Micelle-enhanced bioorthogonal labeling of genetically-encoded azido groups on the lipid-embedded surface of a GPCR

摘要

Genetically-encoded p-azido-phenylalanine (azF) residues in G protein-coupled receptors (GPCRs) can be targeted with dibenzocyclooctyne (DIBO) modified fluorescent probes using strain-promoted [3+2] azide–alkyne cycloaddition (SpAAC). Here we show that azF residues situated on the transmembrane surface of detergent-solubilized receptors exhibit up to 1000-fold rate enhancement compared with azF residues on water-exposed surfaces. We show that the amphipathic moment of the labeling reagent, consisting of hydrophobic DIBO coupled to hydrophilic Alexa dye, results in strong partitioning of the DIBO group into the hydrocarbon core of the detergent micelle and consequently high local reactant concentrations. The observed rate constant for the micelle-enhanced SpAAC is comparable with the fastest bioorthogonal labeling reactions known. Targeting hydrophobic regions of membrane proteins using the micelle-enhanced SpAAC reaction should expand the utility of bioorthogonal labeling strategies.