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

Jamshad M, Charlton J, Lin YP, Routledge SJ, Bawa Z, Knowles TJ, Overduin M, Dekker N, Dafforn TR, Bill RM, Poyner DR, Wheatley M - Biosci. Rep. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: *School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.

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.

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Thermostability of A2aR–SMALP, DDM-solubilized A2aR and membrane-bound A2aR prepared from HEK293T cellsA2aR–SMALP (●), DDM-solubilized A2aR (▲) or membranes (○), prepared from HEK293T cells transfected with A2aR, were incubated for 30 min at the stated temperatures, then chilled on ice, before specific binding of [3H]ZM241385 was determined as described in ‘Methods’. Data are expressed as specific binding relative to the 20°C data point (mean ± S.E.M. of three separate experiments performed in triplicate).
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Figure 5: Thermostability of A2aR–SMALP, DDM-solubilized A2aR and membrane-bound A2aR prepared from HEK293T cellsA2aR–SMALP (●), DDM-solubilized A2aR (▲) or membranes (○), prepared from HEK293T cells transfected with A2aR, were incubated for 30 min at the stated temperatures, then chilled on ice, before specific binding of [3H]ZM241385 was determined as described in ‘Methods’. Data are expressed as specific binding relative to the 20°C data point (mean ± S.E.M. of three separate experiments performed in triplicate).

Mentions: HEK293T cells, expressing A2AR, were directly solubilized with 2.0% (w/v) SMA, in the total absence of detergent, to generate a functional A2AR–SMALP preparation of 2.0±0.24 pmol/mg protein (n=3), equivalent to 23.3±2.75% (n=3) recovery of A2AR. Characterization of this A2AR–SMALP by competition radioligand-binding using a range of ligands with [3H]ZM241385 as tracer confirmed retention of typical A2AR pharmacology (Table 2). The A2AR–SMALP from HEK293T cells exhibited an increase in thermostability of 4°C compared with the corresponding detergent (DDM)-solubilized A2AR (Figure 5), with the T50 value increasing from T50=36.2±0.52°C (n=3) for A2AR–DDM to T50=40.2±0.44°C (n=3) for A2AR–SMALP. The A2AR–SMALP was not as stable as A2AR embedded in the native HEK293T cell membrane however (Figure 5).


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

Jamshad M, Charlton J, Lin YP, Routledge SJ, Bawa Z, Knowles TJ, Overduin M, Dekker N, Dafforn TR, Bill RM, Poyner DR, Wheatley M - Biosci. Rep. (2015)

Thermostability of A2aR–SMALP, DDM-solubilized A2aR and membrane-bound A2aR prepared from HEK293T cellsA2aR–SMALP (●), DDM-solubilized A2aR (▲) or membranes (○), prepared from HEK293T cells transfected with A2aR, were incubated for 30 min at the stated temperatures, then chilled on ice, before specific binding of [3H]ZM241385 was determined as described in ‘Methods’. Data are expressed as specific binding relative to the 20°C data point (mean ± S.E.M. of three separate experiments performed in triplicate).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400634&req=5

Figure 5: Thermostability of A2aR–SMALP, DDM-solubilized A2aR and membrane-bound A2aR prepared from HEK293T cellsA2aR–SMALP (●), DDM-solubilized A2aR (▲) or membranes (○), prepared from HEK293T cells transfected with A2aR, were incubated for 30 min at the stated temperatures, then chilled on ice, before specific binding of [3H]ZM241385 was determined as described in ‘Methods’. Data are expressed as specific binding relative to the 20°C data point (mean ± S.E.M. of three separate experiments performed in triplicate).
Mentions: HEK293T cells, expressing A2AR, were directly solubilized with 2.0% (w/v) SMA, in the total absence of detergent, to generate a functional A2AR–SMALP preparation of 2.0±0.24 pmol/mg protein (n=3), equivalent to 23.3±2.75% (n=3) recovery of A2AR. Characterization of this A2AR–SMALP by competition radioligand-binding using a range of ligands with [3H]ZM241385 as tracer confirmed retention of typical A2AR pharmacology (Table 2). The A2AR–SMALP from HEK293T cells exhibited an increase in thermostability of 4°C compared with the corresponding detergent (DDM)-solubilized A2AR (Figure 5), with the T50 value increasing from T50=36.2±0.52°C (n=3) for A2AR–DDM to T50=40.2±0.44°C (n=3) for A2AR–SMALP. The A2AR–SMALP was not as stable as A2AR embedded in the native HEK293T cell membrane however (Figure 5).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: *School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.

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.

Show MeSH
Related in: MedlinePlus