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Adding an unnatural covalent bond to proteins through proximity-enhanced bioreactivity

Nature Methods volume 10, pages 885–888 (2013)Cite this article

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An Erratum to this article was published on 30 January 2014

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Abstract

Natural proteins often rely on the disulfide bond to covalently link side chains. Here we genetically introduce a new type of covalent bond into proteins by enabling an unnatural amino acid to react with a proximal cysteine. We demonstrate the utility of this bond for enabling irreversible binding between an affibody and its protein substrate, capturing peptide-protein interactions in mammalian cells, and improving the photon output of fluorescent proteins.

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Figure 1: Covalent bond formation through proximity-enhanced bioreactivity.
Figure 2: Covalent bond formation at the interface of the affibody and the Z protein resulted in irreversible binding.
Figure 3: Covalent bond formation between Ucn-1 and CRF-R1 site-specifically captured ligand-receptor interaction in mammalian cells.

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  • 21 November 2013

    In the version of this article initially published, in Figure 2e, lanes 6 and 8 should have been labeled "Fact," not "Ffact." In the legend for this figure, the sentence "N3 indicates cysteine..." should have read "C3 indicates cysteine...." The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank J. Xu for help with the NMR measurements, M. Beyermann (Leibniz Institute of Molecular Pharmacology, Germany) for synthesizing the Cys-Ucn-1 analogs, and the Vale laboratory (Salk Institute) for the polyclonal rabbit anti-urocortin. H.R. was partially funded by the Nomis Postdoctoral Fellowship. I.C. was supported by a Marie Curie fellowship from the European Commission within the 7th framework program. L.W. acknowledges support from the California Institute for Regenerative Medicine (RN1-00577-1) and US National Institutes of Health (1DP2OD004744-01, P30CA014195).

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Author notes

  1. Zheng Xiang and Haiyan Ren: These authors contributed equally to this work.

Authors and Affiliations

  1. The Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, California, USA

    Zheng Xiang, Haiyan Ren, Irene Coin & Lei Wang

  2. Waitt Advanced Biophotonics Center, The Salk Institute for Biological Studies, La Jolla, California, USA

    Ying S Hu & Hu Cang

  3. Jadebio, Inc., La Jolla, California, USA

    Jing Wei

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  1. Zheng Xiang
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Contributions

Z.X. designed and synthesized the Uaa, tested the reaction, analyzed the data and wrote the manuscript; H.R. performed affibody-Z expression and complex formation, expressed and purified fluorescent proteins, measured quantum yields, analyzed the data and wrote the method section; Y.S.H. and H.C. performed single-molecule imaging, analyzed the data and wrote the single-molecule section. I.C. performed the CRF-R1 experiments and analyzed the data. J.W. characterized Uaa incorporation by MS, analyzed the data and wrote the MS section; L.W. conceived and directed the project, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Lei Wang.

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Competing interests

J.W. is an employee of Jadebio, Inc., which is a contract research organization providing mass spectrometry–based protein analysis.

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Supplementary Figures 1–4, Supplementary Table 1, Supplementary Note, Supplementary Results and Supplementary Methods (PDF 3679 kb)

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Xiang, Z., Ren, H., Hu, Y. et al. Adding an unnatural covalent bond to proteins through proximity-enhanced bioreactivity. Nat Methods 10, 885–888 (2013). https://doi.org/10.1038/nmeth.2595

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