An Engineered Aryl Azide Ligase for Site-Specific Mapping of Protein-Protein Interactions via Photocrosslinking

We re-engineered the small-molecule binding site of E. coli lipoic acid ligase (LplA) to accept a fluorinated aryl azide probe in place of lipoic acid. The mutated ligase can covalently attach the aryl azide to recombinant proteins fused to a 17-amino acid recognition sequence for LplA (called "LAP"). Labeling is highly specific, modifying LAP fusion proteins to the exclusion of all endogenous mammalian proteins. In cell lysate, we labeled FKBP with aryl azide and demonstrated rapamycin-dependent photocrosslinking to FRB.