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Site-specific incorporation of phosphotyrosine using an expanded genetic code

Nature Chemical Biology volume 13, pages 842–844 (2017)Cite this article

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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.

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Figure 1: Site-specific incorporation of phosphotyrosine into proteins.
Figure 2: Phosphorylation of Tyr59 in Ub impacts its conformation and function.

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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).

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Authors and 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
  1. Christian Hoppmann
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  2. Allison Wong
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  3. Bing Yang
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  4. Shuwei Li
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  5. Tony Hunter
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  6. Kevan M Shokat
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  7. Lei Wang
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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.

Corresponding author

Correspondence to Lei Wang.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Table 1, Supplementary Figures 1–12 and Supplementary Note 2 (PDF 2942 kb)

Supplementary Note 1

Synthesis and characterization of Uaa 1 including experimental procedure, NMR and MS spectra. (PDF 267 kb)

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Hoppmann, C., Wong, A., Yang, B. et al. Site-specific incorporation of phosphotyrosine using an expanded genetic code. Nat Chem Biol 13, 842–844 (2017). https://doi.org/10.1038/nchembio.2406

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