Limits...
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.

Show MeSH

Related in: MedlinePlus

Thermostability of A2AR–SMALP and DDM-solubilized A2AR from P. pastorisA2AR–SMALP (●) and DDM-solubilized A2AR (▲) prepared from P. pastoris overexpressing A2aR were incubated for 30 min at the stated temperatures, 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
getmorefigures.php?uid=PMC4400634&req=5

Figure 2: Thermostability of A2AR–SMALP and DDM-solubilized A2AR from P. pastorisA2AR–SMALP (●) and DDM-solubilized A2AR (▲) prepared from P. pastoris overexpressing A2aR were incubated for 30 min at the stated temperatures, 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: An acknowledged common problem with detergent solubilization of GPCRs is destabilization of the receptor in the detergent micelle compared with the native plasma membrane. As the A2AR–SMALP preserves the annular lipid in close contact with the receptor in the membrane, it was reasoned that A2AR would be more thermostable in a SMALP than when detergent-solubilized by DDM, a widely employed detergent for GPCR solubilization. The antagonist [3H]ZM241385 is widely used to quantify functional A2AR-binding capability using radioligand-binding assays. A2AR–SMALP and DDM-solubilized A2AR from P. pastoris membranes were incubated in parallel at various temperatures and then the residual specific binding of [3H]ZM241385 was determined. From data presented in Figure 2, it can be seen that the SMALP conferred a marked increase in A2AR thermostability of ∼5.5°C over the detergent micelle, with the T50 value increasing from T50=44.4±0.27°C (n=3) for A2AR–DDM to T50=49.9±1.19°C (n=3) for A2AR–SMALP.


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 and DDM-solubilized A2AR from P. pastorisA2AR–SMALP (●) and DDM-solubilized A2AR (▲) prepared from P. pastoris overexpressing A2aR were incubated for 30 min at the stated temperatures, 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 2: Thermostability of A2AR–SMALP and DDM-solubilized A2AR from P. pastorisA2AR–SMALP (●) and DDM-solubilized A2AR (▲) prepared from P. pastoris overexpressing A2aR were incubated for 30 min at the stated temperatures, 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: An acknowledged common problem with detergent solubilization of GPCRs is destabilization of the receptor in the detergent micelle compared with the native plasma membrane. As the A2AR–SMALP preserves the annular lipid in close contact with the receptor in the membrane, it was reasoned that A2AR would be more thermostable in a SMALP than when detergent-solubilized by DDM, a widely employed detergent for GPCR solubilization. The antagonist [3H]ZM241385 is widely used to quantify functional A2AR-binding capability using radioligand-binding assays. A2AR–SMALP and DDM-solubilized A2AR from P. pastoris membranes were incubated in parallel at various temperatures and then the residual specific binding of [3H]ZM241385 was determined. From data presented in Figure 2, it can be seen that the SMALP conferred a marked increase in A2AR thermostability of ∼5.5°C over the detergent micelle, with the T50 value increasing from T50=44.4±0.27°C (n=3) for A2AR–DDM to T50=49.9±1.19°C (n=3) for A2AR–SMALP.

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