Structural equilibrium underlying ligand-dependent activation of β-adrenoreceptor.

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins mediating cellular signals in response to extracellular stimuli. Although three-dimensional structures showcase snapshots that can be sampled in the process and nuclear magnetic resonance detects conformational equilibria, the mechanism by which agonist-activated GPCRs interact with various effectors remains elusive. Here, we used paramagnetic nuclear magnetic resonance for leucine amide resonances to visualize the structure of β-adrenoreceptor in the full agonist-bound state, without thermostabilizing mutations abolishing its activity.

Deuteration and selective labeling of alanine methyl groups of β-adrenergic receptor expressed in a baculovirus-insect cell expression system.

G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl-CH-labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES.