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Genetically encoded bioorthogonal tryptophan decaging in living cells

Nature Chemistry volume 16, pages 533–542 (2024)Cite this article

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Abstract

Tryptophan (Trp) plays a critical role in the regulation of protein structure, interactions and functions through its π system and indole N–H group. A generalizable method for blocking and rescuing Trp interactions would enable the gain-of-function manipulation of various Trp-containing proteins in vivo, but generating such a platform remains challenging. Here we develop a genetically encoded N1-vinyl-caged Trp capable of rapid and bioorthogonal decaging through an optimized inverse electron-demand Diels–Alder reaction, allowing site-specific activation of Trp on a protein of interest in living cells. This chemical activation of a genetically encoded caged-tryptophan (Trp-CAGE) strategy enables precise activation of the Trp of interest underlying diverse important molecular interactions. We demonstrate the utility of Trp-CAGE across various protein families, such as catalase-peroxidases and kinases, as translation initiators and posttranslational modification readers, allowing the modulation of epigenetic signalling in a temporally controlled manner. Coupled with computer-aided prediction, our strategy paves the way for bioorthogonal Trp activation on more than 28,000 candidate proteins within their native cellular settings.

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Fig. 1: Design of the bioorthogonal Trp-caging/decaging chemistry.
Fig. 2: Development of the bioorthogonal Trp-caging/decaging chemistry.
Fig. 3: Validating Trp-CAGE in vitro and in living cells.
Fig. 4: Establishment of the Trp-CAGE-based general protein activation platform.
Fig. 5: Applying Trp-CAGE for manipulating epigenetic reader proteins in living cells.

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Data availability

Data relating to the materials and methods, optimization studies, experimental procedures, DFT calculations, crystallographic data for vyW, NMR spectra and mass spectrometry are available in the Article or its Supplementary Information. Uncropped gels/blots and MS–MS data are provided in the related source data. Proteomics data are available via ProteomeXchange with the identifier PXD048817. Crystallographic data for vyW have been deposited at the Cambridge Crystallographic Data Centre, under the deposition number CCDC 2327816. Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/. Source data are provided with this paper.

Code availability

The source codes for Python used for measuring Trp–ligand distances are available in Supplementary Note 1.

References

  1. Khemaissa, S., Sagan, S. & Walrant, A. Tryptophan, an amino-acid endowed with unique properties and its many roles in membrane proteins. Crystals 11, 1032 (2021).

    Article  CAS  Google Scholar 

  2. Barik, S. The uniqueness of tryptophan in biology: properties, metabolism, interactions and localization in proteins. Int. J. Mol. Sci. 21, 8776 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Cuello, L. G. et al. Structural basis for the coupling between activation and inactivation gates in K+ channels. Nature 466, 272–275 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Spiro, K. R. R. A. T. G. Nanosecond dynamics of the R–T transition in hemoglobin: ultraviolet Raman studies. Science 265, 1697–1699 (1994).

    Article  PubMed  Google Scholar 

  5. Foda, Z. H., Shan, Y., Kim, E. T., Shaw, D. E. & Seeliger, M. A. A dynamically coupled allosteric network underlies binding cooperativity in Src kinase. Nat. Commun. 6, 5939 (2015).

    Article  CAS  PubMed  Google Scholar 

  6. Loewen, P. C., Carpena, X., Vidossich, P., Fita, I. & Rovira, C. An ionizable active-site tryptophan imparts catalase activity to a peroxidase core. J. Am. Chem. Soc. 136, 7249–7252 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Norimatsu, Y., Hasegawa, K., Shimizu, N. & Toyoshima, C. Protein–phospholipid interplay revealed with crystals of a calcium pump. Nature 545, 193–198 (2017).

    Article  CAS  PubMed  Google Scholar 

  8. Chen, K.-M. et al. Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G. Nature 452, 116–119 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Xue, Y. et al. Cu(I) recognition via cation–π and methionine interactions in CusF. Nat. Chem. Biol. 4, 107–109 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Xu, R. et al. A recurring motif for antibody recognition of the receptor-binding site of influenza hemagglutinin. Nat. Struc. Mol. Biol. 20, 363–370 (2013).

    Article  CAS  Google Scholar 

  11. Wilson, K. A., Kellie, J. L. & Wetmore, S. D. DNA–protein π-interactions in nature: abundance, structure, composition and strength of contacts between aromatic amino acids and DNA nucleobases or deoxyribose sugar. Nucleic Acids Res. 42, 6726–6741 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Dougherty, D. A. & Gallivan, J. P. Cation–π interactions in structural biology. Proc. Natl Acad. Sci. USA 96, 9459–9464 (1999).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Knox, H. J., Rego Campello, H., Lester, H. A., Gallagher, T. & Dougherty, D. A. Characterization of binding site interactions and selectivity principles in the α3β4 nicotinic acetylcholine receptor. J. Am. Chem. Soc. 144, 16101–16117 (2022).

    Article  CAS  PubMed  Google Scholar 

  14. Zhao, H. et al. Manipulating cation–π interactions with genetically encoded tryptophan derivatives. J. Am. Chem. Soc. 144, 6742–6748 (2022).

    Article  CAS  PubMed  Google Scholar 

  15. Krall, N., da Cruz, F. P., Boutureira, O. & Bernardes, G. J. Site-selective protein-modification chemistry for basic biology and drug development. Nat. Chem. 8, 103–113 (2016).

    Article  CAS  PubMed  Google Scholar 

  16. Li, J. & Chen, P. R. Development and application of bond cleavage reactions in bioorthogonal chemistry. Nat. Chem. Biol. 12, 129–137 (2016).

    Article  CAS  PubMed  Google Scholar 

  17. Wang, J., Wang, X., Fan, X. & Chen, P. R. Unleashing the power of bond cleavage chemistry in living systems. ACS Cent. Sci. 7, 929–943 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wu, Y. I. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Toettcher, J. E., Weiner, O. D. & Lim, W. A. Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell 155, 1422–1434 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Taslimi, A. et al. An optimized optogenetic clustering tool for probing protein interaction and function. Nat. Commun. 5, 4925 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A. & Liu, D. R. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat. Chem. Biol. 11, 316–318 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wang, H. et al. LOVTRAP: an optogenetic system for photoinduced protein dissociation. Nat. Methods 13, 755–758 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhou, X. X., Fan, L. Z., Li, P., Shen, K. & Lin, M. Z. Optical control of cell signaling by single-chain photoswitchable kinases. Science 355, 836–842 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Wang, J. et al. Time-resolved protein activation by proximal decaging in living systems. Nature 569, 509–513 (2019).

    Article  CAS  PubMed  Google Scholar 

  25. Wang, J. et al. Palladium-triggered chemical rescue of intracellular proteins via genetically encoded allene-caged tyrosine. J. Am. Chem. Soc. 138, 15118–15121 (2016).

    Article  CAS  PubMed  Google Scholar 

  26. Arbely, E., Torres-Kolbus, J., Deiters, A. & Chin, J. W. Photocontrol of tyrosine phosphorylation in mammalian cells via genetic encoding of photocaged tyrosine. J. Am. Chem. Soc. 134, 11912–11915 (2012).

    Article  CAS  PubMed  Google Scholar 

  27. Deiters, A., Groff, D., Ryu, Y., Xie, J. & Schultz, P. G. A genetically encoded photocaged tyrosine. Angew. Chem. Int. Ed. 45, 2728–2731 (2006).

    Article  CAS  Google Scholar 

  28. Li, J., Jia, S. & Chen, P. R. Diels–Alder reaction-triggered bioorthogonal protein decaging in living cells. Nat. Chem. Biol. 10, 1003–1005 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Wang, X. et al. Copper-triggered bioorthogonal cleavage reactions for reversible protein and cell surface modifications. J. Am. Chem. Soc. 141, 17133–17141 (2019).

    Article  CAS  PubMed  Google Scholar 

  30. Li, J. et al. Palladium-triggered deprotection chemistry for protein activation in living cells. Nat. Chem. 6, 352–361 (2014).

    Article  CAS  PubMed  Google Scholar 

  31. Cheung, J. W. et al. Genetic encoding of a photocaged histidine for light‐control of protein activity. Chembiochem. 24, e202200721 (2023).

  32. Ling, X. et al. Genetic encoding of a photocaged glutamate for optical control of protein functions. CCS Chem. 5, 1301–1307 (2023).

  33. Zhang, G. et al. Bioorthogonal chemical activation of kinases in living systems. ACS. Cent. Sci. 2, 325–331 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Huguenin-Dezot, N. et al. Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid. Nature 565, 112–117 (2019).

    Article  CAS  PubMed  Google Scholar 

  35. Luo, J., Liu, Q., Morihiro, K. & Deiters, A. Small-molecule control of protein function through Staudinger reduction. Nat. Chem. 8, 1027–1034 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ngai, W. S. C. et al. Bioorthogonally activatable base editing for on-demand pyroptosis. J. Am. Chem. Soc. 144, 5411–5417 (2022).

    Article  CAS  PubMed  Google Scholar 

  37. Fan, X. et al. Optimized tetrazine derivatives for rapid bioorthogonal decaging in living cells. Angew. Chem. Int. Ed. 55, 14046–14050 (2016).

    Article  CAS  Google Scholar 

  38. Versteegen, R. M., Rossin, R., ten Hoeve, W., Janssen, H. M. & Robillard, M. S. Click to release: instantaneous doxorubicin elimination upon tetrazine ligation. Angew. Chem. Int. Ed. 52, 14112–14116 (2013).

    Article  CAS  Google Scholar 

  39. Carlson, J. C. T., Mikula, H. & Weissleder, R. Unraveling tetrazine-triggered bioorthogonal elimination enables chemical tools for ultrafast release and universal cleavage. J. Am. Chem. Soc. 140, 3603–3612 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Gordon, C. G. et al. Reactivity of biarylazacyclooctynones in copper-free click chemistry. J. Am. Chem. Soc. 134, 9199–9208 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Jimenez-Moreno, E. et al. Vinyl ether/tetrazine pair for the traceless release of alcohols in cells. Angew.Chem. Int.Ed. 56, 243–247 (2017).

    Article  CAS  Google Scholar 

  42. Ding, W. et al. Chimeric design of pyrrolysyl-tRNA synthetase/tRNA pairs and canonical synthetase/tRNA pairs for genetic code expansion. Nat. Commun. 11, 3154 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Zhao, H. et al. Directed-evolution of translation system for efficient unnatural amino acids incorporation and generalizable synthetic auxotroph construction. Nat. Commun. 12, 7039 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Marcotrigiano, J., Gingras, A.-C., Sonenberg, N. & Burley, S. K. Cocrystal structure of the messenger RNA 5′ cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell 89, 951–961 (1997).

    Article  CAS  PubMed  Google Scholar 

  45. Truitt, M. L. & Ruggero, D. New frontiers in translational control of the cancer genome. Nat. Rev. Cancer 16, 288–304 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wang, G. G. et al. Haematopoietic malignancies caused by dysregulation of a chromatin-binding PHD finger. Nature 459, 847–851 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Dong, C. et al. Structural basis for the binding selectivity of human CDY chromodomains. Cell. Chem. Biol. 27, 827–838 e827 (2020).

    Article  CAS  PubMed  Google Scholar 

  48. Fan, H. et al. BAHCC1 binds H3K27me3 via a conserved BAH module to mediate gene silencing and oncogenesis. Nat. Genet. 52, 1384–1396 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Cai, L. et al. An H3K36 methylation-engaging Tudor motif of polycomb-like proteins mediates PRC2 complex targeting. Mol. Cell 49, 571–582 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. Villasenor, R. et al. ChromID identifies the protein interactome at chromatin marks. Nat. Biotechnol. 38, 728–736 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Frisch, M. J. T. et al. Gaussian 09, revision A.01 (Gaussian, 2009).

  52. Becke, A. D. Density‐functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652 (1993).

    Article  CAS  Google Scholar 

  53. Yang, J., Liang, Y., Šečkutė, J., Houk, K. N. & Devaraj, N. K. Synthesis and reactivity comparisons of 1-methyl-3-substituted cyclopropene mini-tags for tetrazine bioorthogonal reactions. Chem. Eur. J. 20, 3365–3375 (2014).

    Article  CAS  PubMed  Google Scholar 

  54. Kim, D. E., Chivian, D. & Baker, D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 32, W526–W531 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Meiler, J. & David, B. ROSETTALIGAND: protein–small molecule docking with full side-chain flexibility. Proteins 65, 538–548 (2006).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Ministry of Science and Technology (2021YFA1302603 to P.R.C., 2019YFA09006600 to S.L., 2019YFA0904201, 2022YFA1304700 to X.F.), the National Natural Science Foundation of China (21937001, 22137001, 91753000 to P.R.C., 22222705, 92253302, 22361142828 to S.L., 22222701, 22077004, 92253301 to X.F., 22207095 to W.D. and 22207005 to Y.Z.), the Beijing Natural Science Foundation (Z200010 to P.R.C.), the Postdoctoral Program for Innovative Talents (BX20200005 to Y.Z.), the Feng Foundation of Biomedical Research to S.L., and the New Cornerstone Science Foundation through the New Cornerstone Investigator Program and the XPLORER Prize to P.R.C.

Author information

Author notes

  1. These authors contributed equally: Yuchao Zhu, Wenlong Ding.

Authors and Affiliations

  1. New Cornerstone Science Laboratory, Synthetic and Functional Biomolecules Center, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Yuchao Zhu, Ye Shan, Xinyuan Fan & Peng R. Chen

  2. Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

    Wenlong Ding, Yulin Chen, Chao Liu & Shixian Lin

  3. Center for Life Sciences, Shaoxing Institute, Zhejiang University, Shaoxing, China

    Yulin Chen, Chao Liu & Shixian Lin

  4. Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Ye Shan & Peng R. Chen

  5. Department of Medical Oncology, State Key Laboratory of Transvascular Implantation Devices, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Shixian Lin

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Contributions

P.R.C., S.L., X.F., Y.Z. and W.D. conceived the study. Y.Z. conducted chemistry development, protein decaging validation, and RLuc, src, KatG and eIF4E decaging experiments. W.D. developed the computational pipeline, aaRS direct evolution, and mCherry and epigenetic reader decaging experiments. Y.C. and C.L. assisted with computational design and decaging experiments. Y.S. contributed to the chemical synthesis and biochemical experiments. Y.Z., W.D., X.F., S.L. and P.R.C. wrote the paper with inputs from all authors.

Corresponding authors

Correspondence to Xinyuan Fan, Shixian Lin or Peng R. Chen.

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Extended data

Extended Data Fig. 1 Design and Establishment of Trp-CAGE.

A, Overview of caging and decaging strategies. Strategies for π system caging and decaging remain undeveloped. B, Trp interactions. Caging π interaction is necessary for covering all Trp interactions. C, Caging group evaluation for Trp π system. Unlike previous reported caging groups, vinyl group weakened Trp π system via conjugation. D, Nonlinear regressions of one phase decay was used during kinetic fitting. The calculation formula of fitted curve, half-life, and second-order rate constants K2 were shown. E, F, G, H, I, Kinetic fitted curve for iEDDA reaction between vyW and Tz 4, 10, 12, 13, 14, respectively. Tzs (1 mM) was treated with a ten-fold excess of vyW in PBS. Characteristic Tz peak absorbance at 520 nm disappears upon reaction, and was tracked over time (30 min) by absorbance scans using a Synergy H4 microplate reader. Reaction data were processed using GraphPad Prism 9.0 and fit to nonlinear regressions of one phase decay.

Source data

Extended Data Fig. 2 Screening, optimization, and validation of Trp-CAGE.

A, The amber suppression efficiency of vyW by the indicated chPheRSs was tested by a GFP reporter assay (n = 2). B, The chPheRS mutants by rational design for vyW recognition. Rational mutations were labelled light brown. C, In vitro optimization of decaging conditions on GFP protein. 100 μL of GFP variant (10 μM) in PBS was treated with Tz at 37 °C for 60 min. The yields were calculated based on relative intensity of substrate and product peaks in LC-MS. D, the activity of wild-type Rluc-WT, RLuc-261-W variant and RLuc-261-vyW variant with/without Tz treatment. Error bars represent ± standard error of the mean (n = 3). Two-tailed t test was used. ****P < 0.0001. E, The decaging of RLuc-261-vyW in different incubation times. The activation signal can be observed after 1 min of Tz treatment, and the signal reached to 50% of maximum activation within 10–15 min. Error bars represent ± standard error of the mean (n = 3 biologically independent samples). Two-tailed t test was used. ****P < 0.0001. F, Summarized Km of RLuc-WT, RLuc-261-W and RLuc-261-vyW. The Km were generated by Michaelis–Menten analysis with Graphpad Prism 9.0. G, RLuc catalysed luminescence process. The peroxide intermediate is crucial for the final product generation. H, The crystal structure of coelenterazine binding to RLuc (PDB code: 7QXR). F261 is close to the heterocycle core of coelenterazine. I, the incorporated vyW may react with peroxide intermediate, thus preventing the formation of the final luminescent product. J, The Prilezhaev oxidation reaction.

Source data

Extended Data Fig. 3 Investigation of potential cross reactivity of strained intermediate and the rationale of choosing tetrazines.

A, Protein pull-down experiment. GFP-D190-vyW was expressed in E. coli, 100 μM Tz 4 was added to trigger the formation of the strained intermediate. The proteins were pulled down with His beads after decaging for 1 h. B, The intact-MS analysis of pull-downed protein samples in the 5000 to 80000 Da range. Around 70% of GFP-190-vyW was rescued in living E. coli cells. C, Gel analysis of pull-downed protein samples. No obvious cross-linked band was detected (n = 2 biologically independent experiments). D, MS/MS analysis of pull-downed samples. The non-linked, decaged vyW>W mutant peptide was detected with high confidence. E, The rationale of choosing tetrazines on various proteins.

Source data

Extended Data Fig. 4 Computer-aided screening of proteins with key tryptophan residue(s).

A, The top10 ligands families from the screening. The X-axis represent the ligand types in every family. The counts of every family were shown in right. B, The function analysis of proteins from screening. The top15 functions of name were shown. C, The detailed categories of ligand in amino acids and its modifications family. The counts of every ligand were shown. D, The detailed categories of ligand in cofactors family. The counts of every ligand were shown.

Extended Data Fig. 5 Optimization and application of Trp-CAGE as a general protein activation platform.

A, LC–MS characterization of mCherry-vyW before and after decaging with Tz 10. The LC-MS peak for mCherry-vyW was labelled in the figure, which indicated that the chromophore of mCherry-92-vyW was not formed (Calculated MW of unmatured mCherry-92-vyW is 27641 Da, and the observed MW is 27644 Da). When decaging with Tz, the major peak was shifted. The molecular weight of this peak was identical with maturated mCherry (decgaing vinyl group and formatting chromophore, the calculated and observed MWs are both 27593 Da). B, Time dependent fluorescence intensity increase of rescued mCherry. C, Evaluating katG decaing efficiency of different Tzs. Asymmetric Tz 13 is the most efficient one among tested. Error bars represent ±standard error of the mean (n = 3 biologically independent samples). D, Schematic of allosteric regulation of Src. Created with BioRender.com. E, Evaluating src decaing efficiency of different Tzs. Asymmetric Tz 12 is the most efficient (n = 2 biologically independent experiments). F, Western blotting analysis of in vivo src-428-vyW decaging (n = 2 biologically independent experiments). G, Schematic of eIF4E activation mediated c-myc upregulation. H, Western blotting analysis of eIF4E-46-vyW, eIF4E-56-vyW decaging in HEK 293T (n = 2 biologically independent experiments). I, Western blotting analysis of eIF4E-102-vyW, eIF4E-166-vyW decaging in HEK 293T cells (n = 2 biologically independent experiments).

Source data

Extended Data Fig. 6 Appling Trp-CAGE for manipulating epigenetic reader proteins.

A, LC-MS validation of reader domain decaging. B, The live cell imaging of CDYL1-chromo domain variants in HEK 293 T cells (n = 3). Scale bar: 10 μm. C, The live cell imaging of BAHCC1-BAH domain variants in HEK 293T cells (n = 3 biologically independent experiments). Scale bar: 10 μm. D-G, Appling Trp-CAGE to recusing function of caged CDY1-chromo domains (D, F) and CBX1-chromo domains (E, G) in vitro (D, E) and in live cells (F, G). MST analysis of affinity of CDY1 (D) and CBX1 (E) variants for H3K9me3 peptide. The data were fitted with a Kd model by MO.Affinity.Analysis Software (v2.3). Fraction bound = 1/(1 + (Kd/[D])), where [D] is the total concentration of the ligand peptide. WT proteins were used as a positive control. In the MST assay, H3K9me3 peptides were labelled by a fluorescent dye molecule, fluorescein isothiocyanate (FITC). Error bars represent ± standard error of the mean (n = 3 biologically independent samples). The live cell imaging of CDY1-chromo domain variants (F) and CBX1-chromo domains (G) in HEK 293T cells (n = 3 biologically independent experiments). Scale bar: 10 μm.

Source data

Extended Data Fig. 7 Quantification on the distribution of reader proteins and colocalization imaging with inhibitors.

A, C the procedure for quantifying the distribution of CDYL1 reader protein of H3K9me3 (A) and BAH reader protein of H3K27me3 (C). B, D the quantification results for CDYL1 (B) and BAH (D) at indicated conditions. Error bars represent ± standard error of the mean (n = 16, 13 and 13 cells for CDYL1-WT, CDYL1-vyW and CDYL1-vyW decaging; n = 11, 11 and 9 cells for BAH-WT, BAH-vyW and BAH-vyW decaging). E, the representative image of BAH variants in presence of H3K27me3 inhibitors of EED226 (20 μM) and EPZ6438 (5 μM), the reader proteins distribution was changed by inhibitors incubation. F, the quantification of distribution pattern in presence or absence of H3K27me3 inhibitors. One-Way ANOVA test was used. Error bars represent ± standard error of the mean (n = 11, 11 and 9 cells for BAH-WT, BAH-vyW and BAH-vyW decaging in absence of inhibitor; n = 13, 11 and 8 cells for BAH-WT, BAH-vyW and BAH-vyW decaging in presence of inhibitors respectively). Scale bar: 10 μm.

Source data

Extended Data Fig. 8 The logic flowchart of Trp-CAGE for protein activation in living systems.

To predict if one POI could be blocked and rescued by Trp-CAGE, an optimized protein–ligand complex structure was obtained from the PDB, and comparative modelling and/or ligand-docking were used when necessary. Trp–ligand distance (d) was calculated by Rosetta and used as filters to differentiate Trp interaction types. Hits with d > 5 A is sent to allosteric verification. DipyTz (Tz 4) is recommended to be firstly tested in the decaging step, further screening and treatment with less-hindered asymmetric Tzs (Tz 10, 12, 13, 14) may improve decaging efficiency.

Supplementary information

Supplementary Information

Supplementary materials and methods, Tables 1–3, Note 1 and Figs. 1–10.

Reporting Summary

Supplementary Data 1

CIF report for for the crystallographic data of vyW.

Supplementary Data 2

MS–MS analysis of non-linked, decaged vyW > W mutant peptides.

Supplementary Data 3

MS–MS analysis of pull-downed proteins/

Supplementary Data 4

The cross-linked peptides analysed by pLink.

Supplementary Data 5

The predicted proteins containing conserved Trp residues.

Supplementary Data 6

The primers and plasmids.

Supplementary Data 7

Crystallographic data for vyW.

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Zhu, Y., Ding, W., Chen, Y. et al. Genetically encoded bioorthogonal tryptophan decaging in living cells. Nat. Chem. 16, 533–542 (2024). https://doi.org/10.1038/s41557-024-01463-7

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