Surfactant Effects on the Activity and Structure of the Human Adenosine A2a G-Protein Coupled Receptor
Michelle O'Malley University of Delaware
The G-protein coupled receptors (GPCRs) constitute the largest family of mammalian membrane proteins, and are targets for nearly half of all pharmaceuticals on the market. Despite their importance, structural and conformational studies of these proteins have been hindered by their relatively low abundance in native tissues, their low levels of expression in recombinant systems, and difficulties associated with their isolation and stabilization in membrane-mimetic environments. An understanding of receptor behavior in a reconstituted micellar environment is necessary prior to biophysical characterization of these important proteins. We have developed a heterologous yeast system that enables high-level expression and purification of the human adenosine A2a receptor (A2aR), a model GPCR. In this work, we assess the ability of protein detergent complexes (PDCs) to stabilize the activity and structure of A2aR, as measured through ligand binding, neutron scattering, and various biophysical techniques. Protein stability is studied in a wide range of surfactants including maltosides, thiomaltosides, and glucosides. We find that the addition of a mammalian cholesterol analog (CHS) to the PDC is crucial to the maintenance of proper structure of A2aR. This interaction may hold the 7-alpha helical domains of the GPCR more rigid in an active conformation within the micelle. Small angle neutron scattering (SANS) measurements on empty micelles also indicate a morphological transition upon the addition of cholesterol. Furthermore, surfactants which share common structure and differ only by one carbon length show marked differences in the ability to stabilize A2aR. Thus, we conclude that protein stability may be correlated with both micelle morphology and chemical environment. We will further explore the effects of surfactant type, chain length, and other parameters on the activity of purified A2aR and on the protein detergent complex.
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