Genetic incorporation of seven ortho-substituted phenylalanine derivatives

Abstract

Seven phenylalanine derivatives with small ortho substitutions were genetically encoded in Escherichia coli and mammalian cells at an amber codon using a previously reported, rationally designed pyrrolysyl-tRNA synthetase mutant (PylRS(N346A/C348A)) coupled with tRNA_CUA^Pyl. Ortho substitutions of the phenylalanine derivatives reported herein include three halides, methyl, methoxy, nitro, and nitrile. These compounds have the potential for use in multiple biochemical and biophysical applications. Specifically, we demonstrated that o-cyano-phenylalanine could be used as a selective sensor to probe the local environment of proteins and applied this to study protein folding/unfolding. For six of these compounds this constitutes the first report of their genetic incorporation in living cells. With these compounds the total number of substrates available for PylRS(N346A/C348A) is increased to nearly 40, which demonstrates that PylRS(N346A/C348A) is able to recognize phenylalanine with a substitution at any side-chain aromatic position as a substrate. To our knowledge, PylRS(N346A/C348A) is the only aminoacyl-tRNA synthetase with such a high substrate promiscuity.

Figure 1

Superimposed structures of the OmeRS complex with O-methyltyrosine and the PylRS complex with pyrrolysyl-adenosyl monophosphate (Pyl-AMP). The structures are based on PDB entries 2Q7H and 3QTC. The PylRS complex with Pyl-AMP is shown in orange for the protein carbon atoms and pink for the Pyl-AMP carbon atoms. Four mutated residues in OmeRS and the p-methoxy-phenylalanine ligand are shown in cyan for the carbon atoms. Letters in parentheses indicate the four mutated residues in OmeRS.

Figure 2

(A) Structures of 1–7. (B) Site-specific incorporation of 1–7 into sfGFP at its S2 site. N/A indicates no NCAA was available in the medium. ND represents nondetected. (C) Deconvoluted ESI-MS of sfGFP with NCAAs 1–7 incorporated at the S2 position. sfGFP-X in which X is one of 1–7 represents the specific sfGFP variant with X incorporated at its S2 site.

Figure 3

Expression of EGFP-N128→X in HEK293T cells cotransfected with the plasmid containing genes encoding PylRS(N346A/C348A) and tRNA_CUA^Pyl. Cell cultures were supplemented with 5 mM of 1–7 or no NCAA (NA). Supplementing the cultures with 5 mM of compound 2 resulted in nearly 100% cell mortality. The relative ratio of expressed EGFP proteins compared to control based on the fluorescent intensity is 2.6, 4.3, 3.2, 4.1, 2.2, and 4.9 for NCAAs 1, 2, 4, 5, 6, and 7, respectively.

Figure 4

Fluorescence spectra of sfGFP-F27→7 (red line), sfGFP-N135→7 (blue line), and wild-type sfGFP (black line) excited at 240 nm. Proteins used were 20 μM in phosphate buffered saline at pH 7.5. The difference in fluorescence is due to the increased solvent exposure of 7 in sfGFP-N135→7. The structure of sfGFP is presented in the left side of the figure with F27 and N135 labeled.

Figure 5

(A) The structure of MPP8 (based on the PDB entry 3QO2). F59 is labeled. (B) Fluorescence spectra of the MPP8-F59→7 at varied concentrations of urea. (C) Fluorescence intensity of MPP8-F59→7 measured at 297 nm as a function of the urea concentration. Proteins used were 2.5 μM in phosphate buffered saline at pH 7.5. The excitation wavelength was 240 nm. A seven point, second order Savitsky–Golay filter was applied to Figure 4B to refine curves.