Site-Specific Fluorescent Labeling of Purified G-Protein-Coupled Receptors Using Genetically-Encoded Unnatural Amino Acids

Abstract

The introduction of unique chemical groups into proteins by means of site-directed mutagenesis with unnatural amino acids has numerous applications in protein engineering and functional studies. We first introduced p-acetyl-L-phenylalanine (Acp) or p-azido-L-phenylalanine (Azp) into the prototypical G protein-coupled receptor (GPCR) rhodopsin at specific sites. We employed an amber codon suppression system where the mutant opsin gene was co-expressed with the appropriate orthogonal pair of engineered tRNA and amino-acyl tRNA synthetase. We then used hydrazone (hydrazide) or Staudinger-Bertozzi (phosphine) ligation chemistry for the keto group (in Acp) or azido group (in Azp), respectively, to link a fluorophore at various solvent accessible sites in rhodopsin. In side-by-side comparisons of the two chemical ligation chemistries, which were carried out under physiological conditions where receptor function is maintained, we conclude that the azido group serves as a more satisfactory chemical handle than the keto moiety. The fully-functional fluorescently-labeled receptor should prove useful for kinetic studies of ligand-receptor interaction.View Large Image | View Hi-Res Image | Download PowerPoint Slide