Figure 1.
Summary of experimental strategy.
A, Testing stabilizing effects of detergents, ligands and lipids. •Efficient solubilization of the fusion CB2 protein from E. coli membranes. Over 40 different detergents and mixtures of detergents were compared for their efficiency in solubilizing fusion CB2 from membranes, and the detergent mixture that performed best was selected for a routine receptor purification protocol. •Optimization of the liposome-reconstitution procedure. A screening of lipid-solubilizing detergents was performed with a goal of maximizing the yield of functionally reconstituted receptor. •Application of the G protein activation test to the analysis of the structural stability of CB2 in micelles following its reconstitution into liposomes. •Screening for stabilizers for CB2 and characterization of stability of the purified receptor. (i)Stabilization of CB2 in micelles by CHS, ligands and phospholipids. (ii)Ligand binding studies by solid-state NMR. (iii)Characterization of stability of CB2 in lipid bilayers. B, Comparison of stabilizing effects of two lipids at two different temperatures.
Figure 2.
Recovery of functional CB2 in proteoliposomes prepared from various dominant detergents.
Activity is presented as % of the maximal activity in the series (1% LDAO). The results shown represent data ± S.D. (error bars) of duplicate determinations from single representative experiments (out of three independently performed experiments, n = 3)
Figure 3.
Activation of G proteins by recombinant CB2 in E. coli membranes expressing CB2-130 and in CHO membranes expressing CB2.
4 ng of CB2 was used in the assay and the figure depicts data ± S.D. (error bars) of duplicate determinations from representative experiments (n = 3).
Figure 4.
Functional activity of CB2 in proteoliposomes.
Dependence of functional activity of CB2 on CHS content in detergent micelles and in proteoliposomes. The sample with the highest activity within this series (0.1% CHS in micelles) is shown as 100% of activity. The figure depicts data ± SD (error bars) of duplicate measurements from representative experiments (n = 3).
Figure 5.
Effect of CHS on stability of CB2 in detergent micelles at 4°C and 37°C.
Activity of CB2 recovered from micelles supplemented with 0.1% CHS at 4°C was set as 100%. Figure presents results of a typical experiment (out of a total of 3), each point is an average of two measurements of the same sample with SD as indicated.
Figure 6.
Stabilizing effects of cannabinoid ligands on CB2 in detergent micelles.
Ligands (CP-55,940 or SR-144,528) at concentration 10 µM were introduced into buffers either through an entire purification procedure or just prior to the reconstitution of the purified CB2 into proteoliposomes as indicated. E. coli BL21(DE3) membranes expressing fusion CB2-130 were used as an activity standard. A quantity of 2 µg of E. coli membranes expressing CB2-130 or liposomes containing 6 ng of purified, reconstituted CB2 were used per reaction. Each point represents an average of duplicate measurements ± SD (error bars) of activity of a representative set of proteoliposome preparation (n = 3).
Figure 7.
Effect of concentration of CP-55,940 on stability of CB2 in micelles.
CB2-130 dissolved at 1 µM concentration in DDM/CHAPS/CHS (0.1%/0.5%/0.1% w/v) micelles was incubated in the presence of CP-55,940 (at indicated concentrations) at either at 4°C or 37°C, reconstituted into liposomes and its functional activity analyzed as described in the text. The activity of protein recovered after incubation at 4°C with 30 µM CP-55,940 is set to 100%. Each point represents an average of duplicate measurements ± SD (error bars) of a representative proteoliposome preparation (n = 2).
Figure 8.
Functional activity of CB2 in liposomes.
A, Effect of 2.5 µM CP-55,940 in growth media of E. coli BL21 (DE3) expressing CB2-130 on functional activity of purified and liposome-reconstituted CB2. Purified protein was reconstituted into liposomes containing POPC:POPS:CHS (60∶15:25 w/w/w) at a protein-to-lipid ratio 1∶500, and activity measured in the presence of 2 µM CP-55,940 by the G protein activation assay. Membranes of E. coli BL21 (DE3) expressing CB2-130 fusion protein served as a positive control. Data represents duplicate measurements ± S.D. (error bars) of a representative set of samples (n = 3). 6 ng of the receptor was introduced into the reaction and normalization was performed assuming concentration of CB2 of 3 ng per 1 µg of total protein in the E. coli membrane preparation. The concentration of CB2-130 in membrane preparations was calculated based on a quantitative Western blot probed with anti-CB2 antibody, by comparing intensity of the band of fusion MBP-CB2 with that of known amounts of purified CB2-130 electroblotted onto the same nitrocellulose membrane (not shown). The concentration of CB2 in proteoliposome preparations was determined by fluorescence of Alexa-488-labeled CB2 added at a ratio of 2∶98 (labeled: unlabeled receptor) to purified CB2-130 prior to its reconstitution into liposomes as described in Materials and Methods and in [19]. B, Activation of G proteins by liposome-reconstituted CB2 as a function of ligand concentration. Effects of agonist CP-55,940 and inverse agonist SR-144,528. Purified CB2-130 stabilized with 2.3-fold molar excess of CP-55,940 was reconstituted into POPC/POPS/CHS (60∶15:25) proteoliposomes. The concentration of CB2 in the reaction was 2 nM, and the G protein activation assay performed as described in Materials and Methods. The figure shows duplicate measurements ± S.D. of representative proteoliposome/membrane preparations (n = 3). C, Gαi1 saturation of CB2-catalyzed GDP/35S-γ GTP exchange. 35S-γ GTP binding was measured in reactions containing 1.2 nM of CB2 in E. coli membranes or 1 nM of purified CB2-130 reconstituted into proteoliposomes. The contribution of spontaneous nucleotide exchange at a given Gαi1 concentration estimated in the absence of CB2 was subtracted from total binding. Average of two measurements is presented with S.D. indicated.
Figure 9.
Stabilizing effects of CHS and phospholipids in DDM/CHAPS micelles.
Detergent micelles supplemented with lipids as indicated below the graph were incubated either for 2 hours at 4°C (light-shaded bars) or for 1.5 hours at 4°C followed by 30 min at 37°C (dark-shaded bars). The composition of micelles was adjusted as indicated (above the graph) and the protein reconstituted into proteoliposomes on mini-spin detergent absorbent columns (Pierce). The functional activity was measured by the G protein activation assay as described in Materials and Methods. Purified CB2 reconstituted from CHS/POPC/DDM/CHAPS micelles (final composition of liposomes: CHS/POPC 25∶75 mol/mol) was used as an activity standard. Presented are average values of duplicate measurements ± S.D. (error bars) from representative experiments (n = 3).
Figure 10.
Stabilizing effects of POPC:POPS (1∶1, w/w) in micelles.
A, Lipids were added to DDM/CHAPS (0.1%/0.5%) micelles at concentrations indicated. Upon incubation samples were supplemented with POPC:POPS dissolved in 1% CHAPS so that the final protein-to-lipid ratio was the same in all samples (1∶500 mol/mol). Upon reconstitution on a mini-spin detergent-absorbent columns the activity of CB2 was determined by the G protein activation assay and reported as % of the maximal activity measured for this series of samples. The liposomes-reconstituted receptor (0.1% CHS in micelles, POPC/POPS/CHS 60∶15:25 in liposomes) exhibiting the highest levels of activation in this experiment was used as an activity standard. B, Purified CB2 in DDM/CHAPS/CHS micelles was captured on Ni-NTA, detergent buffer rapidly exchanged to DDM/CHAPS containing 0.4% of lipids of indicated composition, protein eluted with imidazole and liposome-reconstituted by rapid dilution. Functional activity of CB2 reconstituted into POPC/POPS/CHS matrix is set as 100% of activity. Figures depict data ± SD (error bars) of duplicate determinations from representative experiments (n = 2-3).
Figure 11.
Stability of CB2 in lipid bilayers.
A, Temperature-induced unfolding of CB2 in detergent micelles and lipid bilayers. For stability studies in micelles the purified CB2-130 in TD buffer supplemented with 10 µM CP-55,940 was subjected to a temperature gradient from 4°C to 74°C at a rate of 1°C/min, 10 µg protein samples withdrawn at indicated time points, mixed with 100 µg lipids POPC/POPS (4∶1 w/w) in 1% CHAPS and diluted 110-fold into cold 10 mM MOPS buffer under vigorous stirring. The activity of CB2 was analyzed by measuring the G protein activation rates as described in Materials and Methods. For measurement of thermostability in lipid bilayers either CB2-proteoliposomes or membrane preparations harboring fusion CB2-130 were suspended in 10 mM MOPS buffer at a concentration of CB2 0.5 ng/µL, subjected to treatment with linear temperature gradient, and analyzed by G protein activation assay. Dotted line depicts the temperature gradient profile. Figure depicts data ± S.D (error bars) of duplicate measurements from representative experiments (n = 3). B, Temperature stability of CB2 in proteoliposomes and E. coli membranes. Either purified CB2 receptor reconstituted into POPC:POPS:CHS bilayers or fusion CB2-130 in E. coli membranes was incubated for 30 min at the temperatures indicated, and the G protein activation assay performed. 4 ng of CB2 was used in every reaction and measurements were performed upon addition of 2 µM of CP-55,940 to all samples. Data ± S.D. (error bars) of duplicate measurements from representative experiments (n = 3) are presented.
Table 1.
Stabilization of CB2 (summary). Expression and purification of functional CB2.
Table 2.
Reconstitution of purified CB2 into proteoliposomes.
Table 3.
Stabilization of CB2 in DDM/CHAPS micelles.
Table 4.
Stability of CB2 in liposomes and in E. coli membranes.