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Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking
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Protein Crosslinking by Genetically Encoded Noncanonical Amino Acids with Reactive Aryl Carbamate Side Chains
Weimin Xuan1, Sida Shao1, Peter G Schultz1
1Department of Chemistry, the Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA, 92037, USA.
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.
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