title: Unnatural amino acid mutagenesis of GPCRs using amber codon suppression and bioorthogonal labeling.
abstract: To advance dynamic, temporal, and kinetic studies of the G protein-coupled receptor (GPCR) signalosome, new approaches are required to introduce non- or minimally perturbing labels or probes into expressed receptors. We report here a series of methods that are based on unnatural amino acid mutagenesis of GPCRs using amber codon suppression technology. We show that labeling reactions at genetically introduced keto moieties (p-acetyl-L-Phe/AcF and p-benzoyl-L-Phe/BzF) are not completely bioorthogonal due to protein oxidation, which causes high background. However, labeling reactions that target p-azido-L-Phe (azF) using the Staudinger-Bertozzi ligation and the strain-promoted alkyne-azide cycloaddition are bioorthogonal and are satisfactory for introducing labels or probes at near quantitative efficiency under mild labeling conditions. To our knowledge, this is the first report of a site-specific modification of an azF residue with a dibenzocyclooctyne-derivatized fluorophore. The methodologies we discuss are general, in that they can be applied in principle to any amino acid position in any expressed GPCR.
authors: Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, USA. hubert@rockefeller.edu
authors: Thomas
authors: T
authors: Huber
authors: Saranga
authors: S
authors: Naganathan
authors: He
authors: H
authors: Tian
authors: Shixin
authors: S
authors: Ye
authors: Thomas P
authors: TP
authors: Sakmar
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D018388:Codon, Terminator / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D016296:Mutagenesis; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*
chemical_list: D000596:Amino Acids; D018388:Codon, Terminator; D043562:Receptors, G-Protein-Coupled