Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A protein functionalization platform based on selective reactions at methionine residues

Abstract

Nature has a remarkable ability to carry out site-selective post-translational modification of proteins, therefore enabling a marked increase in their functional diversity1. Inspired by this, chemical tools have been developed for the synthetic manipulation of protein structure and function, and have become essential to the continued advancement of chemical biology, molecular biology and medicine. However, the number of chemical transformations that are suitable for effective protein functionalization is limited, because the stringent demands inherent to biological systems preclude the applicability of many potential processes2. These chemical transformations often need to be selective at a single site on a protein, proceed with very fast reaction rates, operate under biologically ambient conditions and should provide homogeneous products with near-perfect conversion2,3,4,5,6,7. Although many bioconjugation methods exist at cysteine, lysine and tyrosine, a method targeting a less-explored amino acid would considerably expand the protein functionalization toolbox. Here we report the development of a multifaceted approach to protein functionalization based on chemoselective labelling at methionine residues. By exploiting the electrophilic reactivity of a bespoke hypervalent iodine reagent, the S-Me group in the side chain of methionine can be targeted. The bioconjugation reaction is fast, selective, operates at low-micromolar concentrations and is complementary to existing bioconjugation strategies. Moreover, it produces a protein conjugate that is itself a high-energy intermediate with reactive properties and can serve as a platform for the development of secondary, visible-light-mediated bioorthogonal protein functionalization processes. The merger of these approaches provides a versatile platform for the development of distinct transformations that deliver information-rich protein conjugates directly from the native biomacromolecules.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: The development of a methionine-selective protein functionalization strategy.
Fig. 2: Evolution of a methionine-selective bioconjugation strategy.
Fig. 3: Scope of the methionine-selective bioconjugation strategy.
Fig. 4: Exploiting the multi-faceted reactivity of the protein–sulfonium conjugate.

Data availability

The data that support the findings of this study are available within the paper and its Supplementary Information. Raw data are available from the corresponding author on reasonable request.

References

  1. 1.

    Walsh, C. T., Garneau-Tsodikova, S. & Gatto, G. J. Jr. Protein posttranslational modifications: the chemistry of proteome diversifications. Angew. Chem. Int. Ed. 44, 7342–7372 (2005).

    CAS  Article  Google Scholar 

  2. 2.

    Sletten, E. M. & Bertozzi, C. R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. 48, 6974–6998 (2009).

    CAS  Article  Google Scholar 

  3. 3.

    Spicer, C. D. & Davis, B. G. Selective chemical protein modification. Nat. Commun. 5, 4740 (2014).

    ADS  CAS  Article  Google Scholar 

  4. 4.

    Koniev, O. & Wagner, A. Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation. Chem. Soc. Rev. 44, 5495–5551 (2015).

    CAS  Article  Google Scholar 

  5. 5.

    Dawson, P. E. & Kent, S. B. H. Synthesis of native proteins by chemical ligation. Annu. Rev. Biochem. 69, 923–960 (2000).

    CAS  Article  Google Scholar 

  6. 6.

    Lang, K. & Chin, J. W. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. 114, 4764–4806 (2014).

    CAS  Article  Google Scholar 

  7. 7.

    Wang, L., Xie, J. & Schultz, P. G. Expanding the genetic code. Annu. Rev. Biophys. Biomol. Struct. 35, 225–249 (2006).

    Article  Google Scholar 

  8. 8.

    Vinogradova, E. V., Zhang, C., Spokoyny, A. M., Pentelute, B. L. & Buchwald, S. L. Organometallic palladium reagents for cysteine bioconjugation. Nature 526, 687–691 (2015).

    ADS  CAS  Article  Google Scholar 

  9. 9.

    Wright, T. H. et al. Posttranslational mutagenesis: a chemical strategy for exploring protein side-chain diversity. Science 354, aag1465 (2016).

    Article  Google Scholar 

  10. 10.

    Yang, A. et al. A chemical biology route to site-specific authentic protein modifications. Science 354, 623–626 (2016).

    ADS  CAS  Article  Google Scholar 

  11. 11.

    Abegg, D. et al. Proteome-wide profiling of targets of cysteine reactive small molecules by using ethynyl benziodoxolone reagents. Angew. Chem. Int. Ed. 54, 10852–10857 (2015).

    CAS  Article  Google Scholar 

  12. 12.

    Levine, R. L., Moskovitz, J. & Stadtman, E. R. Oxidation of methionine in proteins: roles in antioxidant defense and cellular regulation. IUBMB Life 50, 301–307 (2000).

    CAS  Article  Google Scholar 

  13. 13.

    Cowie, D. B., Cohen, G. N., Bolton, E. T. & De Robichon-Szulmajster, H. Amino acid analog incorporation into bacterial proteins. Biochim. Biophys. Acta 34, 39–46 (1959).

    CAS  Article  Google Scholar 

  14. 14.

    Lin, S. et al. Redox-based reagents for chemoselective methionine bioconjugation. Science 355, 597–602 (2017).

    ADS  CAS  Article  Google Scholar 

  15. 15.

    Gross, E. & Witkop, B. Nonenzymatic cleavage of peptide bonds: the methionine residues in bovine pancreatic ribonuclease. J. Biol. Chem. 237, 1856–1860 (1962).

    CAS  PubMed  Google Scholar 

  16. 16.

    Gundlach, H. G., Stein, W. H. & Moore, S. The nature of the amino acid residues involved in the inactivation of ribonuclease by iodoacetate. J. Biol. Chem. 234, 1754–1760 (1959).

    CAS  PubMed  Google Scholar 

  17. 17.

    Vithayathil, P. J. & Richards, F. M. Modification of the methionine residue in the peptide component of ribonuclease-S. J. Biol. Chem. 235, 2343–2351 (1960).

    CAS  Google Scholar 

  18. 18.

    Kramer, J. R. & Deming, T. J. Preparation of multifunctional and multireactive polypeptides via methionine alkylation. Biomacromolecules 13, 1719–1723 (2012).

    CAS  Article  Google Scholar 

  19. 19.

    Kramer, J. R. & Deming, T. J. Reversible chemoselective tagging and functionalization of methionine containing peptides. Chem. Commun. 49, 5144–5146 (2013).

    CAS  Article  Google Scholar 

  20. 20.

    Stang, P. J. & Zhdankin, V. V. Organic polyvalent iodine compounds. Chem. Rev. 96, 1123–1178 (1996).

    CAS  Article  Google Scholar 

  21. 21.

    Weiss, R., Seubert, J. & Hampel, F. α-Aryliodonio diazo compounds: SN reactions at the α-C atom as a novel reaction type for diazo compounds. Angew. Chem. Int. Edn Engl. 33, 1952–1953 (1994).

    Article  Google Scholar 

  22. 22.

    Schnaars, C., Hennum, M. & Bonge-Hansen, T. Nucleophilic halogenations of diazo compounds, a complementary principle for the synthesis of halodiazo compounds: experimental and theoretical studies. J. Org. Chem. 78, 7488–7497 (2013).

    CAS  Article  Google Scholar 

  23. 23.

    Kim, Y. et al. Efficient site-specific labeling of proteins via cysteines. Bioconjug. Chem. 19, 786–791 (2008).

    CAS  Article  Google Scholar 

  24. 24.

    Mülhberg, M. et al. Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds. Beilstein J. Org. Chem. 11, 784–791 (2015).

    Article  Google Scholar 

  25. 25.

    Staudinger, H. & Lüscher, G. Über darstellung und reaktionen von phosphazinen. Helv. Chim. Acta 5, 75–86 (1922).

    CAS  Article  Google Scholar 

  26. 26.

    Prier, C. K., Rankic, D. A. & MacMillan, D. W. C. Visible light photoredox catalysis with transition metal complexes: applications in organic synthesis. Chem. Rev. 113, 5322–5363 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Chen, Y., Kamlet, A. S., Steinman, J. B. & Liu, D. R. A biomolecule-compatible visible-light-induced azide reduction from a DNA-encoded reaction-discovery system. Nat. Chem. 3, 146–153 (2011).

    Article  Google Scholar 

  28. 28.

    Huang, W. & Cheng, X. Hantzsch esters as multifunctional reagents in visible-light photoredox catalysis. Synlett 28, 148–158 (2017).

    CAS  Google Scholar 

  29. 29.

    Fukuzumi, S., Hironaka, K. & Tanaka, T. Photoreduction of alkyl halides by an NADH model compound. An electron-transfer chain mechanism. J. Am. Chem. Soc. 105, 4722–4727 (1983).

    CAS  Article  Google Scholar 

  30. 30.

    Hedstrand, D. M., Kruizinga, W. H. & Kellog, R. M. Light induced and dye accelerated reductions of phenacyl onium salts by 1,4-dihydropyridines. Tetrahedron Lett. 19, 1255–1258 (1978).

    Article  Google Scholar 

  31. 31.

    Krause, G., Lundström, J., Barea, J. L., Pueyo de la Cuesta, C. & Holmgren, A. Mimicking the active site of protein disulfide-isomerase by substitution of proline 34 in Escherichia coli thioredoxin. J. Biol. Chem. 266, 9494–9500 (1991).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Nappi and C. Guerot for advice and useful discussions. We thank the Marie Curie Actions program (M.T.T. and M.G.S.), AstraZeneca and EPRSC (J.E.N.), and the European Research Council (ERC-SRG-259711), EPSRC (EP/100548X/1) and the Royal Society (Wolfson Merit Award) for fellowships (M.J.G.). We are grateful to J. Chin, N. Huguen, M. Skehel, H. Lewis and M. Edgeworth for assistance with protein purification and mass spectrometry experiments.

Reviewer information

Nature thanks A. Spokoyny and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

Authors

Contributions

M.J.G., M.T.T., J.E.N. and M.G.S. conceived the project and designed the experiments. M.J.G., M.T.T., J.E.N. and M.G.S. performed and analysed the experiments. M.J.G., M.T.T. and J.E.N. wrote the paper.

Corresponding author

Correspondence to Matthew J. Gaunt.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

This file contains Supplementary Text, which includes Supplementary Figs. S1–S83 and Supplementary Tables S1–S8.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Taylor, M.T., Nelson, J.E., Suero, M.G. et al. A protein functionalization platform based on selective reactions at methionine residues. Nature 562, 563–568 (2018). https://doi.org/10.1038/s41586-018-0608-y

Download citation

Keywords

  • Methionine Residues
  • Hypervalent Iodine Reagents
  • Bioconjugation Strategies
  • Sulfonate Conjugation
  • Exenatide

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing