Pressure resolved DEER to define the conformational landscape governing ligand-mediated β2-adrenergic receptor signal transduction.

Abstract

The β-adrenergic receptors (βARs) are a subfamily of G protein-coupled receptors (GPCRs) that are expressed by most cell types in humans; as such, signaling through βARs regulates a wide variety of physiological processes. Upon binding extracellular ligands, these receptors couple to one of several subtypes of G-protein which reside at the intracellular side of the plasma membrane to trigger signaling events. During receptor signaling, ligand binding is converted into an intracellular signal via conformational changes in the GPCR. The ligand shifts the conformational equilibrium of the receptor toward active or inactive states, dependent on whether it is an agonist, inverse agonist, or antagonist. The prevailing conformational selection model suggests that receptors exist as an ensemble of distinct conformations in dynamic equilibrium where each conformation translates functionally by possessing distinct affinities and catalytic activities for the different intracellular transducer proteins. Experimental techniques that characterize structural differences of functionally distinct receptor conformations and their equilibrium populations are paramount in furthering our understanding of ligand-mediated GPCR signal transduction. Pressure induces a reversible shift in conformational equilibria to populate excited states, allowing for the characterization of sparsely-populated states and the identification of their functional relevance. Pressure perturbation grants the ability to systematically populate states that may otherwise be invisible. With pressure-resolved double electron-electron resonance (DEER), we are able to capture the relative populations of each conformation under different hydrostatic pressures. I have demonstrated a pressure dependence for the β2-adrenergic receptor. These data indicate we can populate the active conformation of the β2AR without a ligand, but with sufficient pressure of which we have the autonomy to adjust, which allows for us to populate otherwise spectroscopically “invisible members” of the β2AR conformational ensemble.