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 Cu(I)-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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