Nonproteinogenic deep mutational scanning of linear and cyclic peptides

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 115; no. 43; pp. 10959 - 10964
• Main Authors: Rogers, Joseph M., Passioura, Toby, Suga, Hiroaki
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 23-10-2018
• Subjects: Aliphatic compounds; Amino Acid Sequence; Amino acids; Amino Acids - genetics; Binding; Biological Sciences; Free energy; Gene mapping; Genetic code; Genetic Code - genetics; Interfaces; Interrogation; Mutagenesis; Mutation; Optimization; Peptides; Peptides, Cyclic - genetics; Physical properties; Physical Sciences; Polypeptides; Protein Binding - genetics; Protein interaction; Proteins; Proteins - genetics; Scanning; Structure-Activity Relationship; Thermodynamics; BH3 domains; macrocyclic peptides; noncanonical amino acids; structure–activity relationships; intrinsically disordered proteins
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1809901115

High-resolution structure–activity analysis of polypeptides requires amino acid structures that are not present in the universal genetic code. Examination of peptide and protein interactions with this resolution has been limited by the need to individually synthesize and test peptides containing nonproteinogenic amino acids. We describe a method to scan entire peptide sequences with multiple nonproteinogenic amino acids and, in parallel, determine the thermodynamics of binding to a partner protein. By coupling genetic code reprogramming to deep mutational scanning, any number of amino acids can be exhaustively substituted into peptides, and single experiments can return all free energy changes of binding. We validate this approach by scanning two model protein-binding peptides with 21 diverse nonproteinogenic amino acids. Dense structure–activity maps were produced at the resolution of single aliphatic atom insertions and deletions. This permits rapid interrogation of interaction interfaces, as well as optimization of affinity, fine-tuning of physical properties, and systematic assessment of nonproteinogenic amino acids in binding and folding.