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Site-specific incorporation of phosphotyrosine using an expanded genetic code

Nature Chemical Biology volume 13, pages 842844 (2017) | Download Citation

Abstract

Access to phosphoproteins with stoichiometric and site-specific phosphorylation status is key to understanding the role of protein phosphorylation. Here we report an efficient method to generate pure, active phosphotyrosine-containing proteins by genetically encoding a stable phosphotyrosine analog that is convertible to native phosphotyrosine. We demonstrate its general compatibility with proteins of various sizes, phosphotyrosine sites and functions, and reveal a possible role of tyrosine phosphorylation in negative regulation of ubiquitination.

  • Compound

    (S)-2-amino-3-(4-((bis(dimethylamino)phosphoryl)oxy)phenyl)propanoic acid

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Acknowledgements

We thank M. Kelly (UCSF NMR Core) for help with NMR measurements and helpful discussions. L.W. acknowledges the support of the NIH (R01GM118384).

Author information

Affiliations

  1. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA.

    • Christian Hoppmann
    • , Bing Yang
    •  & Lei Wang
  2. Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA.

    • Allison Wong
    •  & Kevan M Shokat
  3. Institute for Bioscience and Biotechnology Research, University of Maryland College Park, Rockville, Maryland, USA.

    • Shuwei Li
  4. The Salk Institute for Biological Studies, Molecular and Cell Biology Laboratory, La Jolla, California, USA.

    • Tony Hunter

Authors

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Contributions

C.H. conducted experiments and characterized data; A.W. assigned data for NMR; B.Y. prepared UBE2D3; S.L., T.H., and K.M.S. provided helpful discussions; L.W. conceived and directed the project; and C.H. and L.W. wrote the manuscript with inputs from S.L., T.H., and K.M.S.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Lei Wang.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Table 1, Supplementary Figures 1–12 and Supplementary Note 2

  2. 2.

    Supplementary Note 1

    Synthesis and characterization of Uaa 1 including experimental procedure, NMR and MS spectra.

About this article

Publication history

Received

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DOI

https://doi.org/10.1038/nchembio.2406

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