Quantitative single-residue bioorthogonal labeling of G protein-coupled receptors in live cells

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Item Type:	Article
Title:	Quantitative single-residue bioorthogonal labeling of G protein-coupled receptors in live cells
Creators Name:	Serfling, R., Seidel, L., Bock, A., Lohse, M.J., Annibale, P. and Coin, I.
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 β(2)-adrenergic and the muscarinic M(2) 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.
Keywords:	Fluorescence Microscopy, Fluorescent Dyes, G-Protein-Coupled Receptors, HEK293 Cells, Lysine
Source:	ACS Chemical Biology
ISSN:	1554-8929
Publisher:	American Chemical Society
Volume:	14
Number:	6
Page Range:	1141-1149
Date:	21 June 2019
Official Publication:	https://doi.org/10.1021/acschembio.8b01115
PubMed:	View item in PubMed

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