G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent

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G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent. / Jamshad, Mohammed; Charlton, Jack; Lin, Yu-Pin; Routledge, Sarah J; Bawa, Zharain; Knowles, Timothy J; Overduin, Michael; Dekker, Niek; Dafforn, Timothy; Bill, Roslyn M; Poyner, David R; Wheatley, Mark.

In: Bioscience Reports, Vol. 35, No. 2, e00188, 01.04.2015.

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@article{b1321d32c3cb4812a2cb065c6b48d88b,
title = "G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent",
abstract = "G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([(3)H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.",
keywords = "adenosine receptor, detergent-free, G-protein coupled receptor (GPCR), protein thermostability, structure",
author = "Mohammed Jamshad and Jack Charlton and Yu-Pin Lin and Routledge, {Sarah J} and Zharain Bawa and Knowles, {Timothy J} and Michael Overduin and Niek Dekker and Timothy Dafforn and Bill, {Roslyn M} and Poyner, {David R} and Mark Wheatley",
year = "2015",
month = apr
day = "1",
doi = "10.1042/BSR20140171",
language = "English",
volume = "35",
journal = "Bioscience Reports",
issn = "0144-8463",
publisher = "Portland Press",
number = "2",

}

RIS

TY - JOUR

T1 - G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent

AU - Jamshad, Mohammed

AU - Charlton, Jack

AU - Lin, Yu-Pin

AU - Routledge, Sarah J

AU - Bawa, Zharain

AU - Knowles, Timothy J

AU - Overduin, Michael

AU - Dekker, Niek

AU - Dafforn, Timothy

AU - Bill, Roslyn M

AU - Poyner, David R

AU - Wheatley, Mark

PY - 2015/4/1

Y1 - 2015/4/1

N2 - G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([(3)H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

AB - G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([(3)H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

KW - adenosine receptor

KW - detergent-free

KW - G-protein coupled receptor (GPCR)

KW - protein thermostability

KW - structure

U2 - 10.1042/BSR20140171

DO - 10.1042/BSR20140171

M3 - Article

C2 - 25720391

VL - 35

JO - Bioscience Reports

JF - Bioscience Reports

SN - 0144-8463

IS - 2

M1 - e00188

ER -