Exploring Conformational Changes and Folding for a Model G-Protein Coupled Receptor Using Biophysical Techniques

Membrane proteins are vital molecules for signal transduction and maintenance of cellular homeostasis.  Despite their importance, relatively little information is known pertaining to their structure and folding compared to soluble proteins.  Difficulties associated with characterization of membrane proteins generally stem from an inability to purify them at high yields, while shielding their hydrophobic domains with a suitable membrane-mimetic system.  Even when sample preparation issues are resolved, techniques traditionally employed to study protein folding (ie. thermal or chemical denaturation) may translate poorly to the characterization of membrane proteins and oftentimes lead to sample aggregation.  

G-protein coupled receptors (GPCRs) are membrane proteins that constitute one major signal transduction system in all eukaryotic cells, yet relatively little information is known pertaining to their structure, folding, and stability. In this work, we describe several approaches to characterize conformational stability of the human adenosine A₂a receptor (hA₂aR). Thermal and chemical denaturation were not completely reversible, yet differences in the unfolding behavior were observed upon ligand binding via circular dichroism, and using the extrinsic fluorophore, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). We find that the stability of hA₂aR is sensitive to relative agonist (CHA) or antagonist (theophylline) concentrations. When extracellular disulfide bonds were reduced with a chemical reducing agent, the activity decreased by ~40%, but reduction of these bonds did not compromise the unfolding transition observed via urea denaturation. Overall, these approaches offer a general strategy for characterizing the effect of solvent and ligand effects on the stability of hA₂aR.