Considerable effort has been devoted to mapping the complex interactions that make up the molecular circuitry of living cells. For weak or transient interactions that are not easily identified through affinity-based approaches, methods such as yeast two-hybrid screening, protein-fragment complementation, chemical crosslinking, and photo-crosslinking are particularly useful. Photo-crosslinkers can be directly incorporated into proteins, nucleic acids, and carbohydrates by exogenously supplying cells with appropriate precursors. Alternatively, photo-crosslinking moieties can be site-specifically introduced into proteins chemically or enzymatically, or in response to nonsense codons by means of orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pairs. For example, amino acids containing benzophenone, diazirine, and aryl azide side chains have all been genetically encoded in prokaryotic and eukaryotic organisms, and used to crosslink protein-protein and protein-DNA interactions in vitro and in living cells. However, the size and photochemical reactivity of these photo-crosslinkers might not be ideal for a given site in a target protein of interest. Recently, an aliphatic diazirine amino acid, 3'-azibutyl-N-carbamoyl-lysine (AbK, Figure ) was introduced into proteins by using the wild-type M. barkeri pyrrolysyl tRNA/tRNA synthetase pair (wt-mbPylRS/tRNA Pyl ). Site-specific incorporation of AbK into glutathione S-transferase allowed covalent crosslinking of the two subunits of the dimeric protein in E. coli by using UV light. Inspired by this success, we sought to solve another limitation: in mammalian cells, photo-crosslinking amino acids typically have a low efficiency of incorporation in response to amber (TAG) codons. Here we report that a new aminoacyl-tRNA synthetase engineered from wt-mbPylRS, when coupled with tRNA Pyl , can significantly increase the coding efficiency of AbK in both E. coli and mammalian cells. Moreover, when Abk was substituted for Asp144 in cyclin-dependent kinase 5 (Cdk5), the diazirine moiety photo-crosslinked Cdk5 to its substrate, p21-activated kinase 1 (Pak1).