A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function
A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function
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DOI：
10.1073/pnas.1209751109
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发表时间：
2012-10-16
影响因子：
11.1
通讯作者：
Fields, Stanley
中科院分区：
文献类型：
--
作者：
Araya, Carlos L.;Fowler, Douglas M.;Fields, Stanley
蛋白质执行给定功能的能力来自基本的物理化学性质，包括蛋白质的结构、作用机制和热力学稳定性。研究这些性质的传统方法通常需要直接测量感兴趣的性质，这通常是一项艰巨的任务。虽然蛋白质性质可以通过诱变来探测，但这种方法受到其低通量的限制。最近的技术发展使得能够快速定量蛋白质的功能，例如针对该蛋白质的许多变体与配体的结合。在这里，我们测量了WW结构域的47，000个变体与肽配体结合的能力，并使用这些功能测量来鉴定稳定突变，而不直接测定稳定性。我们的方法植根于一个公认的概念，即蛋白质功能与稳定性密切相关。蛋白质功能通常会因不稳定突变而降低，但这种降低可以通过稳定突变来挽救。基于这一观察结果，我们介绍了合作伙伴的增强作用，一个度量，使用这种救援能力，以确定稳定的突变，并确定15个候选稳定的突变在WW域。我们通过热变性测试了六个候选人，发现了两个高度稳定的突变，一个比任何以前已知的突变更稳定。因此，物理化学性质，如稳定性是潜在的这些大规模的蛋白质功能数据，可以通过系统分析揭示。这种方法应该允许发现其他蛋白质特性。
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 through-put. 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.