title: Ligand-assisted dual-site click labeling of EGFR on living cells.
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.
authors.affiliation: Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, 202, Chengfu Road, Beijing 100871 (China).
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.forename: Shixian
authors.initials: S
authors.lastname: Lin
authors.forename: Wei
authors.initials: W
authors.lastname: Lin
authors.forename: Peng R
authors.initials: PR
authors.lastname: Chen
mesh_terms: D002470:Cell Survival; D057930:Click Chemistry; D003300:Copper / Q000737:chemistry; D003500:Cyclization; D066246:ErbB Receptors / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D005815:Genetic Code; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D009942:Organometallic Compounds / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D008024:Ligands; D009942:Organometallic Compounds; D003300:Copper; D066246:ErbB Receptors
keywords: EGFR; FRET; click chemistry; dual-site labeling; genetic code expansion