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

Robert Serfling, Lisa Seidel, Andreas Bock, Martin J. Lohse, Paolo Annibale*, Irene Coin

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1141-1149
Number of pages9
JournalACS Chemical Biology
Volume14
Issue number6
DOIs
Publication statusPublished - 21 Jun 2019

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