛋白质稳定性具有挑战性。在这里，我们报告了一种体内稳定性生物传感器，其中将感兴趣的蛋白质（POI）插入微生物酶（CysGA）中，催化内源荧光化合物的形成，从而将 POI 稳定性与简单的荧光读数结合起来。我们证明了生物传感器在定向进化中的实用性，以获得两个 POI 的稳定的、不易聚集的变体（包括人胰岛淀粉样多肽的非淀粉样变体）。除了工程应用之外，我们还利用我们的生物传感器进行深度突变扫描，以实验描绘组蛋白 H3K4 甲基转移酶催化域中所有残基的稳定性相关贡献，从而揭示其具有科学信息的稳定性景观。因此，我们用于体内监测不同蛋白质稳定性的高度易于使用的方法将促进基础研究和应用蛋白质工程工作。
Significance Protein stability is central to the pathogenesis of several major human diseases and is the key to extensive research. Improved methods are needed for protein stability engineering. Here, we present a high-throughput screening strategy to stabilize proteins by linking their stabilities to the fluorescent readout of cells expressing an engineered bacterial enzyme. This strategy is generally applicable to proteins of different sources, sizes, and structural characteristics, including industrial-related enzymes and disease-related proteins. We also combined this strategy with deep mutational scanning to comprehensively understand how mutations shape the stability landscape of an important epigenetic enzyme. Our strategy expands the current toolbox of protein research and will also facilitate a variety of protein design. Protein stability affects the physiological functions of proteins and is also a desirable trait in many protein engineering tasks, yet improving protein stability is challenging because of limitations in methods for directly monitoring protein stability in cells. Here, we report an in vivo stability biosensor wherein a protein of interest (POI) is inserted into a microbial enzyme (CysGA) that catalyzes the formation of endogenous fluorescent compounds, thereby coupling POI stability to simple fluorescence readouts. We demonstrate the utility of the biosensor in directed evolution to obtain stabilized, less aggregation-prone variants of two POIs (including nonamyloidogenic variants of human islet amyloid polypeptide). Beyond engineering applications, we exploited our biosensor in deep mutational scanning for experimental delineation of the stability-related contributions of all residues throughout the catalytic domain of a histone H3K4 methyltransferase, thereby revealing its scientifically informative stability landscape. Thus, our highly accessible method for in vivo monitoring of the stability of diverse proteins will facilitate both basic research and applied protein engineering efforts.
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影响因子：
64.8
作者：
Taylor JP;Brown RH Jr;Cleveland DW
通讯作者：
Cleveland DW
影响因子：
6.8
作者：
Magliery TJ
通讯作者：
Magliery TJ
影响因子：
48
作者：
Fowler, Douglas M.;Fields, Stanley
通讯作者：
Fields, Stanley
影响因子：
4.3
作者：
Akter R;Cao P;Noor H;Ridgway Z;Tu LH;Wang H;Wong AG;Zhang X;Abedini A;Schmidt AM;Raleigh DP
通讯作者：
Raleigh DP
影响因子：
2.9
作者：
Fox, Ayano;Snollaerts, Thibaut;Casanova, Camille Errecart;Calciano, Anastasia;Nogaj, Luiza A.;Moffet, David A.
通讯作者：
Moffet, David A.