Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells

Abstract

The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call dual encoding and labeling (DEAL), allowed us to efficiently encode tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically defined protein-protein cross-linking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro. By enabling the efficient dual modification of proteins in vivo, this DEAL approach provides a tool for the characterization and engineering of proteins in vivo.

Figure 1.

Schematic of in vivo dual encoding and labeling. Noncanonical amino acids (ncAAs) para-azidophenylalanine (pAzF; blue) and a meta-substituted tetrazine-containing phenylalanine derivative (Tet3.0; orange) are encoded into proteins using genetic code expansion via mutually orthogonal, dual nonsense suppression systems. Controlled bioorthogonal labeling at both sites within proteins enables site-specific dual labeling, topologically-defined intermolecular crosslinking and intramolecular stapling in vivo.

Figure 2.

Validation of dual suppression. (A) Normalized SUMO-sfGFP fluorescence for single and dual UAG-UAA suppression at 24 hours in the presence of both pAzF and Tet3.0 at 1.0 and 0.5 mM (hashed blue and orange), pAzF alone (blue), Tet3.0 alone (orange), and in the absence of ncAAs (gray). (B) Overlaid ESI mass spectra of sfGFP containing either pAzF (blue) or Tet3.0 (pink) at site 150, or together at sites 134 (pAzF) and 150 (Tet3.0) (purple), or neither (WT; green). Observed masses are indicated above each major peak. Peaks corresponding to the loss of N-terminal methionine are indicated by “*”, while peaks corresponding to pAzF reduction are indicated by “†”. Mass measurement error is ± 1 Da, see Table S6 for expected masses.

Figure 3.

In vitro dual labeling of dual encoded SUMO-sfGFP. (A) Reaction scheme of SUMO^pAzF-sfGFP^Tet3.0 dual labeling. (B) SDS-PAGE of SUMO^pAzF-sfGFP^Tet3.0 (10 μM) exposed to DBCO-TAMRA and/or sTCO-PEG₅₀₀₀ (100 μM) imaged by Coomassie staining (top) and in-gel fluorescence (bottom). In these experiments, pAzF is incorporated at position 35 (in the SUMO domain), and Tet3.0 at position 253 (in the sfGFP domain). The contents of each lane are indicated above.

Figure 4.

Quantification of dual labeling reactions in vitro and in vivo using two step blocking labeling (see methods). (A) SDS-PAGE of in vitro reactions on sfGFP (10 μM) variants labeled with sTCO-OH and/or DBCO-NH₂ (67 μM each for 15 min, and 24 h, respectively) followed by sTCO-JF669 and/or DBCO-TAMRA (667 μM, 2 h) and off-target reactions quenched with excess pAzF/Tet3.0 prior to in-gel fluorescence imaging; top panel is the overlay of TAMRA and JF669 signals followed by each individual channel, and Coomassie staining. Encoding positions for sfGFP^Tet3.0 and sfGFP^pAzF are at site 150, while encoding of sfGFP^Dual includes Tet3.0 at site 134, and pAzF at site 150. (B) Densitometry quantification of the fluorescent gels in panel A (red bars are quantifications of the TAMRA channel, and green bars are quantifications of the JF669 channel). See the materials and methods section for details about how these quantifications were performed and presented. (C) In vivo labeling analysis analogously presented to panel A. (D) Densitometry quantification of fluorescent gel channels in panel C, processed and presented analogously to panel B.

Figure 5.

In vitro and in vivo intermolecular protein-protein crosslinking through DEAL. (A) Reaction scheme of crosslinking between sfGFP^Tet3.0 and mTagBFP2^pAzF via an sTCO-DBCO linker. (B) SDS-PAGE analysis of in vitro reaction between purified sfGFP^Tet3.0 and mTagBFP2^pAzF (10 μM each) at 2 and 24 hours after sTCO-DBCO addition (10 μM). The lower mobility crosslinked product, sfGFP-mTagBFP2*, matches the mass loss for photolyzed mTagBFP2* known to accumulate over time. (C) Size exclusion chromatogram of crosslinking reaction mixture at 24 hours post sTCO-DBCO addition and monitored by absorbance at 280 nm (black), 399 nm (mTagBFP2 λ_max; blue), 485 nm (sfGFP λ_max; green). Dashed lines are elution profiles of purified sfGFP^WT (dashed green) and mTagBFP2^WT (dashed blue). (D) SDS-PAGE in-gel fluorescence image of in vivo crosslinking reaction time course following the addition of sTCO-DBCO (concentration empirically determined, see materials and methods) to E. coli cells containing expressed either sfGFP^Tet3.0 and mTagBFP2^pAzF, or sfGFP^WT and mTagBFP2^WT. (E) Densitometry quantification of panel D. The percent crosslinked was estimated as the percentage of fluorescence that the upper, crosslinked product band constitutes of the total fluorescence in each lane at each time point. The contents of each lane are indicated above each gel.

Figure 6.

In vitro and in vivo intramolecular protein stapling via DEAL. (A) Reaction scheme of intramolecular stapling of sfGFP^Tet3.0-mTagBFP2^pAzF (10 μM) with sTCO-PEG₄-DBCO (10 μM) and subsequent TEV cleavage. (B) Analysis of in vitro protein stapling of sfGFP^Tet3.0-mTagBFP2^pAzF and mTagBFP2^pAzF-sfGFP^Tet3.0 with sTCO-PEG₄-DBCO, using SDS-PAGE fluorescent imaging. *refers to both sfGFP-mTagBFP2 (starting material) and sfGFP (stapled product), since they have similar mobility. (C) SDS-PAGE in-gel fluorescence image of in vivo reaction time course following the addition of sTCO-PEG₄-DBCO (20 μM) to E. coli cells containing expressed sfGFP^Tet3.0-mTagBFP2^pAzF or sfGFP^WT-mTagBFP2^WT. (D) Densitometry quantification of panel C. The percent stapled was estimated as the percentage of fluorescence that the upper, stapled product band constitutes of the total fluorescence in each lane at each time point. The contents of each lane are indicated above each gel.

Figure 7.

In vivo dual fluorophore labeling of dual encoded protein in E. coli. (A) Reaction scheme of dual fluorophore labeling of sfGFP^Tet3.0-mTagBFP2^pAzF with sTCO-JF669 and DBCO-TAMRA. (B) SDS-PAGE of resulting E. coli cell lysate after in vivo reactions in cells containing expressed sfGFP^Tet3.0-mTagBFP2^pAzF labeled with sTCO-JF669 and DBCO-TAMRA (1 μM for 5 and 60 min, respectively); top panel is the overlay of TAMRA and JF669 channels followed by each individual channel, and Coomassie staining. Cells exposed to fluorescent labels were quenched with excess pAzF and/or Tet3.0 prior to sample preparation.

Figure 8.

Characterization of ScRPA labeling and FRET analysis of DNA binding. (A) Structural model of S. cerevisiae RPA complex (PDB: 6I52) highlighting the location of encoded pAzF and Tet3.0 in DBD-A and DBD-D, respectively. (B) Scheme depicting RPA binding ssDNA and the predicted FRET changes associated with these conformational states. (C) SDS-PAGE in-gel fluorescence analysis of ScRPA after labeling by DBCO-Cy3 and TCO-Cy5. Each dye signal is imaged individually and as an overlay. (D) Resulting Cy5 fluorescence intensity from dual Cy3/Cy5 labeled ScRPA^Dual as a function of DNA concentration.