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This content will become publicly available on May 27, 2017

Title: Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

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. Furthermore, the studies presented in this paper also underline the importancemore » 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.« less
Authors:
 [1] ;  [1] ;  [2] ;  [3]
  1. Univ. of Delaware, Newark, DE (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
  3. Univ. of Delaware, Newark, DE (United States); Tulane Univ., New Orleans, LA (United States)
Publication Date:
OSTI Identifier:
1279449
Grant/Contract Number:
AC05-00OR22725
Type:
Accepted Manuscript
Journal Name:
Protein Expression and Purification
Additional Journal Information:
Journal Volume: 124; Journal ID: ISSN 1046-5928
Publisher:
Elsevier
Research Org:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES GPCR; lipids; detergents; ligand binding; cholesterol