You are here

Unnatural amino acid mutagenesis of GPCRs using amber codon suppression and bioorthogonal labeling.

TitleUnnatural amino acid mutagenesis of GPCRs using amber codon suppression and bioorthogonal labeling.
Publication TypeJournal Article
Year of Publication2013
AuthorsHuber, T, Naganathan, S, Tian, H, Ye, S, Sakmar, TP
JournalMethods Enzymol
Volume520
Pagination281-305
Date Published2013
ISSN1557-7988
KeywordsAmino Acids, Animals, Codon, Terminator, Humans, Mutagenesis, Receptors, G-Protein-Coupled
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.

DOI10.1016/B978-0-12-391861-1.00013-7
Alternate JournalMeth. Enzymol.
PubMed ID23332705
Grant ListR01 EY012049 / EY / NEI NIH HHS / United States