title: Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids.
abstract: The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, or DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. The studies presented in this paper also underline the importance of the protein's purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19711, United States.
authors.forename: Andrea N
authors.initials: AN
authors.lastname: Naranjo
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19711, United States.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: McNeely
authors.affiliation: Biology and Soft Matter Division, Neutron Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6100, United States; Department of Physics and Astronomy, The University of Tennessee, Knoxville, TN 37996-1200, United States; Joint Institute for Neutron Sciences, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6453, United States.
authors.forename: John
authors.initials: J
authors.lastname: Katsaras
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19711, United States; Department of Chemical and Biomolecular Engineering, 300 Lindy Boggs Laboratory, Tulane University, New Orleans, LA 70118, United States. Electronic address: asr@tulane.edu.
authors.forename: Anne Skaja
authors.initials: AS
authors.lastname: Robinson
mesh_terms: D002788:Cholesterol Esters / Q000737:chemistry*; D002793:Cholic Acids / Q000737:chemistry*; D006801:Humans; D010742:Phospholipid Ethers / Q000737:chemistry*; D017433:Protein Structure, Secondary; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry*
chemical_list: C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D002788:Cholesterol Esters; D002793:Cholic Acids; D010742:Phospholipid Ethers; D043705:Receptor, Adenosine A2A; D011994:Recombinant Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C013440:cholesteryl succinate
keywords: Cholesterol; Detergents; GPCR; Ligand binding; Lipids