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Ligand-Assisted Dual-Site Click Labeling of EGFR on Living Cells

Authors

  • Yi Yang,

    1. Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, 202, Chengfu Road, Beijing 100871 (China)
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  • Shixian Lin,

    1. Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, 202, Chengfu Road, Beijing 100871 (China)
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  • Wei Lin,

    1. Peking-Tsinghua Center for Life Sciences, Peking University, 5, Yiheyuan Road, Beijing 100871 (China)
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  • Prof. Dr. Peng R. Chen

    Corresponding author
    1. Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, 202, Chengfu Road, Beijing 100871 (China)
    2. Peking-Tsinghua Center for Life Sciences, Peking University, 5, Yiheyuan Road, Beijing 100871 (China)
    • Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, 202, Chengfu Road, Beijing 100871 (China)===

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Abstract

We have developed a dual-site click labeling strategy for the simultaneous installation of a FRET donor–acceptor pair onto the extracellular domains of epidermal growth factor receptor (EGFR) on living cells. Our method integrates the genetic code expansion strategy, enzyme-mediated protein labeling, and ligand-assisted CuI-catalyzed azide–alkyne cycloaddition (CuAAC) into a tri-step labeling procedure. This enabled cis-membrane FRET imaging of EGFR under living conditions. This procedure might be generally applicable for dual-site labeling and cis-membrane FRET analysis of the domain–domain dynamics of important mammalian cell-surface receptors.

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