Exploring Conformational Changes and Folding for a Model GPCR 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.  

In this work, we will describe several biophysical approaches to characterize the human adenosine A₂a receptor, a model G-protein coupled receptor which binds to adenosine analogs.  Binding of an adenosine agonist to purified A₂aR led to a slight, yet distinct change in the intrinsic tryptophan emission spectra, while no change was observed in protein secondary structure.  Overall, we find that unfolding was achieved through both thermal and chemical means, although both techniques led to irreversible aggregation of micelle-reconstituted A₂aR.  Monitoring tertiary structure changes, caused by thermal denaturation, via intrinsic fluorescence spectroscopy yielded unclear folding transitions, likely due to decreases in intrinsic fluorescence incurred with temperature increase.  Addition of a thiol-reactive fluorescent dye, whose quantum yield increases as A₂aR unfolds, largely eliminated this effect and made thermal unfolding transitions clear.  In an effort to reduce the effects of aggregation, we have also employed hydrostatic pressure to unfold the tertiary structure of A₂aR, yet complete unfolding of the sample was not observed using this technique alone.  Although reversible unfolding of the protein proved elusive, we compared unfolding behavior under a variety of conditions.  In summary, we find that the stability of A₂aR is not appreciably affected by ligand addition, although stability is decreased when disulfide bonds located in the extracellular loop regions are reduced with a chemical reducing agent.  Further, we have confirmed that loss of these disulfide bonds also leads to a decrease in receptor ligand-binding activity.