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 journalJournal articleResearchpeer-review

Harvard

Tian, H, Naganathan, S, Kazmi, MA, Schwartz, TW, Sakmar, TP & Huber, T 2014, 'Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors', ChemBioChem, vol. 15, no. 12, pp. 1820-9. https://doi.org/10.1002/cbic.201402193

APA

Tian, H., Naganathan, S., Kazmi, M. A., Schwartz, T. W., Sakmar, T. P., & Huber, T. (2014). Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors. ChemBioChem, 15(12), 1820-9. https://doi.org/10.1002/cbic.201402193

Vancouver

Tian H, Naganathan S, Kazmi MA, Schwartz TW, Sakmar TP, Huber T. Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors. ChemBioChem. 2014 Aug 18;15(12):1820-9. https://doi.org/10.1002/cbic.201402193

Author

Tian, He ; Naganathan, Saranga ; Kazmi, Manija A ; Schwartz, Thue W. ; Sakmar, Thomas P ; Huber, Thomas. / Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors. In: ChemBioChem. 2014 ; Vol. 15, No. 12. pp. 1820-9.

Bibtex

@article{fe3ca9d674e94cb8906114ed7c0dc535,
title = "Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors",
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 = "Fluorescence, Fluorescent Dyes, Genetic Code, Kinetics, Models, Molecular, Receptors, G-Protein-Coupled, Spectrometry, Fluorescence, Staining and Labeling",
author = "He Tian and Saranga Naganathan and Kazmi, {Manija A} and Schwartz, {Thue W.} and Sakmar, {Thomas P} and Thomas Huber",
note = "{\circledC} 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.",
year = "2014",
month = "8",
day = "18",
doi = "10.1002/cbic.201402193",
language = "English",
volume = "15",
pages = "1820--9",
journal = "ChemBioChem",
issn = "1439-4227",
publisher = "Wiley - V C H Verlag GmbH & Co. KGaA",
number = "12",

}

RIS

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

VL - 15

SP - 1820

EP - 1829

JO - ChemBioChem

JF - ChemBioChem

SN - 1439-4227

IS - 12

ER -

ID: 137293988