A Genetically Encoded Diazirine Photocrosslinker in Escherichia coli

Photoaffinity labels and crosslinkers have been used to map biomolecular interactions, as well as to identify the biological targets of small molecules. Benzophenones are among the most useful photocrosslinking agents and preferentially insert into C H bonds upon excitation with UV light. Aryl azides and [3-(trifluoromethyl)-3H-diazin-3yl]phenones generate reactive nitrenes and carbenes, respectively, that undergo relatively nonspecific insertion and addition reactions. 6] To facilitate the selective incorporation of photocrosslinking agents into proteins in living cells, we recently genetically encoded para-benzoyl-l-phenylalanine (pBpa) and para-azido-l-phenylalanine (pAzpa) in response to the amber nonsense codon in E. coli, yeast and mammalian cells. These photocrosslinkers were site-specifically incorporated into proteins by means of heterologous amber suppressor tRNA/aminoacyl–tRNA synthetase (aaRS) pairs that recognize the unnatural amino acid, but do not cross-react with endogenous host cell tRNAs, aaRSs or amino acids. To expand the photoaffinity label repertoire, we now report that 4’-[3-(trifluoromethyl)-3H-diazirin-3-yl]-l-phenylalanine (TfmdPhe; Figure 1A) can also be genetically encoded with excellent efficiency and fidelity in bacteria. TfmdPhe has useful photochemical properties and is stable under physiological conditions. Upon excitation by ~350 nm light, TfmdPhe undergoes fragmentation to N2 and a reactive carbene, which readily inserts into C H or O H bonds. In contrast, pAzpa requires relatively short wavelength UV excitation and can rearrange to less reactive secondary products prior to crosslinking. TfmdPhe is also somewhat smaller than pBpa, which can facilitate its incorporation into proteins at sites that are involved in biomolecular interactions. TfmdPhe has been incorporated into proteins previously by a chemically misacylated amber suppressor tRNA in a cell-free protein expression system. However, this method severely limits the protein yield, and is not amenable to studies directly in living cells. TfmdPhe was synthesized by using a previously reported method. To selectively incorporate TfmdPhe into proteins in E. coli, an orthogonal amber suppressor tRNA/aaRS was generated from a Methanococcus jannaschii amber suppressor tRNA (MjtRNACUA)/para-bromo-l-phenylalanyl-tRNA synthetase (BrPheRS) pair by using a previously reported, directed evolution strategy. BrPheRS was used as a template for mutagenesis because para-bromo-l-phenylalanine (BrPhe) is structurally similar to TfmdPhe, and BrPheRS has polypeptide backbone ACHTUNGTRENNUNGrearrangements that enlarge the substrate-binding pocket. Based on the structure of the BrPheRS–BrPhe complex, a library of aaRS active-site mutants was generated, in which residues L32, L65, H70, Q109, H160 and Y161 of BrPheRS were randomized by overlap extension polymerase chain reaction with synthetic oligonucleotide primers; the intended mutations were encoded by NNK (N=A+T+C+G; K=T+G). This library of aaRS mutants (on pBK plasmids under the control of the E. coli GlnRS promoter and terminator) was then passed through rounds of alternating positive and negative selections. In the positive selection, cell survival is dependent on the suppression of an amber codon that was introduced at a permissive site in the chloramphenicol acetyl transferase (CAT) gene in the presence of chloramphenicol and 1 mm TfmdPhe. The negative selection utilizes the toxic barnase gene with amber mutations at two permissive sites, and is carried out in the absence of TfmdPhe. Cells that contain MjTyrRS variants that acylate MjtRNACUA with TfmdPhe, but not any endogenous amino acids survive both positive and negative selections, whereas cells that contain MjTyrRS variants that acylate MjtRNACUA with endogenous amino acids express barnase and die in the negative selection. Five rounds of selection afforded five colonies that survived on chloramphenicol only in the presence of TfmdPhe. DNA sequencing of MjTyrRS mutants in these five colonies revealed one unique TfmdPheRS that had three new mutation sites : Y32I, H70F, and Q109M as well as four mutations (E107S, D158P, I159L, and L162E) that were inherited from BrPheRS. To determine the efficiency and fidelity of the incorporation of TfmdPhe into proteins, an amber codon was substituted for [a] Dr. E. M. Tippmann , Dr. W. Liu , Dr. D. Summerer, A. V. Mack, Prof. Dr. P. G. Schultz Department of Chemistry and the Skaggs Institute for Chemical Biology The Scripps Research Institute 10550 N. Torrey Pines Rd, La Jolla, CA 92037 (USA) Fax: (+1)858-784-9440 E-mail : schultz@scripps.edu [b] Dr. E. M. Tippmann + Present address: Cardiff School of Chemistry, Cardiff University Cardiff, Wales, CF10 3XQ (UK) [c] Dr. W. Liu + Present address: Department of Chemistry, Texas A&M University College Station, TX 77842 (USA) [] These authors contributed equally to this work. Figure 1. A) Structure of 4’-[3-(trifluoromethyl)-3H-diazin-3-yl]-l-phenylalanine (TfmdPhe); B) Specificity and efficiency of TfmdPhe incorporation by TfmdPheRS–MjtRNA CUA pair. TfmdPhe was incorporated in response to an amber codon at position 7 in Z domain, analyzed by SDS-PAGE, and stained by gel code blue. Proteins were purified by Ni-affinity chromatography.