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

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.

Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins

“…In GCE-based bioorthogonal labeling of proteins, more than one type of Fl-dye can be applied at the labeling step, resulting in competitive labeling of the protein with different Fl-dyes. 6,41 This can potentially be used for performing SPT and live-SMLM (that require Fl-dyes with different fluorescent properties) in the same cell. To this end, we expressed EGFR 128BCNK in COS7 cells and applied both Tet-Cy3 and Tet-AF647 at the labeling step (as described in materials and methods).…”

Dual Bioorthogonal Labeling of the Amyloid-β Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products

“…Fluorescence of the free dye is significantly quenched by the tetrazine moiety, and the quenching is relieved after undergoing cycloaddition with TCO*K. The fluorescence intensity of the uncharged and lipophilic dye is increased 15 times upon reacting with the dienophile of the ncAA [22]. As Serfling et al show, high intracellular labeling background can be a problem for TAMRA and SiR-tetrazines because of excessive TCO*K and tRNA-TCO*K inside the cells [23]. However, we detect background labeling only when we express the PylRS AF /tRNA pair.…”

Capturing Peptide–GPCR Interactions and Their Dynamics

“…Importantly, this approach does not require working in a Cys-free background. In the first examples of GPCR labeling on ncAA anchors, the same p -azido-Phe (Azi) that we have discussed as a photo-crosslinker was applied for either copper-catalyzed or catalyst-free (strain promoted) azide-alkyne cycloaddition to label rhodopsin in vitro (CuAAC and SPAAC, respectively) [ 199 , 200 ]. Other ncAAs bear biophysical probes suitable for studies of GPCR dynamic directly on the side chain, as for instance EPR probes [ 201 , 202 , 203 ].…”