Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors
Research output: Contribution to journal › Journal article › Research › peer-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.
|Number of pages||10|
|Publication status||Published - 18 Aug 2014|
- Fluorescence, Fluorescent Dyes, Genetic Code, Kinetics, Models, Molecular, Receptors, G-Protein-Coupled, Spectrometry, Fluorescence, Staining and Labeling