(545a) Characterization of Maltoside-Based Mixed Micelles for the Reconstitution of a Model G-Protein Coupled Receptor

Authors: 
O'Malley, M. A. - Presenter, Massachusetts Institute of Technology
Helgeson, M. E. - Presenter, Massachusetts Institute of Technology
Wagner, N. J. - Presenter, University of Delaware
Robinson, A. S. - Presenter, University of Delaware

G-protein coupled receptors (GPCRs) represent the largest
group of integral membrane proteins, and are attractive drug targets.  However,
GPCRs prove difficult to study due to problems with their purification and
stability outside of the membrane environment.  In vitro isolation of
GPCRs generally requires solubilization in surfactant micelles to form
so-called protein-detergent complexes (PDCs).  PDCs stabilize protein conformation
in vitro, but may also impact folding or activity of the solubilized
protein.  Currently, selection of appropriate surfactants and micellar
compositions for solubilization of GPCRs is done in an ad hoc fashion. 
The role that specific surfactants play during crystallization of PDCs is also
poorly understood.  In this work, we seek to understand the critical parameters
governing stability and structure of PDCs for more rational selection of
surfactants to enable physical characterization and structure determination for
this important class of proteins.

Mixed micelles comprised of n-alkyl β-D-maltosides (CiβG2)
and β-D-thiomaltosides (CiSβG2),
3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS), and cholesterol
hemisuccinate (CHS) provide a model micellar system in which to study the in
vitro
stability of membrane proteins, where the overall morphology and
hydrophobic environment of the micelles can be tuned by adjusting the ratio of
surfactants in solution as well as the surfactant tail length.  Small angle
neutron scattering measurements are used to characterize the structure and
composition of CiβG2/CHAPS/CHS micelles at different
surfactant ratios, and show that the micelles adopt an oblate ellipsoid
morphology.  Furthermore, the addition of CHS results in an overall reduction
in micellar curvature and provides a more favorable membrane-mimetic environment.

The effects of micellar structure and composition on in
vitro
protein stability are studied on a model GPCR, the human adenosine A2a
(hA2aR) receptor, for which high-level expression and purification have
been accomplished from a heterologous yeast system.  The ability of different micellar
compositions to stabilize the active conformation of hA2aR was
measured through radio-ligand binding and biophysical methods, providing a
sensitive probe of protein structure.  The incorporation of CHS in the mixed
micelles is found to be crucial to the maintenance of proper structure of hA2aR. 
Furthermore, activity depends significantly on the alkyl chain length of the CiβG2
and CiSβG2 surfactants and surfactant/lipid ratio.  These
effects are due to both structural changes of the micelle as well as specific
protein-surfactant interactions.  These results are expected to apply to other
related GPCRs, and may be more generally applicable to the study of membrane
proteins.