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