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

Andrea N. Naranjo; Patrick M. McNeely; John Katsaras; Anne Skaja Robinson

doi:10.1016/j.pep.2016.05.015

Impact of purification conditions and history on A_2A adenosine receptor activity: The role of CHAPS and lipids

Andrea N. Naranjo, Patrick M. McNeely, John Katsaras, Anne Skaja Robinson

Research output: Contribution to journal › Article › peer-review

11 Scopus citations

Abstract

The adenosine A_2A receptor (A_2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A_2AR 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 A_2AR. 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 A_2AR 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 A_2AR 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.

Original language	English
Pages (from-to)	62-67
Number of pages	6
Journal	Protein Expression and Purification
Volume	124
DOIs	https://doi.org/10.1016/j.pep.2016.05.015
State	Published - Aug 1 2016

Funding

This project was supported by grants from the National Institutes of Health (National Center for Research Resources ( 5P30RR031160-03 ) and the National Institute of General Medical Sciences ( 8 P30 GM103519-03 ), and from the National Science Foundation ( 1033268/1249200 ) and NSF Graduate Research Fellowship Program (ANN). JK is supported through the Scientific User Facilities Division of the DOE Office of Basic Energy Sciences (BES) , under contract no. DE-AC05 00OR2275 . We also thank Dr. K. Dane Wittrup (Massachusetts Institute of Technology, Cambridge, MA) for the pITy plasmid, Dr. Marlene Jacobson (Merck) for the human adenosine hA 2A R gene, Dr. Kelvin Lee (University of Delaware) for assistance with mass spectrometry, Dr. William Wimley (Tulane University) for the use of the circular dichroism equipment, and Dr. Robert F. Standaert (Oak Ridge National Laboratory) for discussions.

Keywords

Cholesterol
Detergents
GPCR
Ligand binding
Lipids

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10.1016/j.pep.2016.05.015

Cite this

@article{809c60c176e24a17858e3cc75b641e11,

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.",

keywords = "Cholesterol, Detergents, GPCR, Ligand binding, Lipids",

author = "Naranjo, \{Andrea N.\} and McNeely, \{Patrick M.\} and John Katsaras and Robinson, \{Anne Skaja\}",

note = "Publisher Copyright: {\textcopyright} 2016 Elsevier Inc.",

year = "2016",

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doi = "10.1016/j.pep.2016.05.015",

language = "English",

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T1 - Impact of purification conditions and history on A2A adenosine receptor activity

T2 - The role of CHAPS and lipids

AU - Naranjo, Andrea N.

AU - McNeely, Patrick M.

AU - Katsaras, John

AU - Robinson, Anne Skaja

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N2 - 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.

AB - 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.

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KW - Detergents

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