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

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

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

Journal Article · Fri May 27 04:00:00 EDT 2016 · Protein Expression and Purification

DOI:https://doi.org/10.1016/j.pep.2016.05.015· OSTI ID:1279449

Naranjo, Andrea N. ^[1]; McNeely, Patrick M. ^[1]; Katsaras, John ^[2]; Skaja Robinson, Anne ^[3]

Univ. of Delaware, Newark, DE (United States)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
Univ. of Delaware, Newark, DE (United States); Tulane Univ., New Orleans, LA (United States)

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. Furthermore, 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.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1279449

Alternate ID(s):: OSTI ID: 1325368

Journal Information:: Protein Expression and Purification, Journal Name: Protein Expression and Purification Vol. 124; ISSN 1046-5928

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

References (31)

Structure-function studies with G protein-coupled receptors as a paradigm for improving drug discovery and development of therapeutics McNeely, Patrick M.; Naranjo, Andrea N.; Robinson, Anne S. Biotechnology Journal, Vol. 7, Issue 12 https://doi.org/10.1002/biot.201200076	journal	December 2012
Solution NMR of membrane proteins: practice and challenges Sanders, Charles R.; Sönnichsen, Frank Magnetic Resonance in Chemistry, Vol. 44, Issue S1 https://doi.org/10.1002/mrc.1816	journal	January 2006
“Bicellar” Lipid Mixtures as used in Biochemical and Biophysical Studies Katsaras, John; Harroun, Thad A.; Pencer, Jeremy Naturwissenschaften, Vol. 92, Issue 8 https://doi.org/10.1007/s00114-005-0641-1	journal	July 2005
Differential solubilization of lipids along with membrane proteins by different classes of detergents Banerjee, Probal; Joo, J. B.; Buse, John T. Chemistry and Physics of Lipids, Vol. 77, Issue 1 https://doi.org/10.1016/0009-3084(95)02455-R	journal	August 1995
Magnetically-oriented phospholipid micelles as a tool for the study of membrane-associated molecules Sanders, Charles R.; Hare, Brian J.; Howard, Kathleen P. Progress in Nuclear Magnetic Resonance Spectroscopy, Vol. 26 https://doi.org/10.1016/0079-6565(94)80012-X	journal	January 1994
Short-chain phospholipids as detergents Hauser, Helmut Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1508, Issue 1-2 https://doi.org/10.1016/S0304-4157(00)00008-3	journal	November 2000
Arresting developments in heptahelical receptor signaling and regulation Perry, Stephen J.; Lefkowitz, Robert J. Trends in Cell Biology, Vol. 12, Issue 3 https://doi.org/10.1016/S0962-8924(01)02239-5	journal	March 2002
Solution- and solid-state NMR studies of GPCRs and their ligands Tapaneeyakorn, Satita; Goddard, Alan D.; Oates, Joanne Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1808, Issue 6 https://doi.org/10.1016/j.bbamem.2010.10.003	journal	June 2011
The Morphology and Composition of Cholesterol-Rich Micellar Nanostructures Determine Transmembrane Protein (GPCR) Activity O'Malley, Michelle A.; Helgeson, Matthew E.; Wagner, Norman J. Biophysical Journal, Vol. 100, Issue 2 https://doi.org/10.1016/j.bpj.2010.12.3698	journal	January 2011
Toward Rational Design of Protein Detergent Complexes: Determinants of Mixed Micelles That Are Critical for the In Vitro Stabilization of a G-Protein Coupled Receptor O'Malley, Michelle A.; Helgeson, Matthew E.; Wagner, Norman J. Biophysical Journal, Vol. 101, Issue 8 https://doi.org/10.1016/j.bpj.2011.09.018	journal	October 2011
Solubilization of human serotonin1A receptors expressed in neuronal cells Jafurulla, Md.; Chattopadhyay, Amitabha Chemistry and Physics of Lipids, Vol. 150, Issue 2 https://doi.org/10.1016/j.chemphyslip.2007.08.003	journal	December 2007
High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor O’Malley, Michelle A.; Lazarova, Tzvetana; Britton, Zachary T. Journal of Structural Biology, Vol. 159, Issue 2 https://doi.org/10.1016/j.jsb.2007.05.001	journal	August 2007
Solubilization and reconstitution of the mu-opioid receptor expressed in human neuronal SH-SY5Y and CHO cells Talmont, Franck; Moulédous, Lionel; Mollereau, Catherine Peptides, Vol. 55 https://doi.org/10.1016/j.peptides.2014.02.009	journal	May 2014
A crystal clear solution for determining G-protein-coupled receptor structures Tate, Christopher G. Trends in Biochemical Sciences, Vol. 37, Issue 9 https://doi.org/10.1016/j.tibs.2012.06.003	journal	September 2012
Detergents for the stabilization and crystallization of membrane proteins Privé, Gilbert G. Methods, Vol. 41, Issue 4 https://doi.org/10.1016/j.ymeth.2007.01.007	journal	April 2007
Analysis of Adenosine A ₂ a Receptor Stability: Effects of Ligands and Disulfide Bonds O’Malley, Michelle A.; Naranjo, Andrea N.; Lazarova, Tzvetana Biochemistry, Vol. 49, Issue 43 https://doi.org/10.1021/bi101155r	journal	November 2010
Predictions for Cholesterol Interaction Sites on the A _2A Adenosine Receptor Lee, Ji Young; Lyman, Edward Journal of the American Chemical Society, Vol. 134, Issue 40 https://doi.org/10.1021/ja307532d	journal	September 2012
Comprehensive Examination of Mesophases Formed by DMPC and DHPC Mixtures Harroun, Thad A.; Koslowsky, Martin; Nieh, Mu-Ping Langmuir, Vol. 21, Issue 12 https://doi.org/10.1021/la050018t	journal	June 2005
Formation of Kinetically Trapped Nanoscopic Unilamellar Vesicles from Metastable Nanodiscs Nieh, Mu-Ping; Dolinar, Paul; Kučerka, Norbert Langmuir, Vol. 27, Issue 23 https://doi.org/10.1021/la2023314	journal	December 2011
Solubilization of Serotonin _1A Receptors Heterologously Expressed in Chinese Hamster Ovary Cells Chattopadhyay, Amitabha; Jafurulla, Md.; Kalipatnapu, Shanti Cellular and Molecular Neurobiology, Vol. 24, Issue 2 https://doi.org/10.1023/B:CEMN.0000018623.81954.39	journal	April 2004
Ice breaking in GPCR structural biology Zhao, Qiang; Wu, Bei-li Acta Pharmacologica Sinica, Vol. 33, Issue 3 https://doi.org/10.1038/aps.2011.187	journal	January 2012
Crystal structure of the human β2 adrenergic G-protein-coupled receptor Rasmussen, Søren G. F.; Choi, Hee-Jung; Rosenbaum, Daniel M. Nature, Vol. 450, Issue 7168 https://doi.org/10.1038/nature06325	journal	October 2007
Fiji: an open-source platform for biological-image analysis Schindelin, Johannes; Arganda-Carreras, Ignacio; Frise, Erwin Nature Methods, Vol. 9, Issue 7 https://doi.org/10.1038/nmeth.2019	journal	June 2012
Purification and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli: Purification and characterization of A2a receptor Weiß, H. Markus; Grisshammer, Reinhard European Journal of Biochemistry, Vol. 269, Issue 1 https://doi.org/10.1046/j.0014-2956.2002.02618.x	journal	January 2002
Designer short peptide surfactants stabilize G protein-coupled receptor bovine rhodopsin Zhao, X.; Nagai, Y.; Reeves, P. J. Proceedings of the National Academy of Sciences, Vol. 103, Issue 47 https://doi.org/10.1073/pnas.0607167103	journal	November 2006
Cholesterol increases kinetic, energetic, and mechanical stability of the human 2-adrenergic receptor Zocher, M.; Zhang, C.; Rasmussen, S. G. F. Proceedings of the National Academy of Sciences, Vol. 109, Issue 50 https://doi.org/10.1073/pnas.1210373109	journal	November 2012
Rarely Observed Phase Transitions in a Novel Lyotropic Liquid Crystal System Katsaras, J.; Donaberger, R. L.; Swainson, I. P. Physical Review Letters, Vol. 78, Issue 5 https://doi.org/10.1103/PhysRevLett.78.899	journal	February 1997
Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions Liu, W.; Chun, E.; Thompson, A. A. Science, Vol. 337, Issue 6091 https://doi.org/10.1126/science.1219218	journal	July 2012
Stabilization of Functional Recombinant Cannabinoid Receptor CB2 in Detergent Micelles and Lipid Bilayers Vukoti, Krishna; Kimura, Tomohiro; Macke, Laura PLoS ONE, Vol. 7, Issue 10 https://doi.org/10.1371/journal.pone.0046290	journal	October 2012
Over-Expression, Solubilization, and Purification of G Protein-Coupled Receptors for Structural Biology Chiu, Mark; Tsang, Cindy; Grihalde, Nelson Combinatorial Chemistry & High Throughput Screening, Vol. 11, Issue 6 https://doi.org/10.2174/138620708784911456	journal	July 2008
Solution NMR studies of peptide-lipid interactions in model membranes Mäler, Lena Molecular Membrane Biology, Vol. 29, Issue 5 https://doi.org/10.3109/09687688.2012.683456	journal	May 2012

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The A _2a R C-terminus provides improved total and active expression yields for adenosine receptor chimeras Jain, Abhinav R.; Stradley, Steven H.; Robinson, Anne S. AIChE Journal, Vol. 64, Issue 12 https://doi.org/10.1002/aic.16398	journal	October 2018

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
GPCR
cholesterol
detergents
ligand binding
lipids