(626d) 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.