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

The effect of temperature on A2aR structure revealed by CD spectra of purified A2aR–SMALPCD spectra were collected using a 1-mm path length cuvette and averaged over eight scans in the far-UV region (195–260 nm). Spectra were corrected for the buffer signal.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400634&req=5

Figure 4: The effect of temperature on A2aR structure revealed by CD spectra of purified A2aR–SMALPCD spectra were collected using a 1-mm path length cuvette and averaged over eight scans in the far-UV region (195–260 nm). Spectra were corrected for the buffer signal.

Mentions: The vast majority of membrane proteins stabilized in nanodiscs to-date have used the MSP-based system developed by Sligar and colleagues [12]. In this MSP method, the lipid disc is stabilized by an annulus of scaffolding proteins which can interfere with spectrophotometric studies on the embedded membrane protein of interest. In contrast, the lipid disc in our SMALP system is stabilized by a non-proteinacious polymer, which we have already shown in a previous study, does not suffer from the same limitations [15]. This allowed the conformation of the SMALP-encapsulated A2AR to be studied using CD spectroscopy. It is known from solved crystal structures that the A2AR possesses a high α-helix content due to the seven transmembrane helical bundle that is a conserved feature of GPCR architecture [31,32]. The far-UV (195–260 nm) CD spectrum of purified A2AR–SMALP showed negative minima at 208 and 222 nm consistent with a folded protein containing a high degree of α-helix (Figure 4), consistent with the known secondary structure content of the A2AR. In contrast, an unfolded protein would have a negative minimum at 200 nm. CD was also used to assess the thermal stability of the encapsulated A2AR. These data show that as the temperature increases from 25°C to 95°C the intensity of the 208 and 222 nm features reduces until at 95°C the intensity is less than 50% of that observed at low temperature. This indicates that the protein is steadily losing secondary structure as the temperature increases. Closer examination of the CD spectra shows a single isodichroic point at ∼201 nm. This indicates that the thermal denaturation process is a two state process most probably correlating with a folded to unfolded transition. Comparison of the CD spectra in Figure 4 to the thermostability binding data (Figure 2) revealed that the change in the CD spectrum observed between 25°C and 65°C reflected the structural changes underlying the complete loss of [3H]ZM241385 binding by the A2AR. The A2AR was not fully denatured when the radioligand-binding capability was lost; however, as α-helix content was still apparent in the CD spectrum at 65°C. The proportion of α-helix decreased further as the temperature was raised from 65°C to 95°C but even at 95°C the A2AR CD signal had not completely changed to that of a random coil (characterized by an intense negative signal at 200 nm and a positive signal at 218 nm [33]). This indicates that the helical secondary structure of the A2AR is resistant to thermal denaturation in SMALPs. Retention of α-helical content has also been reported for rhodopsin during denaturation studies with combinations of different denaturants. Furthermore, it was concluded that surface elements within the rhodopsin structure were susceptible to denaturation, becoming more flexible [34], whereas a cluster of interconnected segments from multiple transmembrane helices preserved a rigid core [35]. This could explain our observations on the effect of increasing temperature on the A2AR structure and function. Molecular dynamics simulations indicate that the process of a ligand binding to its cognate GPCR progresses through an intermediate state in which the ligand binds initially to an extracellular vestibule prior to docking in the classical ‘orthosteric’-binding site [36,37]. The rhodopsin unfolding studies cited above indicate that surface elements within the A2AR architecture would be more susceptible to structural perturbation than the relatively rigid α-helical core. This suggests a feasible mechanism for our observations that the ligand-binding capability of the A2AR was lost at temperatures which preserved significant α-helical content, albeit lower than native A2AR. If the conformations of extracellular structural elements that contribute to the approach of a ligand to the orthosteric-binding site via the extracellular vestibule were susceptible to thermal disruption, then ligand binding capability would be ablated but core α-helical structure would be largely retained.


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)

The effect of temperature on A2aR structure revealed by CD spectra of purified A2aR–SMALPCD spectra were collected using a 1-mm path length cuvette and averaged over eight scans in the far-UV region (195–260 nm). Spectra were corrected for the buffer signal.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: The effect of temperature on A2aR structure revealed by CD spectra of purified A2aR–SMALPCD spectra were collected using a 1-mm path length cuvette and averaged over eight scans in the far-UV region (195–260 nm). Spectra were corrected for the buffer signal.
Mentions: The vast majority of membrane proteins stabilized in nanodiscs to-date have used the MSP-based system developed by Sligar and colleagues [12]. In this MSP method, the lipid disc is stabilized by an annulus of scaffolding proteins which can interfere with spectrophotometric studies on the embedded membrane protein of interest. In contrast, the lipid disc in our SMALP system is stabilized by a non-proteinacious polymer, which we have already shown in a previous study, does not suffer from the same limitations [15]. This allowed the conformation of the SMALP-encapsulated A2AR to be studied using CD spectroscopy. It is known from solved crystal structures that the A2AR possesses a high α-helix content due to the seven transmembrane helical bundle that is a conserved feature of GPCR architecture [31,32]. The far-UV (195–260 nm) CD spectrum of purified A2AR–SMALP showed negative minima at 208 and 222 nm consistent with a folded protein containing a high degree of α-helix (Figure 4), consistent with the known secondary structure content of the A2AR. In contrast, an unfolded protein would have a negative minimum at 200 nm. CD was also used to assess the thermal stability of the encapsulated A2AR. These data show that as the temperature increases from 25°C to 95°C the intensity of the 208 and 222 nm features reduces until at 95°C the intensity is less than 50% of that observed at low temperature. This indicates that the protein is steadily losing secondary structure as the temperature increases. Closer examination of the CD spectra shows a single isodichroic point at ∼201 nm. This indicates that the thermal denaturation process is a two state process most probably correlating with a folded to unfolded transition. Comparison of the CD spectra in Figure 4 to the thermostability binding data (Figure 2) revealed that the change in the CD spectrum observed between 25°C and 65°C reflected the structural changes underlying the complete loss of [3H]ZM241385 binding by the A2AR. The A2AR was not fully denatured when the radioligand-binding capability was lost; however, as α-helix content was still apparent in the CD spectrum at 65°C. The proportion of α-helix decreased further as the temperature was raised from 65°C to 95°C but even at 95°C the A2AR CD signal had not completely changed to that of a random coil (characterized by an intense negative signal at 200 nm and a positive signal at 218 nm [33]). This indicates that the helical secondary structure of the A2AR is resistant to thermal denaturation in SMALPs. Retention of α-helical content has also been reported for rhodopsin during denaturation studies with combinations of different denaturants. Furthermore, it was concluded that surface elements within the rhodopsin structure were susceptible to denaturation, becoming more flexible [34], whereas a cluster of interconnected segments from multiple transmembrane helices preserved a rigid core [35]. This could explain our observations on the effect of increasing temperature on the A2AR structure and function. Molecular dynamics simulations indicate that the process of a ligand binding to its cognate GPCR progresses through an intermediate state in which the ligand binds initially to an extracellular vestibule prior to docking in the classical ‘orthosteric’-binding site [36,37]. The rhodopsin unfolding studies cited above indicate that surface elements within the A2AR architecture would be more susceptible to structural perturbation than the relatively rigid α-helical core. This suggests a feasible mechanism for our observations that the ligand-binding capability of the A2AR was lost at temperatures which preserved significant α-helical content, albeit lower than native A2AR. If the conformations of extracellular structural elements that contribute to the approach of a ligand to the orthosteric-binding site via the extracellular vestibule were susceptible to thermal disruption, then ligand binding capability would be ablated but core α-helical structure would be largely retained.

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