Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors

TY - JOUR

T1 - Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors

AU - Tian, He

AU - Naganathan, Saranga

AU - Kazmi, Manija A

AU - Schwartz, Thue W.

AU - Sakmar, Thomas P

AU - Huber, Thomas

N1 - © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PY - 2014/8/18

Y1 - 2014/8/18

N2 - 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. GPCRs click to glow: Rhodopsin, a prototypical G-protein-coupled receptor (GPCR), was tagged with an azido group using non-canonical amino-acid mutagenesis and site-specifically labeled with a fluorophore using a bioorthogonal "click" reaction. The ligand-binding ability of the fluorescently labeled rhodopsin was confirmed by using a novel fluorescence-quenching assay.

AB - 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. GPCRs click to glow: Rhodopsin, a prototypical G-protein-coupled receptor (GPCR), was tagged with an azido group using non-canonical amino-acid mutagenesis and site-specifically labeled with a fluorophore using a bioorthogonal "click" reaction. The ligand-binding ability of the fluorescently labeled rhodopsin was confirmed by using a novel fluorescence-quenching assay.

KW - Fluorescence

KW - Fluorescent Dyes

KW - Genetic Code

KW - Kinetics

KW - Models, Molecular

KW - Receptors, G-Protein-Coupled

KW - Spectrometry, Fluorescence

KW - Staining and Labeling

U2 - 10.1002/cbic.201402193

DO - 10.1002/cbic.201402193

M3 - Journal article

C2 - 25045132

SN - 1439-4227

VL - 15

SP - 1820

EP - 1829

JO - ChemBioChem

JF - ChemBioChem

IS - 12

ER -