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. GPCRs click to glow: Rhodopsin, a prototypical G-protein-coupled receptor (GPCR), was tagged with an azido group using non-canonical amino-acid mutagenesis and site-specifically labeled with a fluorophore using a bioorthogonal "click" reaction. The ligand-binding ability of the fluorescently labeled rhodopsin was confirmed by using a novel fluorescence-quenching assay.
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | ChemBioChem |
| Vol/bind | 15 |
| Udgave nummer | 12 |
| Sider (fra-til) | 1820-9 |
| Antal sider | 10 |
| ISSN | 1439-4227 |
| DOI | |
| Status | Udgivet - 18 aug. 2014 |