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. 2013 Feb 15;8(2):405-15.
doi: 10.1021/cb300512r. Epub 2012 Nov 19.

Genetic incorporation of twelve meta-substituted phenylalanine derivatives using a single pyrrolysyl-tRNA synthetase mutant

Yane-Shih Wang  1 , Xinqiang Fang, Hsueh-Ying Chen, Bo Wu, Zhiyong U Wang, Christian Hilty, Wenshe R Liu
Affiliations

Genetic incorporation of twelve meta-substituted phenylalanine derivatives using a single pyrrolysyl-tRNA synthetase mutant

Yane-Shih Wang et al. ACS Chem Biol. .

Abstract

When coexpressed with its cognate amber suppressing tRNACUAPyl(CUA), a pyrrolysyltRNA synthetase mutant N346A/C348A is able to genetically incorporate 12 meta-substituted phenylalanine derivatives into proteins site-specifically at amber mutation sites in Escherichia coli. These genetically encoded noncanonical amino acids resemble phenylalanine in size and contain diverse bioorthogonal functional groups such as halide, trifluoromethyl, nitrile, nitro,ketone, alkyne, and azide moieties. The genetic installation of these functional groups in proteins provides multiple ways to site-selectively label proteins with biophysical and biochemical probes for their functional investigations. We demonstrate that a genetically incorporated trifluoromethyl group can be used as a sensitive 19F NMR probe to study protein folding/unfolding, and that genetically incorporated reactive functional groups such as ketone,alkyne, and azide moieties can be applied to site-specifically label proteins with fluorescent probes. This critical discovery allows the synthesis of proteins with diverse bioorthogonal functional groups for a variety of basic studies and biotechnology development using a single recombinant expression system.

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Figures

Figure 1
Figure 1
(A) Structures of NAAs 10-18. (B) Site-specific incorporation of 10-18 into sfGFP at its S2 position. C indicates a control experiment without the addition of any NAA. ND stands for non-detected. (C) Deconvoluted ESI-MS spectra of sfGFP variants incorporated with 10-18. In sfGFP-X, X is one of 10-18 incorporated at the S2 position.
Figure 2
Figure 2
(A) Unfolding of sfGFP27-15, by stepwise addition of GndCl, characterized by 19F NMR. Titrations were carried out by sequential addition of GndCl, starting from 0 M. (B) Spectra of unfolded sfGFP27-15 upon stepwise reduction of GndCl. Precipitation occurred below 1.6 M GndCl. In A and B, time between each point was 1 h, and measurement temperature was 25 °C. (C) Unfolding of sfGFP2-15, sfGFP8-15, sfGFP130-15, and sfGFP135-15 by stepwise addition of GndCl, characterized by 19F NMR. Time between spectra was 2-3 h, except for the spectra indicated with (●), where additional time of 1-2 days was allowed for transitions to occur.
Figure 3
Figure 3
(A) Structures of 19-21. (B) Site-specific incorporation of 19-21 into sfGFP at its S2 position. C indicates a control experiment without the addition of any NAA. ND stands for non-detected. (C) Deconvoluted ESI-MS spectra of sfGFP variants incorporated with 19-21. In sfGFP-X, X is one of 19-21 represents incorporated at the S2 position.
Figure 4
Figure 4
(A) Structures of dyes D2-D5. (B) Specific labeling of sfGFP-19 with D2. (C) Specific labeling of sfGFP-20 with D3. (D) Specific labeling of sfGFP-20 with D4. (E) Specific labeling of sfGFP-21 with D1. (F) Specific labeling of sfGFP-21 with D5. In B-F, the top panels show denaturing SDS-PAGE analysis of sfGFP proteins with Coomassie blue staining and the bottom panels are from fluorescent imaging of the same gels before Coomassie blue staining. Wt sfGFP stands for wild-type sfGFP.
Figure 5
Figure 5
The superimposed structures of the PylRS complex with pyrrolysyl-AMP (Pyl-AMP) and the OmeRS complex with 9 (Ome). The PylRS complex with pyrrolysyl-AMP is shown in orange for the protein carbon atoms and pink for the pyrrolysyl-AMP carbon atoms. Four mutated residues in OmeRS and the ligand 9 are shown in cyan for the carbon atoms. Letters in the parentheses indicate four mutated residues in OmeRS.
Scheme 1
Scheme 1
Scheme 2
Scheme 2
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