Recent development of methods for genetic incorporation of unnatural amino acids into proteins in live cells enables us to analyze protein interactions by site-specific photocrosslinking. Here we describe a method to incorporatep-benzoyl-l -phenylalanine (pBpa), a photoreactive unnatural amino acid, into defined positions of a target protein in living yeast cells. Photocrosslinking using thepBpa-incorporated proteins has been proven to be a powerful method for analyzing protein–protein interactions at the spatial resolution of amino-acid residues. Since photocrosslinking can be performed forpBpa-incorporated proteins that are properly assembled into a protein complex in living cells, this method will allow us to reveal protein–protein interactions of the target proteins at work.
