Journal
CHEMBIOCHEM
Volume 15, Issue 12, Pages 1820-1829Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201402193
Keywords
bioorthogonal labeling; fluorescence; non-canonical amino acids; receptors; rhodopsin
Funding
- Crowley Family Fund
- Danica Foundation
- NIH [R01 EY012049]
- Tri-Institutional Training Program in Chemical Biology
- Novo Nordisk Foundation
- Sakmar laboratory
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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 strainpromoted [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.
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