Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors

Research output: Contribution to journalJournal articleResearchpeer-review

He Tian, Saranga Naganathan, Manija A Kazmi, Thue W. Schwartz, Thomas P Sakmar, Thomas Huber

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.

Original languageEnglish
JournalChemBioChem
Volume15
Issue number12
Pages (from-to)1820-9
Number of pages10
ISSN1439-4227
DOIs
Publication statusPublished - 18 Aug 2014

    Research areas

  • Fluorescence, Fluorescent Dyes, Genetic Code, Kinetics, Models, Molecular, Receptors, G-Protein-Coupled, Spectrometry, Fluorescence, Staining and Labeling

ID: 137293988