title: Protein Crosslinking by Genetically Encoded Noncanonical Amino Acids with Reactive Aryl Carbamate Side Chains.
abstract: The use of genetically encoded noncanonical amino acids (ncAAs) to construct crosslinks within or between proteins has emerged as a useful method to enhance protein stability, investigate protein-protein interactions, and improve the pharmacological properties of proteins. We report ncAAs with aryl carbamate side chains (PheK and FPheK) that can react with proximal nucleophilic residues to form intra- or intermolecular protein crosslinks. We evolved a pyrrolysyl-tRNA synthetase that incorporates site-specifically PheK and FPheK into proteins in both E. coli and mammalian cells. PheK and FPheK when incorporated into proteins showed good stability during protein expression and purification. FPheK reacted with adjacent Lys, Cys, and Tyr residues in thioredoxin in high yields. In addition, crosslinks could be formed between FPheK and Lys residue of two interacting proteins, including the heavy chain and light chain of an antibody Fab.
authors.affiliation: Department of Chemistry, the Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA, 92037, USA.
authors.forename: Weimin
authors.initials: W
authors.lastname: Xuan
authors.affiliation: Department of Chemistry, the Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA, 92037, USA.
authors.forename: Sida
authors.initials: S
authors.lastname: Shao
authors.affiliation: Department of Chemistry, the Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA, 92037, USA.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D002219:Carbamates / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods; D011506:Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D000596:Amino Acids; D002219:Carbamates; D003432:Cross-Linking Reagents; D011506:Proteins
keywords: carbamate; crosslinks; noncanonical amino acids; protein engineering; protein modifications