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首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >A fundamental protein property thermodynamic stability revealed solely from large-scale measurements of protein function
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A fundamental protein property thermodynamic stability revealed solely from large-scale measurements of protein function

机译：蛋白质的基本特性即热力学稳定性仅从蛋白质功能的大规模测量中获得

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摘要

The ability of a protein to carry out a given function results from fundamental physicochemical properties that include the protein’s structure, mechanism of action, and thermodynamic stability. Traditional approaches to study these properties have typically required the direct measurement of the property of interest, oftentimes a laborious undertaking. Although protein properties can be probed by mutagenesis, this approach has been limited by its low throughput. Recent technological developments have enabled the rapid quantification of a protein’s function, such as binding to a ligand, for numerous variants of that protein. Here, we measure the ability of 47,000 variants of a WW domain to bind to a peptide ligand and use these functional measurements to identify stabilizing mutations without directly assaying stability. Our approach is rooted in the well-established concept that protein function is closely related to stability. Protein function is generally reduced by destabilizing mutations, but this decrease can be rescued by stabilizing mutations. Based on this observation, we introduce partner potentiation, a metric that uses this rescue ability to identify stabilizing mutations, and identify 15 candidate stabilizing mutations in the WW domain. We tested six candidates by thermal denaturation and found two highly stabilizing mutations, one more stabilizing than any previously known mutation. Thus, physicochemical properties such as stability are latent within these large-scale protein functional data and can be revealed by systematic analysis. This approach should allow other protein properties to be discovered.
机译：蛋白质执行给定功能的能力来自于基本的物理化学性质，包括蛋白质的结构，作用机理和热力学稳定性。研究这些特性的传统方法通常需要直接测量感兴趣的特性，这通常是一项艰巨的任务。尽管可以通过诱变来探测蛋白质特性，但该方法因其通量低而受到限制。最近的技术发展已使该蛋白质的众多变体能够快速量化蛋白质的功能，例如与配体的结合。在这里，我们测量了WW域的47,000个变体与肽配体结合的能力，并使用这些功能测量结果来确定稳定的突变而无需直接检测稳定性。我们的方法扎根于公认的蛋白质功能与稳定性密切相关的概念。蛋白质功能通常通过破坏突变来降低，但是这种下降可以通过稳定突变来挽救。基于此观察结果，我们介绍了伙伴增强，这是一种使用该救援能力来识别稳定突变并在WW域中识别15个候选稳定突变的度量。我们通过热变性测试了六个候选基因，发现了两个高度稳定的突变，一个比以前已知的突变更稳定。因此，在这些大规模蛋白质功能数据中潜在的物理化学性质（例如稳定性）是潜在的，并且可以通过系统分析来揭示。这种方法应允许发现其他蛋白质特性。

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