(485aj) 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.