Multiplexed genomic encoding of non-canonical amino acids for labeling large complexes

Abstract

Stunning advances in the structural biology of multicomponent biomolecular complexes (MBCs) have ushered in an era of intense, structure-guided mechanistic and functional studies of these complexes. Nonetheless, existing methods to site-specifically conjugate MBCs with biochemical and biophysical labels are notoriously impracticable and/or significantly perturb MBC assembly and function. To overcome these limitations, we have developed a general, multiplexed method in which we genomically encode non-canonical amino acids (ncAAs) into multiple, structure-informed, individual sites within a target MBC; select for ncAA-containing MBC variants that assemble and function like the wildtype MBC; and site-specifically conjugate biochemical or biophysical labels to these ncAAs. As a proof-of-principle, we have used this method to generate unique single-molecule fluorescence resonance energy transfer (smFRET) signals reporting on ribosome structural dynamics that have thus far remained inaccessible to smFRET studies of translation.

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Fig. 1: General overview of multiplexed, bioorthogonal labeling of MBCs using genomically encoded ncAAs.
Fig. 2: Locations of amino acid residues in ribosomal proteins that were targeted for labeling.
Fig. 3: Site-specific Cy3 and/or Cy5 labeling of ribosomes purified from genomic mutant strains.
Fig. 4: smFRET experiments using Cy3- and Cy5-labeled ribosomal complexes assembled using ribosomes isolated from mutant strains.

Data availability

With the exception of the smFRET data, all other data supporting the findings of this study are presented within this article. Due to the lack of a public repository for smFRET data, the smFRET data supporting the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

The code used to analyze the TIRF movies in this study is associated with a manuscript in preparation (J. Hon, C. Kinz-Thompson, R.L.G.), and is consequently available from the corresponding author upon request.

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Acknowledgements

We thank H. Wang and J. Park for helpful discussions regarding MGE, B. Huang and H. Li for supplying useful reagents, and C. Kinz-Thompson and K. Caban for valuable feedback on the manuscript. This work was supported by funds to R.L.G. from the National Institute of Health (grant nos. R01 GM084288 and R01 GM137608).

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B.J.D. and R.L.G. designed the experiments. B.J.D. performed the experiments. B.J.D. and R.L.G. analyzed the results and wrote the manuscript. Both authors agreed to the final version of the manuscript.

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Correspondence to Ruben L. Gonzalez Jr..

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Extended data

Extended Data Fig. 1 Site-specific Cy3 and/or Cy5 labeling of ribosomes purified from wildtype versus genomic mutant strains.

SDS-PAGE analysis of ribosomal proteins derived from 30 S or 50 S subunits isolated from the wildtype (wt) strain (Lanes 1 and 2) or the IR1, MT1, HS1, and HS2 mutant strains (Lanes 4-8) and reacted with DBCO-derivatized Cy3 and/or Cy5 fluorophores as shown in Fig. 3. Left panel shows visible light scan of Coomassie-stained gel. Middle and right panels show fluorescence emission scans of pre-Coomassie-stained gel using excitation wavelengths of 532 nm for Cy3 (Middle Panel) and 635 nm for Cy5 (Right Panel). The position at which each labeled ribosomal protein is expected to run on the SDS-PAGE gel was determined using a standard protein molecular weight ladder, and is indicated along the right side of the figure. The experiment shown here was replicated a total of three times, with similar results each time.

Extended Data Fig. 2 Idealized EFRET versus time trajectories generated using Bayesian inference-based hidden Markov modeling.

Representative Cy3- and Cy5 fluorescence intensity versus time trajectories (Top Sub-Panel) and corresponding EFRET versus time trajectories (Bottom Sub-Panel) for smFRET experiments performed on ribosomal complexes assembled using Cy3- and/or Cy5-labeled 30 S and/or 50 S subunits isolated from the (a and b) HS1, (c) MT1, and (d) IR1 strains as shown in Fig. 4. In the Cy3 and Cy5 fluorescence intensity versus time trajectories, the Cy3 and Cy5 fluorescence intensities are shown as green and red curves, respectively. In the EFRET versus time trajectories, the EFRET is shown as blue curves. The idealized EFRET versus time trajectory is shown as black lines.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Tables 1–5.

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Source data

Source Data Fig. 3 and Extended Data Fig. 1

Three replicates of: unprocessed visible light scans of Coomassie-stained gels; unprocessed fluorescence scans of unstained gels with a 532-nm excitation source and unprocessed fluorescence scans of unstained gels with a 635-nm excitation source.

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Desai, B.J., Gonzalez, R.L. Multiplexed genomic encoding of non-canonical amino acids for labeling large complexes. Nat Chem Biol 16, 1129–1135 (2020). https://doi.org/10.1038/s41589-020-0599-5

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