Analyses of ProteinProtein Interactions by In Vivo Photocrosslinking in Budding Yeast

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 incorporate p -benzoyl-l -phenylalanine (p Bpa), a photoreactive unnatural amino acid, into defined positions of a target protein in living yeast cells. Photocrosslinking using the p Bpa-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 for p Bpa-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.