Quantitative Single-Residue Bioorthogonal Labeling of G Protein-Coupled Receptors in Live Cells

ACS Chem Biol. 2019 Jun 21;14(6):1141-1149. doi: 10.1021/acschembio.8b01115. Epub 2019 May 23.


High-end microscopy studies of G protein-coupled receptors (GPCRs) require installing onto the receptors bright and photostable dyes. Labeling must occur in quantitative yields, to allow stoichiometric data analysis, and in a minimally invasive fashion, to avoid perturbing GPCR function. We demonstrate here that the genetic incorporation of trans-cyclooct-2-ene lysine (TCO*) allows achieving quantitative single-residue labeling of the extracellular loops of the β2-adrenergic and the muscarinic M2 class A GPCRs, as well as of the corticotropin releasing factor class B GPCR. Labeling occurs within a few minutes by reaction with dye-tetrazine conjugates on the surface of live cells and preserves the functionality of the receptors. To precisely quantify the labeling yields, we devise a method based on fluorescence fluctuation microscopy that extracts the number of labeling sites at the single-cell level. Further, we show that single-residue labeling is better suited for studies of GPCR diffusion than fluorescent-protein tags, since the latter can affect the mobility of the receptor. Finally, by performing dual-color competitive labeling on a single TCO* site, we devise a method to estimate the oligomerization state of a GPCR without the need for a biological monomeric reference, which facilitates the application of fluorescence methods to oligomerization studies. As TCO* and the dye-tetrazines used in this study are commercially available and the described microscopy techniques can be performed on a commercial microscope, we expect our approach to be widely applicable to fluorescence microscopy studies of membrane proteins in general.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescent Dyes / chemistry
  • HEK293 Cells
  • Humans
  • Lysine / analogs & derivatives
  • Microscopy, Fluorescence
  • Receptors, G-Protein-Coupled / metabolism*


  • Fluorescent Dyes
  • Receptors, G-Protein-Coupled
  • Lysine