title: Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors.
abstract: Novel methods are required for site-specific, quantitative fluorescent labeling of G-protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb the 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 owing to post-translational 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 application of this technique, we used Alexa488-rhodopsin to measure the kinetics of ligand uptake and release in membrane-mimetic bicelles using a novel fluorescence-quenching assay.
authors.affiliation: Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (USA).
authors.forename: He
authors.initials: H
authors.lastname: Tian
authors.forename: Saranga
authors.initials: S
authors.lastname: Naganathan
authors.forename: Manija A
authors.initials: MA
authors.lastname: Kazmi
authors.forename: Thue W
authors.initials: TW
authors.lastname: Schwartz
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
mesh_terms: D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D005815:Genetic Code; D007700:Kinetics; D008958:Models, Molecular; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D043562:Receptors, G-Protein-Coupled
keywords: bioorthogonal labeling; fluorescence; non-canonical amino acids; receptors; rhodopsin