The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Nature. 2010 Dec 9;468(7325):790-5. doi: 10.1038/nature09472. Epub 2010 Nov 17.
Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.
半胱氨酸是蛋白质中最具内在亲核性的氨基酸，其反应性可调节以执行多种生化功能。缺乏定义蛋白质中功能性半胱氨酸的共识序列阻碍了它们的发现和表征。在这里，我们描述了一种蛋白质组学方法，可直接在天然生物系统中大规模定量描绘半胱氨酸残基的固有反应性。超反应性在半胱氨酸中是一种罕见的特征，它被发现指定了广泛的活性，包括亲核和还原催化以及氧化修饰的部位。在几种功能未知的蛋白质中鉴定出了超反应性半胱氨酸，包括在真核生物进化过程中保守的一个残基，我们证明该残基对于酵母活力是必需的，并且参与铁硫蛋白生物发生。我们还表明，定量反应性分析可以作为筛选和功能分配计算设计蛋白质中半胱氨酸的基础，其中它可以区分催化活性和非活性半胱氨酸水解酶设计。
