Multi-region mapping of ligand binding and structural changes in the β-1 adrenergic receptor using carbene footprinting and mass spectrometry.

G protein-coupled receptors (GPCRs) control many physiological processes and are major targets for therapeutic intervention. Transmembrane proteins, such as GPCRs, are inherently flexible and dynamic and often challenging to study using conventional structural methods. Here, we report the use of carbene footprinting to investigate ligand binding and structural changes in the turkey β1-adrenergic receptor (tβ1AR), a GPCR and therapeutic target of beta-blocker drugs. The method revealed differences between binding of the agonist, isoprenaline, and the inverse agonist, carazalol, both in terms of their occupancy of the orthosteric ligand binding site and their effects on key regulatory structural features of tβ1AR including the 'ionic lock' between transmembrane (TM) helicies 3 and 6. Addition of nanobodies (Nbs) known to stabilise the activated complex (Nb80) and inactivated complex (Nb60) of tβ1AR induced further structural changes above those seen with the ligands alone.