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.

SUBMITTER: Tian H 

PROVIDER: S-EPMC4215543 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature