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

Methods Enzymol. 2013;520:281-305. doi: 10.1016/B978-0-12-391861-1.00013-7.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / chemistry*
  • Amino Acids / genetics
  • Animals
  • Codon, Terminator / chemistry*
  • Codon, Terminator / genetics*
  • Humans
  • Mutagenesis
  • Receptors, G-Protein-Coupled / genetics*

Substances

  • Amino Acids
  • Codon, Terminator
  • Receptors, G-Protein-Coupled