Bioorthogonal Fluorescent Labeling of Functional G‐Protein‐Coupled Receptors

Abstract

Novel methods are required for site-specific, quantitative fluorescence labeling of G protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb native structure and function as little as possible. We evaluated bioorthogonal reactions to label genetically encoded p-acetyl-l-phenylalanine (AcF) or p-azido-l-phenylalanine (azF) residues in receptors heterologously expressed in mammalian cells. We found that keto-selective reagents were not truly bioorthogonal, possibly due to posttranslational protein oxidation reactions. In contrast, the strain-promoted [3+2] azide–alkyne cycloaddition (SpAAC) with dibenzocyclooctyne (DIBO) reagents yielded stoichiometric conjugates with azF-rhodopsin while undergoing negligible background reactions. As one useful application, we used Alexa488-rhodopsin to measure the kinetics of ligand uptake and release in membrane-mimetic bicelles using a novel fluorescence-quenching assay.

Measurement of Slow Spontaneous Release of 11-cis-Retinal from Rhodopsin

“…Various bicelle systems have been shown to retain the structural and functional integrity of membrane proteins (18). Previously, we have shown that the binding product between 11CR and photobleached Alexa488-labeled Rho in the bicelle system could be repeatedly photoactivated, demonstrating the regeneration of mature pigment (19). In our study, we used Trp fluorescence to show that the photobleached Rho in bicelles bind with 11CR or 9CR with comparable kinetic rate constants.…”

Ghrelin receptor conformational dynamics regulate the transition from a preassembled to an active receptor:Gq complex

“…S3). The azidoF-containing receptor was assembled as a monomer into lipid discs (10) and then labeled with the fluorescence acceptor (Alexa Fluor 488) using the strain-promoted alkyne-azide cycloaddition reaction (21,22). Approximately 90% of labeling was achieved under these conditions, whereas no labeling was observed with the unmodified receptor (SI Appendix, Fig.…”

Conformational Selection and Equilibrium Governs the Ability of Retinals to Bind Opsin

“…Temperatures were controlled with a VWR water bath and constantly monitored using an Omega Thermister to within 0.5°C of the desired temperature. Retinal binding was observed by quenching of intrinsic tryptophan fluorescence measured as a decrease in emission at 330 nm, essentially the inverse of the retinal release assay (21,22,26,29,30). To minimize rhodopsin bleaching, excitation was tempered by a neutral density (optical density 1.7) filter and emission bandwidth was expanded to 20 nm.…”

“…Essentially, this process entails running a retinal release experiment in reverse (26) where instead of observing the relief of quenching of intrinsic tryptophan fluorescence by retinal exit one now monitors the decrease of tryptophan fluorescence emission caused by the tryptophan residues undergoing FRET to the newly bound retinal (21,22,(27)(28)(29)(30)(31). In contrast to traditional absorbance-based assays, this approach allows for the detection of any chromophore in the binding pocket and not just those retinals that have formed a protonated Schiff base, thus enabling the study of both 11CR and ATR binding (the latter of which is "spectroscopically silent" in traditional absorbance binding assays).…”

“…Our approach tracks the quenching of intrinsic tryptophan fluorescence as the ligand binds in the pocket. A similar general approach has been used in several other works (21,22,(27)(28)(29)(30)(31). Here, we established and calibrated our approach to ensure accurate, reproducible measurements so that retinal binding rates measured by fluorescence could be directly compared between different conditions.…”