Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors
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Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors. / Tian, He; Naganathan, Saranga; Kazmi, Manija A; Schwartz, Thue W.; Sakmar, Thomas P; Huber, Thomas.
In: ChemBioChem, Vol. 15, No. 12, 18.08.2014, p. 1820-9.Research output: Contribution to journal › Journal article › Research › peer-review
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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.
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.
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
VL - 15
SP - 1820
EP - 1829
JO - ChemBioChem
JF - ChemBioChem
SN - 1439-4227
IS - 12
ER -
ID: 137293988