Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
HiQScreen, CH-1022 Vésenaz, Geneva, Switzerland.
Nat Chem. 2014 Nov;6(11):1009-16. doi: 10.1038/nchem.2043. Epub 2014 Aug 31.
The disulfide bonds that form between two cysteine residues are important in defining and rigidifying the structures of proteins and peptides. In polypeptides containing multiple cysteine residues, disulfide isomerization can lead to multiple products with different biological activities. Here, we describe the development of a dithiol amino acid (Dtaa) that can form two disulfide bridges at a single amino acid site. Application of Dtaas to a serine protease inhibitor and a nicotinic acetylcholine receptor inhibitor that contain disulfide constraints enhanced their inhibitory activities 40- and 7.6-fold, respectively. X-ray crystallographic and NMR structure analysis show that the peptide ligands containing Dtaas have retained their native tertiary structures. We furthermore show that replacement of two cysteines by Dtaas can avoid the formation of disulfide bond isomers. With these properties, Dtaas are likely to have broad application in the rational design or directed evolution of peptides and proteins with high activity and stability.
形成于两个半胱氨酸残基之间的二硫键对于确定和稳定蛋白质和肽的结构非常重要。在含有多个半胱氨酸残基的多肽中，二硫键异构化可能导致具有不同生物学活性的多种产物。在这里，我们描述了一种二硫醇氨基酸 (Dtaa) 的开发，该氨基酸可以在单个氨基酸位点形成两个二硫键。将 Dtaas 应用于含有二硫键限制的丝氨酸蛋白酶抑制剂和烟碱型乙酰胆碱受体抑制剂，分别将其抑制活性提高了 40 倍和 7.6 倍。X 射线晶体学和 NMR 结构分析表明，含有 Dtaas 的肽配体保留了其天然的三级结构。我们还表明，用 Dtaa 替代两个半胱氨酸可以避免形成二硫键异构体。具有这些特性，Dtaa 很可能在具有高活性和稳定性的肽和蛋白质的合理设计或定向进化中得到广泛应用。
