Two-Step Ligand-Directed Covalent Fluorescent Labeling of the Adenosine A₁‑Receptor That Maintains Its Orthosteric Binding Site’s Availability to Bind Ligands

Genetic tagging of G protein-coupled receptors (GPCRs) with bioluminescent or fluorescent proteins is a well-established method for the study of ligand-binding and protein–protein interactions using resonance energy transfer approaches. Here we present a two-step, ligand-directed covalent labeling (LDCL) method that allows attachment of different fluorescent labels to an untagged adenosine A1 receptor using click chemistry. We also describe a range of biophysical approaches to confirm that the orthosteric binding site remains available to interact with endogenous ligands, agonists and antagonists, and access to the orthosteric binding site is not sterically hindered by the transferred cargo (fluorophore or click-reactive group).