A Genetically Encoded aza-Michael Acceptor for Covalent Cross-Linking of Protein-Receptor Complexes

Selective covalent bond formation at a protein-protein interface potentially can be achieved by genetically introducing into a protein an appropriately tuned electrophilic unnatural amino acid that reacts with a native nucleophilic residue in its cognate receptor upon complex formation. We have evolved orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) pairs that genetically encode three aza-Michael acceptor amino acids, N-epsilon-acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-(S)-phenylalanine (AcrF, 2), and p-vinylsulfonamido-(S)-phenylalanine (VSF, 3), in response to the amber stop codon in Escherichia coli. Using an alpha ErbB2 Fab-ErbB2 antibody-receptor pair as an example, we demonstrate covalent bond formation between an alpha ErbB2-VSF mutant and a specific surface lysine epsilon-amino group of ErbB2, leading to near quantitative cross-linking to either purified ErbB2 in vitro or to native cellular ErbB2 at physiological pH. This efficient biocompatible reaction may be useful for creating novel cell biological probes, diagnostics, or therapeutics that selectively and irreversibly bind a target protein in vitro or in living cells.