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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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Effect of repeated freeze-thaw cycles on A2aR–SMALP binding capabilityA2aR–SMALP was subjected to repeated freeze/thaw cycles and specific binding of [3H]ZM241385 determined after each cycle. Data are expressed as specific binding relative to binding before freezing (mean ± S.E.M. of three separate experiments performed in triplicate).
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Figure 7: Effect of repeated freeze-thaw cycles on A2aR–SMALP binding capabilityA2aR–SMALP was subjected to repeated freeze/thaw cycles and specific binding of [3H]ZM241385 determined after each cycle. Data are expressed as specific binding relative to binding before freezing (mean ± S.E.M. of three separate experiments performed in triplicate).

Mentions: Given the potential utility of the GPCR–SMALP for receptor-based in vitro assays and screens, the stability of the A2AR–SMALP at the physiological temperature of 37°C and the storage temperature of 4°C was investigated together with the resistance to repeated freeze-thaw cycles. A comparison of the loss of ligand binding capability of the A2AR at 37°C with time is presented in Figure 6 for A2AR–DDM, A2AR–SMALP and cell membranes. The improved stability of the A2AR–SMALP over A2AR–DDM at 37°C is very marked, with a 7-fold increase in the half-life of [3H]ZM241385 binding (A2AR–DDM, t½=21±7 min; A2AR–SMALP, t½=148±13 min). This improvement is particularly apparent after 1 h, with no specific binding detected for the detergent-solubilized receptor, whereas 85±5% of the binding was retained in the A2AR–SMALP (Figure 6). Although receptor stability was increased in the SMALP, it did not match receptor stability in the native HEK293T membranes at 37°C, suggesting that not all the stabilizing factors of the plasma membrane are incorporated into SMALPs. In contrast, the stability of A2AR–SMALP at 4°C was essentially indistinguishable from that of membranes with t½ ≥16 days, whereas A2AR–DDM exhibited a t½=1.8±0.3 days (mean ± S.E.M.). The resilience of A2AR–SMALP was also evident from repeated freeze/thaw cycles (Figure 7). Even after five freeze/thaw cycles on the same sample, there was no decrease in [3H]ZM241385 binding capability of the A2AR–SMALP. In contrast, specific binding to the A2AR–DDM preparation was completely lost after a single freeze-thaw cycle. Our data indicate that it is feasible to SMALP solubilize GPCRs and store them at 4°C until required. Interestingly, the increase in thermostability endowed on A2aR by the SMALP compared with DDM was approximately 5°C for both yeast and HEK 293T, suggesting that the increased thermostability is an inherent property of the SMALP rather than due to differences in the lipid composition of the original membrane.


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)

Effect of repeated freeze-thaw cycles on A2aR–SMALP binding capabilityA2aR–SMALP was subjected to repeated freeze/thaw cycles and specific binding of [3H]ZM241385 determined after each cycle. Data are expressed as specific binding relative to binding before freezing (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 7: Effect of repeated freeze-thaw cycles on A2aR–SMALP binding capabilityA2aR–SMALP was subjected to repeated freeze/thaw cycles and specific binding of [3H]ZM241385 determined after each cycle. Data are expressed as specific binding relative to binding before freezing (mean ± S.E.M. of three separate experiments performed in triplicate).
Mentions: Given the potential utility of the GPCR–SMALP for receptor-based in vitro assays and screens, the stability of the A2AR–SMALP at the physiological temperature of 37°C and the storage temperature of 4°C was investigated together with the resistance to repeated freeze-thaw cycles. A comparison of the loss of ligand binding capability of the A2AR at 37°C with time is presented in Figure 6 for A2AR–DDM, A2AR–SMALP and cell membranes. The improved stability of the A2AR–SMALP over A2AR–DDM at 37°C is very marked, with a 7-fold increase in the half-life of [3H]ZM241385 binding (A2AR–DDM, t½=21±7 min; A2AR–SMALP, t½=148±13 min). This improvement is particularly apparent after 1 h, with no specific binding detected for the detergent-solubilized receptor, whereas 85±5% of the binding was retained in the A2AR–SMALP (Figure 6). Although receptor stability was increased in the SMALP, it did not match receptor stability in the native HEK293T membranes at 37°C, suggesting that not all the stabilizing factors of the plasma membrane are incorporated into SMALPs. In contrast, the stability of A2AR–SMALP at 4°C was essentially indistinguishable from that of membranes with t½ ≥16 days, whereas A2AR–DDM exhibited a t½=1.8±0.3 days (mean ± S.E.M.). The resilience of A2AR–SMALP was also evident from repeated freeze/thaw cycles (Figure 7). Even after five freeze/thaw cycles on the same sample, there was no decrease in [3H]ZM241385 binding capability of the A2AR–SMALP. In contrast, specific binding to the A2AR–DDM preparation was completely lost after a single freeze-thaw cycle. Our data indicate that it is feasible to SMALP solubilize GPCRs and store them at 4°C until required. Interestingly, the increase in thermostability endowed on A2aR by the SMALP compared with DDM was approximately 5°C for both yeast and HEK 293T, suggesting that the increased thermostability is an inherent property of the SMALP rather than due to differences in the lipid composition of the original membrane.

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