Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli

Abstract

Benzophenones are among the most useful photocrosslinking agents in biology. We have evolved an orthogonal aminoacyl-tRNA synthetase/tRNA pair that makes possible the in vivo incorporation of p-benzoyl-l-phenylalanine into proteins in Escherichia coli in response to the amber codon, TAG. This unnatural amino acid was incorporated with high translational efficiency and fidelity into the dimeric protein glutathione S-transferase. Irradiation resulted in efficient crosslinking (>50%) of the protein subunits. This methodology may prove useful for discovering and defining protein interactions in vitro and in vivo.

Fig 1.

(A) The chemical structure of pBpa. (B) The active site of B. stearothermophilus (Bs) tyrosyl-tRNA synthetase with bound tyrosine. The homologous residues to those mutated in the library used are shown. (Bs:Y34, N123, D176, F177, and L180 correspond to Mj:Y32, E107, D158, I159, and L162). (C) The selection scheme used to isolate a p-benzoyl-L-phenylalanyl-tRNA synthetase.

Fig 2.

Selection and characterization of a BpaRS. (A) After five rounds of selection, the Bpa-dependent GFP fluorescence of 96 independent clones (48 shown) was screened by replica spotting [DH10B cells cotransformed with individual selected synthetase genes and pREP(2)/YC-JYCUA] onto GMML agar plates in the presence (Upper Left) and absence (Upper Right) of 1 mM pBpa. Upon excitation with a blue laser, amino acid-dependent GFP expression was clearly visible (Lower Right vs. Lower Left). (B) Bpa-dependent expression of sperm whale myoglobin in response to an amber codon at the fourth position in the gene for this protein. The protein yield was similar to that observed when suppressing the amber codon with MjTyrRS and mutRNA

Fig 3.

Photocrosslinking reactions with pBpa. (A) The residues of SjGST mutated to pBpa. Monomers of the dimer are shown in blue and red. The side chain of Phe-52 is shown in orange for each monomer (Left). The side chain of residue Tyr-198 is shown in orange (Right). (B) Surface-specific protein–protein crosslinking. SjGST Phe-52-pBpa or SjGST Tyr-198-pBpa were irradiated for 0, 1, and 5 min with a 360-nm lamp, and monomer and covalent dimer were resolved by SDS/PAGE.