Human adenosine A_2A receptor (A_2aR).

Two different A_2AR constructs were used during this work. For SEC experiments using purified receptor, an A_2AR construct was used that contained an N-terminal thioredoxin fusion protein to increase the molecular weight of the receptor. Without this fusion protein, A_2AR and the mini-G protein had identical mobility on SDS-PAGE, thus making it difficult to visualise the separate components when analyzing a complex. The thioredoxin-A_2AR fusion protein consisted of an N-terminal cleavable leader sequence (gp67), His10 tag and TEV protease cleavage site, followed by thioredoxin, which was connected to wild-type human A_2AR (residues 6–316) through an EAAAKA linker. A_2AR contained the N154A mutation to remove a potential N-linked glycosylation site. For all other experiments, a C-terminally truncated human A_2AR construct was used (residues 1–317), which contained a C-terminal His10 tag and TEV protease cleavage site and the N154A mutation to remove the potential N-linked glycosylation site. Both constructs were expressed using the baculovirus expression system as described previously [20, 26]. Cells were harvested by centrifugation 72 hours post infection, resuspended in hypotonic buffer (20 mM HEPES pH7.5, 1 mM EDTA, 1 mM PMSF), flash-frozen in liquid nitrogen and stored at –80°C until use. Purification of the receptor was performed in DDM using Ni²⁺-affinity chromatography followed by SEC essentially as described previously [20, 26].

Turkey β₁-adrenergic receptor (β₁AR).

A truncated version of wild type turkey β₁AR (construct βAR6; [27]) contained truncations at the N-terminus and the C-terminus and a C-terminal His6 tag for purification [27], and was expressed using the baculovirus expression system at 27°C as described previously [28]. Cells were harvested by centrifugation 48 hours post infection, resuspended in hypotonic buffer (20 mM Tris-HCl pH8, 1 mM EDTA, 1 mM PMSF), flash-frozen in liquid nitrogen and stored at -80°C until use.

Human angiotensin type II receptor 1 (AT₁R).

Wild type AT₁R (residues 1–359) had a C-terminal factor X cleavage site followed by GFP and a His10 tag for purification, and was expressed using the tetracycline-inducible mammalian expression system as a stable cell line in HEK293 cells [29]. Cells were grown in DMEM containing 5% tetracycline-free FBS until they were 80% confluent and then tetracycline was added to a final concentration of 1 μg/ml. Cells were grown for 24 hours and then harvested, and resuspended in PBS, flash frozen in liquid nitrogen and stored at -80°C until use.

Rat neurotensin receptor (NTSR1).

NTSR1 was expressed as described previously [13]. The baculovirus construct NTSR1 consisted of the hemagglutinin signal peptide and the Flag tag, followed by the wild-type rat NTSR1 (residues 43–396) and a C-terminal His10 tag. Recombinant baculovirus was generated using a modified pFastBac1 transfer plasmid (Invitrogen). Trichoplusia ni cells were infected with recombinant virus, and the temperature was lowered from 27°C to 21°C. Cells were harvested by centrifugation 48 hours post infection, resuspended in hypotonic buffer (10 mM HEPES pH 7.5, 10 mM MgCl₂, 20 mM KCl), flash-frozen in liquid nitrogen and stored at -80°C until use.

Human serotonin 5HT_1B receptor (5HT_1BR).

Wild-type 5HT_1BR (residues 34–390) was modified to contain a C-terminal TEV cleavage site and a His10 tag, cloned into plasmid pBacPAK8 and recombinant baculoviruses were prepared using the FlashBAC ULTRA system (Oxford Expression Technologies). Trichoplusia ni cells were grown in ESF921 media (Expression Systems) to a density of 3x10⁶ cells/ml, infected with 5HT_1BR baculovirus and incubated for 48 h at 27°C for expression. Purification of the receptor was performed in either DDM or LMNG using Ni²⁺-affinity chromatography followed by SEC.

(1) A_2AR–mini-G_s complex.

The thioredoxin fusion construct of A_2AR was purified in DDM essentially as described previously [19]. A_2AR and mini-G protein were mixed in a 1:1.2 molar ratio, apyrase (0.1 U/ml final concentration) was added and the sample was incubated overnight on ice before loading onto a Superdex S200 10/300 size exclusion column (10 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl₂, 100 μM NECA, 0.02% DDM; 4°C, 0.5 ml/min). Peak fractions were analysed by SDS-PAGE.

(2) 5HT_1BR–mini-G_o1 complex.

Donitriptan-bound 5HT_1BR purified in DDM was mixed with mini-G_o1 in a 1:1.2 molar ratio. Apyrase (0.1 U/ml final concentration) was added and the sample was incubated 4 h on ice before loading onto a Superdex S200 10/300 size exclusion column (20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl₂, 1 μM donitriptan, 0.03% DDM; 4°C, 0.5 ml/min). Peak fractions were analysed by SDS-PAGE.

(1) A_2AR.

Insect cell membranes containing a total of 20 μg (560 pmol) wild-type A_2AR (20 x 10⁶ cells) were solubilized for 30 min on ice in 40 mM HEPES pH7.5, 500 mM NaCl, 2 mM MgCl₂, 2 U/mL apyrase (Sigma-Aldrich), and 0.5% (v/v) DDM (final volume of 2 ml, concentration of A_2AR 280 nM). Insoluble material was removed by ultracentrifugation (30 min, 4°C, 135,000 xg). The supernatant was divided into aliquots for the subsequent assay. To 900 μl of the supernatant was added either the agonist NECA or the inverse agonist ZM241385 (negative control), both at a final concentration of 60 μM. GFP-mini-G_s (6 μg; 110 pmol, final concentration 122 nM) was then added and allowed to bind for 90 min on ice before loading 200 μl onto a Superdex S200 10/300 size exclusion column (buffer 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 1 μM NECA or ZM241385, 0.03% DDM, 4°C, flow rate 0.45 ml/min). The control sample contained 6 μg GFP-mini-G_s only in 500 μl assay buffer. GFP fluorescence was detected by a Hitachi fluorometer (mV) set to an excitation of 488 nm and an emission of 525 nm.

(2) β₁AR.

Insect cell membranes containing a total of 8 μg (178 pmol) wild-type β₁AR (30 x 10⁶ cells) were solubilized for 30 min on ice in 20 mM Tris-HCl pH8, 500 mM NaCl, 5 mM MgCl₂, 2 U/mL apyrase and 0.5% (v/v) DDM (final volume 2 ml, β₁AR concentration 90 nM). Insoluble material was removed by ultracentrifugation (30 min, 4°C, 135,000 xg). The supernatant was divided into aliquots for the subsequent assay. Isoprenaline (100 μM final concentration) or ICI118551 (10 μM final concentration) were added to 500 μl of the supernatant. GFP-mini-G_s (6 μg, 110 pmol) was then added to give a final concentration of 122 nM and allowed to bind for 90 min on ice before loading 200 μl onto a Superdex S200 10/300 size exclusion column (buffer 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 1 μM isoprenaline or ICI118551, 0.03% DDM, 4°C, flow rate 0.45 ml/min). The control sample contained 6 μg GFP-mini-G_s only in 500 μl assay buffer.

(3) 5HT_1BR.

When detergent-solubilized unpurified receptor was used, insect cells expressing 610 pmol 5HT_1BR (40 x 10⁶ cells) were resuspended in 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 2 U/ml apyrase to a final cell density of 20 x 10⁶ cells/ml and solubilized with 0.5% DDM (45 min, 4°C, final volume 2 ml, 5HT_1BR concentration 305 nM). Insoluble material was removed by ultracentrifugation (30 min, 4°C, 135,000 xg). The supernatant was divided into 900 μl aliquots for the subsequent assay. GFP-mini-G_o1 (5 μg, 100 pmol) was added (final concentration 111 nM) with either donitriptan or SB224289, each to a final concentration of 100 μM, and allowed to bind for 90 min on ice before loading 500 μl onto a Superdex S200 10/300 size exclusion column. The control sample contained 5 μg GFP-mini-G_o1 in 500 μl assay buffer.

In some FSEC experiments, purified 5HT_1BR was used. Donitriptan-bound, purified receptor (120 μg; 3 nmol) in either LMNG or DDM was incubated for 90 min on ice with 4 μg (60–80 pmol) either of GFP-mini-G_i1, GFP-mini-G_o1 or GFP-mini-G_s (negative control) in a final volume of 450 μl. Samples (200 μl) were then loaded onto Superdex S200 10/300 size exclusion column (buffer 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 1 μM donitriptan, 0.03% DDM or 0.001% LMNG buffer, 4°C, flow rate 0.45 ml/min). The final concentration of purified 5HT_1BR was 6.7 μM and the final concentrations of the mini-G proteins were as follows: GFP-mini-G_s/i1, 133 nM; GFP-mini-G_i1, 167 nM; GFP-mini-G_o1, 170 nM; GFP-mini-G_s, 167 nM; GFP-mini-G_i1β₁γ₂, 90 nM.

(1) β₁AR.

Membranes prepared from insect cells expressing β₁AR (50 x 10⁶ cells) were solubilized in 20 mM Tris-HCl pH8, 500 mM NaCl, 3 mM imidazole, 2 U/ml apyrase 0.5% DDM (1 hour, 4°C, final volume 8 ml, β₁AR concentration 37.5 nM). Insoluble material was removed by ultracentrifugation (30 min, 4°C, 135,000 xg) and the supernatant was divided into two aliquots. The agonist isoprenaline was added to one sample (final concentration 10 μM) and the inverse agonist ICI118551 was added to the other (final concentration 1 μM). Samples were then aliquoted 200 μl per well into a black Ni²⁺-coated 96-well plate (Pierce; Thermo Fisher). The receptor was allowed to bind via its His tag for 1 h on ice. The supernatant was then aspirated and 200 μl GFP-mini-G_s at varying concentrations (0 to 2.8 μM) were added and incubated for a further 90 min on ice. The supernatant was then removed by aspiration and each well washed 4 times with buffer A (10 μM isoprenaline (agonist), 20 mM Tris-HCl pH8, 100 mM NaCl, 1 mM MgCl₂, 1 mg/mL BSA, 30 mM imidazole, 0.03% DDM,) or buffer B (1 μM ICI118551 (inverse agonist), 20 mM Tris-HCl pH8, 100 mM NaCl, 1 mM MgCl₂, 1 mg/mL BSA, 30 mM imidazole, 0.03% DDM). Elution of the receptor–GFP-mini-G_s complex from the sides of the well to make a homogeneous solution was performed with 200 μl of the respective wash buffers that contained 300 mM imidazole. Fluorescence was then measured using a Pherastar plate reader (BMG Labtech, Inc.) with excitation at 485 nm and emission at 520 nm. ΔF data (fluorescence agonist condition minus fluorescence antagonist condition) corresponding to specific binding were analysed by non-linear regression using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA) and apparent K_D values derived from one site-specific binding analysis.

(2) A_2AR.

The assay was performed essentially as described above for β₁AR, but the buffer conditions were different. Solubilisation of insect cell membranes (40 x 10⁶ cells) was performed in 10 ml of 20 mM Tris-HCl pH8, 500 mM NaCl, 10 mM imidazole, 2 U/ml apyrase and 0.5% DDM (1 h, 4°C, final volume 10 ml, A_2AR concentration 112 nM). After ultracentrifugation, the agonist NECA (10 μM final concentration) was added to one supernatant sample and the inverse agonist ZM241385 (10 μM final concentration) to the other. Washing buffers for A_2AR were buffer C (10 μM NECA, 20 mM Tris-HCl pH8, 100 mM NaCl, 1 mM MgCl₂, 1 mg/mL BSA, 50 mM imidazole, 0.03% DDM) or buffer D (10 μM ZM241385, 20 mM Tris-HCl pH8, 100 mM NaCl, 1 mM MgCl₂, 1 mg/mL BSA, 50 mM imidazole, 0.03% DDM).

(3) 5HT_1BR.

Insect cells expressing 5HT_1BR (50 x 10⁶ cells) were solubilized with buffer containing 10 μM Donitriptan, 20 mM Tris-HCl pH8; 500 mM NaCl; 10 mM imidazole, 2 U/ml apyrase and 0.5% DDM (1 h, 4°C, final volume 6 ml, 5HT_1BR concentration 127 nM). Insoluble material was removed by ultracentrifugation (30 min, 4°C, 135,000 xg) and 200 μl of supernatant was then aliquoted per well into a black Ni²⁺-coated 96-well plate. The receptor was allowed to bind via its His tag for 1 h on ice. The supernatant was then aspirated and 200 μl either of GFP-mini-G_o1, GFP-mini-G_s/i1 or GFP-mini-G_s (negative control) at varying concentrations (from 0 to 5 μM) were added and incubated for a further 90 min on ice. The supernatant was then removed by aspiration and each well washed 4 times with buffer E (1 μM Donitriptan, 20 mM Tris-HCl pH8, 100 mM NaCl, 1 mM MgCl₂, 1 mg/mL BSA, 50 mM imidazole, 0.03% DDM). Elution was carried out with 200 μL of buffer E containing 300 mM imidazole. ΔF data (fluorescence G_o1 condition minus fluorescence G_s condition) corresponding to specific binding were analysed by non-linear regression using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA) and apparent K_D values derived from one site-specific binding analysis.

(1) A_2AR.

Membranes from Trichoplusia ni cells expressing wild-type human A_2AR were resuspended in T_m buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl₂) and homogenized by ten passages through a 26G needle. Mini-G protein was added at a final concentration of 25 μM. ³H-NECA and unlabeled NECA, prepared in assay buffer containing 2 U/ml apyrase, were mixed in a molar ratio of 1:5 and added to the membranes to give a final concentration of 1 μM (approximately ten-fold above the apparent K_D). The samples were incubated at room temperature for 1 h, then chilled on ice for 30 min. Decylmaltoside (DM) was added to a final concentration of 0.13%, and samples were incubated on ice for 1 h. Cell debris and insoluble material were removed by centrifugation (5 min, 20,000 xg, 4°C) and the supernatant was aliquoted (120 μl) into PCR strips. Samples were heated to the desired temperature for exactly 30 min, then quenched on ice for 30 min. Samples (50 μl) were loaded onto gel-filtration resin (Toyopearl HW-40F) packed into a 96-well filter plate (Millipore), which was centrifuged to separate receptor-bound from free radioligand [31]. Nonspecific binding was determined in the presence of 200 μM unlabelled NECA. Radioactivity was quantified by liquid scintillation counting using a MicroBeta TriLux scintillation counter (PerkinElmer). Data were analysed by nonlinear regression using GraphPad Prism software. Apparent T_m values were derived from sigmoidal dose-response analysis performed by non-liner regression. Results represent the mean ± SEM of two independent experiments, performed in duplicate.

(2) NTSR1.

Cell pellets from 10 ml of insect cell cultures were resuspended in 1.8 ml buffer containing DDM to give a final buffer composition of 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl2, 1% (w/v) DDM. The samples were placed on a rotating mixer at 4°C for 1 hour. Cell debris and non-solubilized material were removed by ultracentrifugation (152,800 xg, 4°C, 30 min), and the supernatant containing detergent-solubilized NTSR1 was used to test for thermal stability in the presence of NTS and mini-G proteins. For thermal denaturation curves, the supernatants were diluted 6.67-fold into assay buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl₂) containing 22.5 μM mini-G protein and 10 nM ³H-NTS and incubated for 1 hour on ice. After addition of apyrase (0.25 units/ml, NEB), the sample was placed on ice for an additional 30 min. Samples (120 μl aliquots) were exposed to different temperatures between 0°C and 60°C for 30 min and placed on ice. Separation of receptor–ligand–mini-G protein complex from free ³H-NTS (100 μl) was achieved by centrifugation-assisted gel filtration (spin assay) using Bio-Spin 30 Tris columns (BioRad), equilibrated with RDB buffer [50 mM Tris-HCl pH7.4, 1 mM EDTA, 0.1% (w/v) DDM, 0.2% (w/v) CHAPS, 0.04% (w/v) CHS], essentially as described previously [32]. Control reactions on ice were recorded at the start and at the end of each denaturation experiment. The percentage of activity remaining after heat exposure was determined with respect to the unheated control. Data were analyzed by nonlinear regression using a Boltzmann sigmoidal equation in the Prism software (GraphPad).

(3) AT₁R.

HEK 293 cells expressing wild type AT₁R were resuspended in a radioligand binding assay buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% BSA, 40 μg/ml bacitracin) and homogenized by sonication (4 sec pulse). Mini-G protein and apyrase were added at a final concentration of 25 μM and 0.1 U/ml, respectively. ¹²⁵I-Ang II and unlabeled Ang II were added at a concentration of 0.5 nM and 25 nM respectively (approximately 50 times the apparent K_D value). The sample was incubated at room temperature (20°C) for an hour, chilled on ice for 10 minutes and then digitonin was added to a final concentration of 1% and incubated on ice for an hour. Insoluble material was removed by centrifugation (2 min, 20,000 xg, 4°C). The reaction mix was split into a number of 115 μl aliquots and each was incubated at various temperatures for exactly 30 minutes. The reactions were then quenched on ice for 5 minutes. ¹²⁵I-Ang II bound to AT₁R was separated from unbound ¹²⁵I-Ang II using centrifugation-assisted gel filtration column, essentially as described previously [29]. Non-specific binding was determined using a 500-fold excess of cold ligand. Radioactivity was measured using liquid scintillation counting. Data was analysed by non-linear regression using GraphPad prism software and apparent T_m values were derived by non-linear regression of the sigmoidal dose-response curve.

(i) Agonist affinity shift assay.

The development of mini-G_s relied on the agonist affinity shift assay to identify those mutants that coupled to β₁AR [24]. It is generally considered that the defining feature of G protein coupling is an increase in the affinity of an agonist for the G protein–GPCR complex compared to the GPCR alone. For example, wild type β₂AR binds an agonist 100-fold more tightly when coupled to a G protein than the receptor alone [34]. However, the shift in agonist affinity in other receptors is often considerably smaller than that observed for β₂AR, such as the 10-fold shift in agonist affinity observed in β₁AR [34]. However, the advantage of this assay is that it can be performed using standard pharmacological procedures in high-throughput, using receptors in either membrane preparations or solubilized in detergent. Assays may use either a radiolabelled agonist in saturation binding experiments or, more usually, a radiolabelled antagonist in competition binding experiments [20, 24] (and see experiments below on the serotonin 5HT_1B receptor). The advantage of this assay is that it is very sensitive and can be performed on membrane-bound receptors i.e. in a format where the receptor is most stable in all conformations. The disadvantage of this assay is that it requires suitable radioligands for the receptor under study and these are not readily available for all GPCRs.

(ii) Thermostability assay.

The thermostability of a detergent-solubilised GPCR depends upon the type of detergent used and whether the receptor is either ligand-free, agonist-bound or antagonist-bound [35, 36]. In addition, the receptor stability tends to be increased by an increase in affinity and/or decrease in the off-rate of the ligand [37]. Often, the agonist bound state is one of the least stable conformations of a receptor, presumably because agonists increase the probability of transitions to a fully active state. In the inactive state there is close packing of the intracellular surface of the transmembrane α-helices. Upon activation, the outward movement of helices 5 and 6 disrupts this close packed structure and creates a crevice where the C-terminus of the G protein binds, thus allowing G protein coupling [38]. The structures of non-rhodopsin GPCRs in the fully active state have been determined only when they have been stabilized through binding of a heterotrimeric G protein [2], a conformation-specific nanobody [15, 18, 19] or a mini-G protein [20]. The interface between a GPCR and a G protein is over 1000 Ų [2, 20], and is therefore predicted to increase the thermostability of the agonist-bound GPCR–G protein complex compared to the agonist-bound GPCR. This was observed for both β₁AR and A_2AR, which were consistently more stable in the agonist-bound state when coupled to mini-G_s in a variety of different detergents compared to when mini-G_s was absent [20, 24].

A typical thermostability assay measures how much of a radiolabelled agonist remains bound to a detergent-solubilised receptor after heating at different temperatures for 30 minutes [36]. The advantage of this assay is that it is fast and high-throughput and can be performed in any detergent of choice. Another advantage is that the agonist–GPCR–mini-G protein complex can be pre-formed in membranes, which may stabilise the receptor upon detergent solubilisation. If there is a shift in thermostability in the presence of a mini-G protein, then this is strongly suggestive of binding or coupling.

(iii) Fluorescence-detection size exclusion chromatography (FSEC).

FSEC is a rapid methodology for assessing whether a membrane protein fused to GFP is stable in detergent by performing SEC on an unpurified detergent solubilisate and monitoring GFP fluorescence in the eluate [39]. A membrane protein stable in detergent gives a symmetrical peak at a size consistent with the molecular weight of the membrane protein plus the mass of specifically bound detergent and lipid. By fusing GFP to the N-terminus of mini-G proteins (S3 Fig), it was possible to use FSEC to monitor whether a stable complex was formed between the mini-G protein and a GPCR. The GFP-mini-G_s fusion protein has a molecular weight of 54 kDa and migrated with a retention volume of 15.1 ml on FSEC. When this was mixed with either unpurified DDM-solubilised β₁AR or A_2AR in the presence of an agonist, then an additional peak was observed at 12.1–12.5 ml (Fig 3A and 3C), which was consistent with the molecular weight of the detergent-solubilised receptor bound to GFP-mini-G_s (~180 kDa). This additional peak was not observed if the receptors were bound to an inverse agonist. An additional peak was sometimes observed at a retention volume of 8 ml, which corresponds to the void volume of the SEC column and was due presumably to aggregates of GFP-mini-G_s.

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Fig 3. The β₁AR−mini-G_s and A_2AR−mini-G_s complexes.

(a) FSEC traces of GFP-mini-G_s with β₁AR (retention volumes are given in parentheses): green, GFP-mini-G_s (15.1 ml); orange, GFP-mini-G_s with β₁AR bound to the inverse agonist ICI118551 (15.1 ml); black, GFP-mini-G_s with β₁AR bound to the agonist isoprenaline (8 ml, 12.1 ml and 15.1 ml). Representative chromatograms from at least two independent experiments are shown. (b) Measurement of GFP-mini-G_s affinity to DDM-solubilized β₁AR using a fluorescent saturation binding assay (FSBA); blue circles, β₁AR bound to the agonist isoprenaline (total binding); orange squares, β₁AR bound to the inverse agonist ICI118551 (non-specific binding); green triangles, specific binding, with an apparent K_D of 200 ± 1 nM (mean ± SEM, n = 2). Curves shown are from a representative experiment. (c) FSEC traces of GFP-mini-G_s with DDM-solubilised A_2AR (retention volumes are given in parentheses): green, GFP-mini-G_s (15.1 ml); orange, GFP-mini-G_s with A_2AR bound to the inverse agonist ZM241385 (15.1 ml); black, GFP-mini-G_s with A_2AR bound to the agonist NECA (12.5 ml and 15.1 ml). Representative chromatograms from at least two independent experiments are shown. (d) Measurement of mini-G_s affinity to DDM-solubilized A_2AR using FSBA: blue circles, A_2AR bound to the agonist NECA (total binding); orange squares, A_2AR bound to the inverse agonist ZM241385 (non-specific binding); green triangles, specific binding, with an apparent K_D of 430 ± 24 nM (mean ± SEM, n = 2). (e) Analytical size exclusion chromatography (SEC) of mini-G_s bound to purified A_2AR (retention volumes are given in parentheses): black, A_2AR–mini-G_s complex, 153 kDa (13 ml); blue, A_2AR, 133 kDa (13.3 ml); green, mini-G_s, 22 kDa (17.2 ml). Three panels to the right of the SEC traces are coomassie blue-stained SDS-PAGE gels of fractions from 3 separate SEC experiments: top panel, mini-G_s; middle panel, A_2AR; bottom panel, mini-G_s mixed with NECA-bound A_2AR (1.2:1 molar ratio).

https://doi.org/10.1371/journal.pone.0175642.g003

The advantage of this assay is that it is a quick assessment of whether a GPCR forms a complex with a mini-G protein, because the receptor does not need to be purified and the SEC experiment takes under an hour. However, a major limitation is that only small amounts of GFP-mini-G_s can be used per experiment to avoid saturation of the detector and producing a very broad peak that would obscure the presence of the complex between the GPCR and GFP-mini-G protein. An additional limitation is the amount of membranes that can be solubilized efficiently using 0.5% DDM (to ensure maximal stability of mini-G_s; see below) and maintaining the small volume (500 μl) required for loading on the FSEC column (24 ml). Thus in the data shown (Fig 3A and Fig 3C) the amount of receptor is limiting and only a proportion of the GFP-mini-G_s is bound to receptor and is shifted in apparent molecular weight. In addition, the receptor–mini-G protein complex must be detergent-stable for a peak to be observed. Many GPCR–G protein complexes are too unstable to be observed in DDM and therefore it is essential to assess milder detergents such as LMNG (see section on the serotonin 5HT_1B receptor).

(iv) Fluorescence-based saturation binding analysis of mini-G protein binding.

To determine the affinity of mini-G protein binding to a receptor, the fluorescence-based saturation binding assay (FSBA) was developed. In this assay, the amount of the GFP-mini-G protein specifically bound to an immobilized receptor was determined using a fluorescent plate reader. As proof of principle, DDM-solubilized β₁AR or A_2AR were immobilized onto Ni²⁺-coated wells of a 96-well plate via their C-terminal poly-histidine tag, in the presence of either an agonist or inverse agonist. GFP-mini-G_s was then added at increasing concentrations. After washing to remove any non-specifically bound GFP-mini-G_s, the amount of GFP-mini-G_s fluorescence was measured (Fig 3B and 3D). GFP-mini-G_s showed a specific saturated binding to the receptor with apparent K_D values of 200 ± 1 nM (n = 2) and 430 ± 24 nM (n = 2) for GFP-mini-G_s binding to β₁AR and A_2AR, respectively.

The FSBA is a simple assay for determining the affinity of mini-G protein binding to a receptor in vitro. However, it must be appreciated that the apparent affinity determined may be specific only for the conditions in the assay. In particular, the type of detergent used may have a profound effect on the affinity, especially if it slightly destabilizes the active state of the receptor. The agonist may also affect the apparent affinity of the mini-G protein, depending on how effective the agonist is in stabilizing the active state of the receptor. However, the FSBA remains a useful tool for biophysical analyses of mini-G protein binding to a receptor.

(v) Size exclusion chromatography (SEC).

The ultimate biochemical assay for observing coupling of mini-G proteins to a receptor is combining the purified components in vitro and then observing the co-elution of the relevant proteins on SEC [24]. Purified A_2AR and purified mini-G_s were mixed at a molar ratio of 1:1.2 in the presence of the agonist NECA, the complex allowed to form and then separation was performed by SEC. The A_2AR–mini-G_s complex resolved as a predominant peak with an apparent molecular weight of 153 kDa compared with 133 kDa for the receptor alone and 22 kDa for mini-G_s alone. SDS-PAGE analysis confirmed the presence of both A_2AR and mini-G_s in fractions from the 153 kDa complex (Fig 3E).

The advantage of using purified components and SEC for analyzing complex formation is that complex formation is observed unambiguously. The conditions for complex formation can be refined and the stability of the complex can be assessed readily after a period of days by repeating the SEC. These data are essential for successful determination of the structure of a GPCR–mini-G protein complex. The disadvantage of this assay is that sufficient quantities of purified receptor are required and this may be limiting in the initial stages of a project. The limitation of the amount of purified receptor required for SEC may be partially overcome by analyzing complex formation using FSEC (see section on 5HT_1BR).

Mini-G_olf couples and stabilizes A_2AR.

The GTPase domains of G_olf and G_s share 87% sequence identity (80% for the full length α subunits) and both G proteins couple to A_2AR [40]. Of the 17 amino acid residues in mini-G_s that make direct contact to residues in A_2AR in the crystal structure of the A_2AR–mini-G_s complex [20], all of these residues are identical except that two Arg residues in G_s are replaced with two Lys residues in G_olf. Despite the high degree of sequence homology between these two isoforms, Gα_olf is far more difficult to overexpress than Gα_s, in fact, the only method reported to produce functional Gα_olf is co-expression with the molecular chaperone RIC8B in insect cells [41]. Therefore, we constructed mini-G_olf to investigate whether the mini-G protein version would be better expressed that native α subunit. Mini-G_olf was constructed by transferring the 7 point mutations and 3 deletions from mini-G_s (Fig 2) and mini-G_olf was highly expressed in E. coli and as stable as mini-G_s. The coupling of mini-G_olf to A_2AR was assessed by SEC of the complex assembled in vitro from purified proteins and a thermostability assay [20, 24]. Purified NECA-bound A_2AR was mixed with mini-G_olf and analysed by SEC and SDS-PAGE (Fig 4A). The apparent molecular weight of mini-G_olf was 23 kDa (17.1 ml; theoretical molecular weight 26 kDa) and the apparent molecular weight of purified A_2AR in DM was 133 kDa (13.3 ml). The complex A_2AR–mini-G_olf resolved as a predominant peak with an apparent molecular weight of 153 kDa (13 ml) and contained both A_2AR and mini-G_olf. Mini-G_olf also stabilized agonist-bound DM-solubilised A_2AR, with mini-G_olf-coupled A_2AR showing an apparent T_m of 32 ± 1°C in comparison with 27 ± 0.3°C for the receptor alone (Fig B). This stability was similar to that obtained with mini-G_s (33°C) under the same conditions [20].

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Fig 4. The A_2AR−mini-G_olf complex.

(a) Analytical SEC of mini-G_olf bound to purified A_2AR (retention volumes are given in parentheses): black, A_2AR–mini-G_olf complex, 153 kDa (13 ml); blue, A_2AR, 133 kDa (13.3 ml); green, mini-G_olf, 23 kDa (17.1 ml). Three panels to the right of the SEC traces are coomassie blue-stained SDS-PAGE gels of fractions from 3 separate SEC experiments: top panel, mini-G_olf; middle panel, A_2AR; bottom panel, mini-G_olf mixed with NECA-bound A_2AR (1.2:1 molar ratio). (b) Thermostability assay (TSA) of unpurified DM-solubilized, ³H-NECA-bound A_2AR. Data were analysed by nonlinear regression and apparent T_m values were determined from analysis of the sigmoidal dose-response curves fitted. T_m values represent mean±SEM of two independent experiments, each performed in duplicate: blue circles, no mini-G_olf (27 ± 0.3°C); green squares, mini-G_olf (33 ± 1°C). Curves shown are from a representative experiment.

https://doi.org/10.1371/journal.pone.0175642.g004

The results with mini-G_olf were very encouraging in terms of both the transferability of the mutations, the expression and stability of the mini-G_olf and the stability of the A_2AR–mini-G_olf complex. Thus where there is a high degree of homology between G proteins, then there is good transferability of the mutations, as was previously observed for the transfer of thermostabilising mutations between GPCRs [42]. These data also suggested that even if the native α subunit is poorly expressed the mini-G protein version may be highly expressed and very stable.

Development of chimeric mini-G_s/q to study G_q-coupled receptors.

The expression of mini-G_q in E. coli was unsuccessful. One possibility to explain this is that efficient folding of G_q in vivo is dependent on the molecular chaperone Ric8 [33] and that mini-G_q had a similar requirement. Indeed, co-expression of Ric8 with mini-G_q in the baculovirus expression system led to the overproduction of mini-G_q. However, upon purification of mini-G_q it was not possible to dissociate Ric8 (results not shown), suggesting that the mini-G_q was perhaps not correctly folded and/or was very unstable. Given the lack of success in transferring the mini-G protein mutations from G_s to G_q, another strategy was developed.

The second strategy used to try and develop mini-G_q was to transfer the specificity determinants of G_q onto mini-G_s. It is well established that the C-terminal region of a Gα subunit forms the main receptor binding site [43] and is one of the main determinants of coupling specificity [44, 45]. Mutating as few as 3–5 amino acids at the C-terminus of the G alpha subunit has been shown to switch the specificity of coupling to some GPCRs [44, 45]. However, the two GPCR–G protein structures published to date [2, 20] revealed an extensive interface between the receptors and Gα, suggesting that other regions of the G protein may also play a role in specificity. Recent in vivo FRET studies suggest that residues within the α5 helix, but distal to the five C-terminal residues, strongly influence specificity [46].

Mini-G_s did not couple to any of the G_q-coupled receptors tested (results not shown). We then evaluated a number of mini-G_s/q chimeras (S4 Fig) for both gain of binding to G_q-coupled receptors (Fig 5A and 5B) and loss of binding to the cognate G_s-coupled receptor A_2AR, predominantly using thermostability assays (Fig 5C) and SEC (S5 Fig). First, the chimera mini-G_s/q57 was constructed in which the five C-terminal amino acids of mini-G_s (Q^H5.22YELL^H5.26) were changed to those found in Gα_q, which required three mutations (Q390E^H5.22, E392N^H5.24 and L394V^H5.26). There was no increase in thermostability of agonist-bound AT₁R or NTSR1 in the presence of mini-G_s/q57 that might suggest coupling (Fig 5A and 5B). In fact, there was a decrease in thermostability of AT₁R in the presence of mini-G_s/q57, suggesting that binding had occurred, but that residues of G_s were clashing with the receptor and causing its destabilisation. Furthermore, a complex between mini-G_s/q57 and A_2AR was still observed (Fig 5C and S5 Fig), suggesting that the mutations were insufficient to change the specificity of G_s to G_q. Therefore, the chimera mini-G_s/q58 was constructed in which the final 19 amino acid residues in the α5 helix of mini-G_s (Phe376^H5.8—Leu394^H5.26) were changed to those in Gα_q; this required 13 mutations (N377A^H5.9, D378A^H5.10, C379V^H5.11, R380K^H5.12, I382T^H5.14, Q384L^H5.16, R385Q^H5.17, M386L^H5.18, H387N^H5.19, R389K^H5.21, Q390E^H5.22, E392N^H5.24 and L394V^H5.26). Mini-G_s/q58 did not couple to A_2AR (Fig 5C and S5 Fig), demonstrating that residues in the α5 helix beyond the C-terminal 5 amino acids are important in G protein specificity. However, there was no significant shift in the thermostability of the G_q-coupled receptor NTSR1 in the presence of mini-G_s/q58 (Fig 5B). We reasoned that this may be because the stability of mini-G_s/q58 was impaired, because mutating the last 19 amino acid residues in mini-G_s would have also changed residues buried in the core of the G protein, thus affecting the stability of the mini-G_s backbone. Therefore, a refined version of this chimera, mini-G_s/q70, was constructed in which residues in the α5 helix whose side chains formed direct contacts (3.9 Å cut-off) with either β₂AR [2] or A_2AR [20] in the G protein-bound structures were mutated to match those in Gα_q (R380K^H5.12, Q384L^H5.16, R385Q^H5.17, H387N^H5.19, E392N^H5.24 and L394V^H5.26; S4 Fig). In addition, the mutation Q390E^H5.22 was included, despite only making contact to A_2AR via its backbone, as it is buried in the receptor–G protein interface and may be important for binding to G_q-coupled receptors. Mini-G_s/q70 gave better binding to both G_q-coupled receptors tested, NTSR1 and AT₁R, and showed no binding to A_2AR (Fig 5 and S5 Fig).

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Fig 5. Thermostability assays of various complexes between mini-G_s/q chimeras and GPCRs.

(a) Thermostability of unpurified digitonin-solubilized, ¹²⁵I-AngII-bound AT₁R (T_m values in parentheses): blue circles, no mini-G_s/q (23 ± 0.4°C); green squares, mini-G_s/q57 (T_m not determined); magenta inverted triangles, mini-G_s/q70 (31 ± 1°C); red triangles, mini-G_s/q71 (30 ± 0.8°C). (b) Thermostability of unpurified DDM-solubilized, ³H-NTS-bound NTSR1: blue, no mini-G_s/q (25 ± 0.4°C); green squares, mini-G_s/q57 (27 ± 0.7°C); black circles, mini-G_s/q58 (25 ± 0.4°C); magenta inverted triangles, mini-G_s/q70 (32 ± 0.3°C); red diamonds, mini-G_s/q71 (29 ± 1.1°C). (c) Thermostability of unpurified DM-solubilized, ³H-NECA-bound A_2AR: blue circles, no mini-G_s/q (27 ± 0.3°C); green squares, mini-G_s/q57 (31 ± 0.3°C); black circles, mini-G_s/q58 (27 ± 0.5°C); magenta inverted triangles, mini-G_s/q70 (27 ± 0.2°C). In all panels, data (n = 3) were analysed by nonlinear regression and apparent T_m values were determined from analysis of the sigmoidal dose-response curves fitted with values shown as mean ± SEM. Curves shown are from a representative experiment.

https://doi.org/10.1371/journal.pone.0175642.g005

Two other chimeras were also constructed to try and improve on mini-G_s/q70. Mini-G_s/q72 contained the additional mutation C379V^H5.11 compared to mini-G_s/q70 and, although the C379^H5.11 side chain does not form direct contacts with either A_2AR or β₂AR, its mutation to Val is predicted to introduce a direct interaction between the Val γ2 carbon and Leu110 from A_2AR. However, the AT₁R–mini-G_s/q72 complex did not have a higher thermostability than AT₁R–mini-G_s/q70 (results not shown). Finally, the chimera mini-G_s/q71 was constructed in which residues from other regions of Gα that form direct contacts with either β₂AR [2] or A_2AR [20] were mutated to match those in Gα_q. This included the seven mutations in mini-G_s/q70 (R380K^H5.12, Q384L^H5.16, R385Q^H5.17, H387N^H5.19, Q390E^H5.22, E392N^H5.24 and L394V^H5.26) and six additional mutations (A39R^HNS1.3, H41L^S1.2, D343K^H4.23, L346V^H4.26, R347D^H4.27 and Y358I ^H4S6.11). D343^H4.23 was the only amino acid residue whose side chain did not interact with either A_2AR or β₂AR, but the mutation to Lys was included because the longer side chain could potentially interact with a receptor and the charge reversal may be important for specificity. Conversely, Thr350^H4S6.3 was not mutated to Pro in mini-G_s/q71 even though its side chain forms direct contacts with β₂AR. Alignment of Gα_s with two independently solved structures of Gα_q [47, 48] showed that this region of the G proteins differ significantly and thus, in Gα_q, this residue is unlikely to interact with the receptor. However, after all these considerations to make an improved version of mini-G_s/q70, mini-G_s/q71 did not improve the thermostability of agonist-bound G_q-coupled receptors compared to mini-G_s/q70 (Fig 5A and 5B).

Mini-G_i1: Tackling stability issues.

Transfer of the 7 point mutations and 3 deletions from mini-G_s into Gα_i1 to make mini-G_i1 was not successful, as the resultant protein was very poorly expressed and had low stability (results not shown). Whilst the work on developing chimeras of mini-G_s/q was underway, we decided to first study the reasons why mini-G_i1 appeared to be so unstable. Therefore, to improve expression, stability and to allow binding of the mini-G_i1 to the βγ subunits, the N-terminus (residues 4–18) was re-inserted, Asp249^H3.8 was mutated back to Leu, and the G217D^H2S4.3 mutation introduced based on a sequence comparison between G_i1 (poorly expressed) and G_s/G_o (highly expressed) (Fig 2 and S1 Fig). The resultant mini-G_i1 (construct 46) yielded only 12 mg of purified protein per litre of culture and was 17°C less stable than mini-G_s, but was suitable for initial studies in GPCR coupling.

The serotonin 5HT_1B receptor (5HT_1BR) was used as a model G_i-coupled receptor for developing mini-G_i1 because it could be expressed and purified in DDM using the baculovirus expression system and its structure determined in the inactive state. Initially, GFP-mini-G_i1 was tested using FSEC for binding to purified 5HT_1BR (in DDM) and bound to the agonist donitriptan. However, the GFP-mini-G_i1 (S3 Fig) migrated at 13.5 ml in the absence of receptor or in the presence of donitriptan-bound 5HT_1BR, indicating that no coupling occurred (Fig 6C). However, when the LMNG-purified 5HT_1BR was used, the FSEC showed two peaks, one corresponding to free GFP-mini-G_i1 with a retention volume of 14.3 ml and the other corresponding to GFP-mini-G_i1 bound to donitriptan-activated 5HT_1BR, with a retention volume of 12.2 ml (Fig 6D). The presence of two peaks, suggested that mini-G_i1 was not stable in detergent when in a complex with 5HT_1BR. The detergent instability of the complex could also have been due to the receptor, but because agonist-bound 5HT_1BR had been crystallised previously [10], we felt this was unlikely. In order to improve the stability of mini-G_i1, the heterotrimer was formed between GFP-mini-G_i146 (S3 Fig) and β₁γ₂ and tested by FSEC. The GFP-mini-trimer in complex with the LMNG-purified 5HT_1BR resolved as a single peak with a retention volume of 11.8 ml compared to 14.3 ml for the free GFP-mini-G_i1β₁γ₂ trimer (Fig 6F). Thus the β₁γ₂ subunits restored the stability of mini-G_i1.

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Fig 6. The 5HT_1BR−mini-G_i1 complexes.

(a) Mini-G_i1 coupling increases agonist affinity to 5HT_1BR. Competition binding curves were performed in duplicate (n = 2) by measuring the displacement of the antagonist ³H-GR125743 with increasing concentration of the agonist sumatriptan (K_i values representing mean ± SEM in parentheses): blue circles, 5HT_1BR (K_i 280 ± 10 nM); green hexagons, 5HT_1BR and mini-G_i1 (K_i 80 ± 13 nM); brown squares, 5HT_1BR and mini-G_s/i1 (K_i 36 ± 2 nM); pale blue triangles, 5HT_1BR and mini-G_i1β₁γ₂ (K_i 15 ± 1 nM); red diamonds, 5HT_1BR and mini-G_s/i1β₁γ₂ (K_i 7.2 ± 0.8 nM). Error bars represent the SEM. (b) Measurement of mini-G_s/i1 chimera affinity to the DDM-solubilized, donitriptan-bound 5HT_1BR using FSBA: brown circles, 5HT_1BR and GFP-mini-G_s/i1 (total binding); purple squares, 5HT_1BR and GFP-mini-G_s (non-specific binding); green triangles, specific binding. The apparent K_D of 390 ± 47 nM represents the mean ± SEM of two independent experiments. Curves shown are from a representative experiment. (c) FSEC traces of GFP-mini-G_i1 with 5HT_1BR in DDM: black, GFP-mini-G_i1 and donitriptan-bound 5HT_1BR purified in DDM (13.5 ml); green GFP-mini-G_i1 (13.5 ml). (d) FSEC traces of GFP-mini-G_i1 with 5HT_1BR in LMNG: black, GFP-mini-G_i1 and donitriptan-bound 5HT_1BR purified in LMNG (12.2 ml and 14.3 ml); green, GFP-mini-G_i1 (14.3 ml). (e) FSEC traces of GFP-mini-G_s/i1 with 5HT_1BR: black, GFP-mini-G_s/i1 and donitriptan-bound 5HT_1BR purified in DDM (13.2 ml); brown, GFP-mini-G_s/i1 (15.1 ml). (f) FSEC traces of GFP-mini-G_i1β₁γ₂ with 5HT_1BR: black, GFP-mini-G_i1β₁γ₂ and donitriptan-bound 5HT_1BR purified in LMNG (11.8 ml); pale blue, GFP-mini-G_i1β₁γ₂ (14.3 ml). In panels c-f, retention volumes are given in parentheses.

https://doi.org/10.1371/journal.pone.0175642.g006

Although the mini-G_i1β₁γ₂ trimer coupled successfully to LMNG-solubilised 5HT_1BR, this is not as desirable for crystallography as a mini-G protein coupled receptor due to the large size of the heterotrimeric G protein. Therefore, following the successful strategy of changing the coupling of mini-G_s to that of G_q by making a mini-G_s/q chimera, the same strategy was applied to engineer a mini-G_s/i1 chimera (S1 Fig and S6 Fig). Therefore 9 mutations (C379V^H5.11, R380T^H5.12, Q384I^H5.16, R385K^H5.17, H387N^H5.19, Q390D^H5.22, Y391C^H5.23, E392G^H5.24 and L394F^H5.26) were introduced into the α5 helix of mini-G_s to change its coupling specificity to that of G_i1. A complex between GFP-mini-G_s/i143 (S3 Fig) with donitriptan-bound DDM-purified 5HT_1BR resolved as a single peak with a retention volume of 13.2 ml compared to 15.1 ml for the free GFP-mini-G_s/i1 (Fig 6E). Thus mini-G_s/i1 was indeed more stable than mini-G_i1. The specificity of mini-G_s compared to mini-G_s/i1 for donitriptan-bound, DDM-solubilised 5HT_1BR was confirmed using FSBA (Fig 6B). No specific coupling of GFP-mini-G_s to 5HT_1BR was observed, although specific coupling to GFP-mini-G_s/i1 (apparent K_D 390 nM; Fig 6B) was confirmed.

In order to compare all of the mini-G_i1 constructs and the role of β₁γ₂, agonist affinity shift assays were performed on 5HT_1BR. The uncoupled receptor showed a K_i for the agonist sumatriptan in this assay of 280 ± 10 nM, which was shifted by mini-G_i146 and mini-G_s/i143 to 80 ± 13 nM and 36 ± 2 nM, respectively (Fig 6A). However, addition of β₁γ₂ to the mini-G proteins resulted in a further increase in agonist affinity to 15 ± 1 nM and 7.2 ± 0.8 nM for mini-G_i146-β₁γ₂ and mini-G_s/i143-β₁γ₂, respectively. Thus despite the successful generation of both mini-G_i1 and mini-G_s/i1, their stability is still not perfect as binding of β₁γ₂ stabilises the mini-G proteins and elicits a greater increase in agonist affinity upon coupling of the mini-trimers.

Coupling of mini-G_o1 to 5HT_1BR.

The GTPase domain of G_o1 and G_i1 are highly conserved (80% identity), but the mini-G proteins derived from them behaved very differently. Unlike the unstable mini-G_i1, mini-G_o1 expressed well (100 mg/L), had high stability comparable to mini-G_s and it was largely insensitive to the presence of mild detergents. Since 5HT_1BR couples to both G_o and G_i family members [49], we tested mini-G_o1 coupling to 5HT_1BR and compared the results to coupling with mini-G_i1 (see above). On FSEC, GFP-mini-G_o112 (S3 Fig) partially coupled to donitriptan-bound, DDM-solubilised 5HT_1BR (unpurified), with the higher molecular weight species (retention volume 13 ml) reduced when the receptor was bound to an antagonist (Fig 7C). This was in contrast to the results with mini-G_i1 under the same conditions where no binding was observed (Fig 6C). The partial coupling probably resulted from the low amount of 5HT_1BR used in the assay, because when the experiment was repeated using purified 5HT_1BR and GFP-mini-G_o1, all of the GFP-mini-G_o1 bound to the receptor (Fig 7E). This is consistent with what was observed for β₁AR and A_2AR (Fig 3). The 5HT_1BR–mini-G_o1 complex was purified by SEC and SDS-PAGE indicated co-elution of 5HT_1BR and mini-G_o1 in a 1:1 molar ratio (Fig 7D). GFP-mini-G_o1 bound to DDM-solubilised 5HT_1BR in the presence of donitriptan with an apparent K_D of 180 ± 24 nM (Fig 7B). In membranes, mini-G_o112 shifted the agonist affinity for 5HT_1BR from 280 ± 10 nM to 32 ± 3 nM (Fig 7A).

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Fig 7. The 5HT_1BR−mini-G_o1 complex.

(a) Competition binding curves were performed on membranes in duplicate (n = 2) by measuring the displacement of the antagonist ³H-GR125743 with increasing concentration of the agonist sumatriptan (apparent K_i values representing mean ± SEM are in parentheses): blue circles, 5HT_1BR (K_i 280 ± 10 nM); green squares, 5HT_1BR and mini-G_o1 (K_i 32 ± 3 nM). Error bars represent SEM. (b) Measurement of GFP-mini-G_o1 affinity to DDM-solubilized, donitriptan-bound 5HT_1BR using the FSBA: blue circles, 5HT_1BR and GFP-mini-G_o1 (total binding); purple squares, 5HT_1BR and GFP-mini-G_s (non-specific binding); green triangles, specific binding. The apparent K_D value (180 ± 24 nM) represents mean ± SEM of two independent experiments. Curves shown are from a representative experiment. (c) FSEC traces of GFP-mini-G_o1 with DDM-solubilized unpurified 5HT_1BR bound to the following (retention volumes are shown in parentheses): orange, the antagonist SB224289 (14.9 ml); black, the agonist donitriptan (11.3 ml and 14.9 ml). Free GFP-mini-G_o1 (green) resolved as a predominant peak with a retention volume of 14.9 ml (d) Mini-G_o1 forms a complex with purified 5HT_1BR. The three panels are coomassie blue-stained SDS-PAGE gels of fractions from 3 separate SEC experiments: top panel, mini-G_o1; middle panel, 5HT_1BR; bottom panel, mini-G_o1 mixed with donitriptan-bound 5HT_1BR (1:1 molar ratio). (e) FSEC traces of GFP-mini-G_o1 with purified 5HT_1BR: black, GFP-mini-G_o1 with 5HT_1BR purified in DDM (13 ml); green, GFP-mini-G_o1 (14.8 ml). (f) FSEC traces of GFP-mini-G_s with purified 5HT_1BR: black, GFP-mini-G_s with 5HT_1BR purified in DDM (negative control; 15.1 ml); purple, GFP-mini-G_s (15.1 ml). Retention volumes are shown in parentheses.

https://doi.org/10.1371/journal.pone.0175642.g007

The properties of mini-G_o1 make this an ideal choice for structural studies of G_o/G_i coupled receptors, rather than using mini-G_s/i, as it is more highly expressed and more tolerant of detergents. This is exemplified by the stability of the donitriptan-bound 5HT_1BR–mini-G_o1 complex made from purified components (S7 Fig).