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

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 (C_iβG₂) and β-D-thiomaltosides (C_iSβG₂), 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 C_iβG₂/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 C_iβG₂ and C_iSβG₂ 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.