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Chembiochem. 2014 Aug 18;15(12):1820-9. doi: 10.1002/cbic.201402193. Epub 2014 Jul 18.

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

Author information

1
Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (USA).

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.

KEYWORDS:

bioorthogonal labeling; fluorescence; non-canonical amino acids; receptors; rhodopsin

PMID:
25045132
PMCID:
PMC4215543
DOI:
10.1002/cbic.201402193
[Indexed for MEDLINE]
Free PMC Article

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