title: Micelle-Enhanced Bioorthogonal Labeling of Genetically Encoded Azido Groups on the Lipid-Embedded Surface of a GPCR.
abstract: Genetically encoded p-azido-phenylalanine (azF) residues in G protein-coupled receptors (GPCRs) can be targeted with dibenzocyclooctyne-modified (DIBO-modified) fluorescent probes by means of strain-promoted [3+2] azide-alkyne cycloaddition (SpAAC). Here we show that azF residues situated on the transmembrane surfaces of detergent-solubilized receptors exhibit up to 1000-fold rate enhancement relative to 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 micelleenhanced SpAAC is comparable with those of the fastest bioorthogonal labeling reactions known. Targeting hydrophobic regions of membrane proteins by use of micelle-enhanced SpAAC should expand the utility of bioorthogonal labeling strategies.
authors.affiliation: Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (USA).
authors.forename: He
authors.initials: H
authors.lastname: Tian
authors.affiliation: Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (USA). sakmar@rockefeller.edu.|Department of Neurobiology, Care Sciences and Society, Division for Neurogeriatrics, Center for Alzheimer Research, Karolinska Institutet, Alfred Nobels Allé 23, 141 57 Huddinge (Sweden). sakmar@rockefeller.edu.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
authors.affiliation: Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (USA). hubert@rockefeller.edu.
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D000818:Animals; D001386:Azides / Q000737:chemistry*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D061565:Cycloaddition Reaction; D003902:Detergents / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D015870:Gene Expression; D008055:Lipids / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000235:genetics; D011487:Protein Conformation; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics
chemical_list: D000480:Alkynes; D001386:Azides; D003902:Detergents; D005456:Fluorescent Dyes; D008055:Lipids; D008823:Micelles; D043562:Receptors, G-Protein-Coupled; D010649:Phenylalanine; D012243:Rhodopsin
keywords: bioorthogonal labeling; cycloaddition; micelles; rhodopsin; unnatural amino acids