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Bioorthogonal Fluorescent Labeling of Functional G‐Protein‐Coupled Receptors
Author(s) -
Tian He,
Naganathan Saranga,
Kazmi Manija A.,
Schwartz Thue W.,
Sakmar Thomas P.,
Huber Thomas
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201402193
Subject(s) - bioorthogonal chemistry , rhodopsin , chemistry , biochemistry , fluorescence , affinity labeling , photoaffinity labeling , g protein coupled receptor , receptor , protein tag , biophysics , combinatorial chemistry , click chemistry , biology , fusion protein , gene , recombinant dna , retinal , physics , quantum mechanics
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.