Dithiol amino acids can structurally shape and enhance the ligand-binding properties of polypeptides

• Published in: Nature chemistry Vol. 6; no. 11; pp. 1009 - 1016
• Main Authors: Chen, Shiyu, Gopalakrishnan, Ranganath, Schaer, Tifany, Marger, Fabrice, Hovius, Ruud, Bertrand, Daniel, Pojer, Florence, Heinis, Christian
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.11.2014; Nature Publishing Group
• Subjects: 140/131; 639/638/309/2144; 639/638/309/2420; 639/638/45/611; Amino acids; Amino Acids - chemistry; Amino Acids - metabolism; Analytical Chemistry; Binding Sites; Biochemistry; Catalytic Domain; Chemistry; Chemistry/Food Science; Conotoxins - chemistry; Conotoxins - metabolism; Crystallography, X-Ray; Disulfides - chemistry; Evolution; Inorganic Chemistry; Isomerism; Ligands; Molecular Dynamics Simulation; Nicotinic Antagonists - chemistry; Nicotinic Antagonists - metabolism; Organic Chemistry; Oxidation; Peptides; Peptides - chemistry; Peptides - metabolism; Physical Chemistry; Polypeptides; Protein Binding; Proteinase inhibitors; Proteins; Residues; Serine Proteinase Inhibitors - chemistry; Serine Proteinase Inhibitors - metabolism; Urokinase-Type Plasminogen Activator - chemistry; Urokinase-Type Plasminogen Activator - metabolism; Switzerland
• ISSN: 1755-4330; 1755-4349
• DOI: 10.1038/nchem.2043

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. Disulfide bonds formed between two cysteine residues are important in the folding and stability of proteins. Now, unnatural amino acids with side-chains that contain two thiol groups are described. Incorporation of these dithiol amino acids into a serine protease inhibitor and a nicotinic acetyl choline receptor antagonist is shown to increase their inhibitory activity.