Supporting Information for High-throughput production and structural characterization of libraries of self-assembly lipidic cubic phase materials Authors: Connie Darmanin, Charlotte E. Conn, Janet Newman, Xavier Mulet, Shane Seabrook, Yi-Lynn Liang, Adrian Hawley, Nigel Kirby, Joseph N. Varghese, Calum J. Drummond Supporting Methods GPCR Expression and Purification Human Histamine H1 receptor (H1R) carrying a 10xhistidine-tag on its C-terminus has been expressed using the insect cell (Spodoptera frugiperda, Sf9) baculovirus expression system. H1R was expressed and purified using a modified version of an established protocol.1 Briefly, the Sf9 cells were infected with pFacBac-H1RHis10 and production tests were routinely performed at different days post infection using ligand-binding assays. Generally, after 72 hrs post infection, the cells were harvested and the cell pellet was resuspended in a buffer containing 50 mM NaHEPES pH 8, 0.1 mM EDTA pH 8.0, 3 mM MgCl2 and protease inhibitors (1 µg/ml DNase, 2.5 µg/ml RNase, 2 µg/ml E64, 5 µM Leupeptin). After sonication of the cells and a series of centrifugation steps, the membrane sample was isolated. The membrane pellet was resuspended in 20 mM Bis-Tris Propane, 1 M NaCl, 1 mM Histidine, 10 % Glycerol, final pH 7.6, containing 2 µg/ml E64, 5 µM Leupeptin and antagonist (2 µM triplenamine). H1R was detergent solubilized using 20 mM n-octyl-β -Dglucopyranoside (nOG). The soluble protein was purified using metal affinity chromatography, which yielded pure protein after elution with imidazole.
The human Dopamine 2 long receptor (D2L) was expressed and purified based on the H1R protocol. The D2L carrying a 6xhistidine-tag on its C-terminus has been expressed using the insect cell, Sf21, baculovirus expression system. The Sf21 cells
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were infected with pFacBac-D2LHis6 and production tests were routinely performed at different days post infection using ligand-binding assays. Generally, after 24 hrs post infection, the cells were harvested and the cell pellet was resuspended in a buffer containing 50 mM NaHEPES pH 7.4, 0.1 mM EDTA pH 8.0, 3 mM MgCl2 and protease inhibitors (1 µg/ml DNase, 2.5 µg/ml RNase, 2 µg/ml E64, 5 µM Leupeptin). After sonication of the cells and a series of centrifugation steps, the membrane sample was isolated. The membrane pellet was resuspended in 20 mM NaHEPES, 1.5
M
NaCl, 10% glycerol and 1 mM EDTA pH 7.4 and 10% Glycerol, final pH 7.4, containing 2 µg/ml E64, 5 µM Leupeptin and an agonist (5 µM butaclamol). D2L was detergent solubilized using 0.8% PMAL-C12 detergent (Affymetrix) in buffer containing 20 mM Bis Tris Propane, 1 M NaCl, 10% glycerol, 2 µg/ml E64 and 5µM butaclamol, pH 7.7. The soluble protein was purified using metal affinity chromatography, which yielded pure protein after elution with imidazole.
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Supporting Results: Average D
QII lattice
Standard
parameter
Deviation
(Å)
(Å)
MO
99.9
0.8
36
MO 4% chol
96.9
1.2
20
MO 8% chol
99.2
1.8
20
MO 12% chol
102.1
2.7
20
MP
110.0
1.3
10
MP 8% chol
106.4
1.2
20
MV
107.0
1.1
20
MV 8% chol
119.3
2.4
10
No. of samples
Table S1. The average lattice parameter obtained for multiple wells containing a range of lipids and lipid mixtures (monoolein (MO), monopalmitolein (MP), monovaccenin (MV) and cholesterol (chol)).
Comparison of the phase behaviour of MO 40% (w/w) H2O samples produced via conventional, robotic, and syringe-based preparation methods We compare the observed phase behaviour for MO samples 40% (w/w) H2O produced robotically as described here, with the phase behaviour of similar samples produced using coupled syringes2, and using the conventional method of cubic phase production in vials. The coupled syringe method has been used previously by us, and others, to produce cubic phase for in meso crystallisation trials.3, 4 Dry lipid is added to one syringe and an appropriate volume of water or protein solution to the other, and the sample mixed by joining the two syringes together through a central coupler and pushing the samples back and forth. With the conventional method of production of bulk cubic phase, dry lipid is weighed directly into a vial and an appropriate volume of water is added. The vial is then sealed and the sample allowed to equilibrate for a
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period of time, typically not less than several days.
Samples produced robotically in
plates were analysed at 28 °C and so we compare with the phase behaviour of alternative production methods in a similar temperature range.
The published phase diagram for MO, produced using a syringe-based mixing device,2 indicates that, in this temperature range, the phase boundary between the gyroid and the diamond cubic phases lies very close to the studied water content of 40% (w/w). At 25 °C, co-existing gyroid (161.5 Å) and diamond (102.1 Å) cubic phases were observed at 36% (w/w) H2O, with the formation of a pure diamond cubic phase (101.8 Å) at 43.6 % (w/w) H2O. As the temperature is increased, the QIIG / QIID phase boundary shifts to lower water content; at 30 °C a pure diamond phase is observed at 36.1% (w/w) H2O. Due to the proximity to the QIIG/ QIID phase boundary, small differences in water content between different samples can result in a phase change within the bulk material.
Using a syringe-based mixing device, MO made up to a nominal water content of 40% (w/w) (calculated as 39.0 % (w/w)) adopted a QIIG phase at 25 °C and 30 °C (calculated lattice parameters of 154.5 Å and 153.3 Å, respectively). The observed QIIG phase is consistent with the slightly reduced water content of this sample. In addition five samples of MO were prepared in vials, using the conventional method, at a nominal water content of 40 % (w/w).
Water contents were calculated accurately
by weighing each vial after addition of water; these lay between 39.3 and 40.4 % (w/w), Table S2. A diamond phase, co-existing with a minority gyroid phase, was observed for four of the five samples, Fig. S1 (A) and (B). For several samples only the √6 reflection was observed for the co-existing QIIG phase, Fig. S2 (B). Assignment was therefore based both on expected phase behaviour, along with adherence of the lattice parameter for this phase to the Bonnet Ratio.5 The Bonnet Ratio predicts that for two coexisting cubic phases in excess water, the ratio of the QIIG lattice parameter to the QIID lattice parameter is 1.576. The calculated ratio for the coexisting phases observed here lie in the range 1.578 – 1.605.
The lattice
parameter of each phase was consistent; for the diamond phase the average lattice parameter was 101.7 Å with a standard deviation of 0.7 Å, for the gyroid phase the average lattice parameter was 161.5 Å with a standard deviation of 0.8 Å. The fifth sample adopted a pure QIIG phase with a lattice parameter of 160.4 Å, Fig. S2 (C). 4
We note that, again, adoption of a pure QIIG phase is consistent with the lower water content associated with this sample (39.4 % w/w). The data indicate that samples prepared in this way are generally consistent in the phase adopted and the associated lattice parameter. However, samples must be produced manually which is rather time-consuming and small differences in water content (< 0.2 %) can lead to phase changes in the bulk material. Due to the proximity to the QIIG / QIID phase boundary, these samples could potentially form a co-existing gyroid cubic phase if the water content is slightly reduced. Therefore, samples produced robotically in plates had a small volume (20 µL) of water added into the reservoir well to maintain environmental hydration levels. The volume of water in the reservoir was minimised to prevent contact with the bulk sample.
Despite the proximity to the gyroid/diamond phase boundary, samples
produced in this way all adopted a QIID phase, indicating that the water reservoir has successfully prevented drying of the samples (Fig S2). The lattice parameter was remarkably similar for all 36 samples; an average lattice parameter of 99.9 Å was observed with a standard deviation of 0.8 Å, similar to the standard deviation of only four samples produced using the conventional method.
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D
Sample No.
% H2O (w/w)
1
39.6
2
39.6
3
39.4
4
40.4
5
39.3
QII lattice parameter (Å)
G
error
101.4 101.1 101.7 101.0
0.2 0.3 0.3
102.9 102.6 101.2 101.7
0.1 0.2 0.3 0.3
QII lattice parameter (Å)
error
162.1 161.2 160.5 162.2 160.4 160.4 162.4 162.8 161.2 161.5
0.7 0.7 0.6 0.2 0.3 0.4
Table S2. The phase adopted and associated lattice parameter for five MO samples, made up using the conventional method in vials, nominally made up to 40 % (w/w) H2O. The accurate water content is given for each sample. Two samples were run from each vial.
√6 10000
√8 Intensity
√22 √20 √24 √16 √14 √26
1000
0.05
0.10
0.15
0.20
0.25
q (Å-1) Figure S1. 1-D diffraction plot of intensity vs q for MO 40 % (w/w) H2O prepared using a coupled syringe device. The √6, √8, √14, √16, √20, √22, √24 and √26 reflections of a QIIG phase (space group Ia3d) are indicated. For ease of visualisation, intensity has been plotted on a logarithmic axis.
6
12000
A
√2
10000
Intensity
8000
6000
√6 √3
4000
√8
2000
0 0.05
√4
0.10
8000
B
√6 0.15
√8 √9 0.20
0.25
-1
q (Å )
√2
6000
Intensity
√6 4000
√3
2000
√4 0 0.05
0.10
√6 √8 √9 0.15
0.20
0.25
-1
10000
C
√6
q (Å )
Intensity
8000
6000
4000
√8
2000
0 0.05
0.10
√22 √20 √24 √14 √16 √26 0.15
0.20
0.25
-1
q (Å )
Figure S2. 1-D diffraction plots of intensity vs q for A) Sample 1, B) Sample 4 and C) Sample 3 of the 5 samples of MO 40 % (w/w) H2O prepared in vials (results tabulated in Table S2). For A) and B) the √2, √3, √4, √6, √8 and √9 reflections of a QIID phase are indicated. For A) the √6 and √8 reflections of a co-existing QIIG phase are indicated in bold font. For B) a single additional reflection is indexed as the √6 reflection of a QIIG phase (bold font). For C) the √6, √8, √14, √16, √20, √22, √24 and √26 reflections of a QIIG phase are indicated. 7
5000
A
√2
Intensity
4000
3000
√3
2000
√4
1000
0 0.05
0.10
√6
√8 √9
0.15
0.20
0.25
-1
q (Å ) 7000
6000
B
√2
Intensity
5000
4000
√3
3000
2000
√4
1000
0 0.05
0.10
√6
√8 √9
0.15
0.20
0.25
-1
q (Å ) 3000
C
√2
2500
Intensity
2000
1500
√3 1000
√4
500
0 0.05
0.10
√6
0.15
√8
√9 0.20
0.25
-1
q (Å )
Figure S3. 1-D diffraction plots of intensity vs q for A) Sample 6, B) Sample 9 and C) Sample 19 of the 36 samples of MO 40 % (w/w) H2O prepared robotically. In each case the √2, √3, √4, √6, √8 and √9 reflections of a QIID phase (space group Pn3m) are indicated.
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Figure S4. The distribution of phases across a two sub-well crystallisation plate 1 day after addition of PACT screen to MO (40 % w/w water). Individual sub-wells are coloured to reflect the observed phase: diamond cubic phase (QIID, green), gyroid cubic phase (QIIG, pink). Well H12 did not have screen added and functions as a water control.
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Figure S5: Saturation binding curve showing 3H-spiperone binding to MO and reconstituted D2L in MO. Specific 3H-spiperone binding for reconstituted D2L–MO (■, dashed line) and control samples containing MO only (▲, solid line) are shown. The data indicate that very little free 3H-spiperone is trapped within the lipid matrix.
Figure S6: Negative control assays showing non-specific binding a) An example of a saturation binding curve for binding of a H1R antagonist (3H-pyrilamine) to the D2L receptor (b) saturation binding curve for 3H- pyrilamine to MO. Both graphs show non-specific binding occurs. Total (■, dashed line) and non-specific (▲, solid line) data is shown in both graphs.
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Figure S7: Saturation binding curve for D2L and H1R receptors reconstituted in MO. a)) D2L bound with 3H-spiperone. b) H1R bound with 3H-pyrilamine pyrilamine binding Both graphs show total (■ ■, dashed line) and non-specific (▲,, solid line) binding to the receptor.
Figure S8: An example of a saturation binding curve with excess cold ligand (spiperone). The total (■ ■, dashed line) and non-specific (▲,, solid line) binding for D2L bound with 10 nM 3H-spiperone H at varying cold ligand (spiperone concentration ranging from 1.1 µM to 8.1 µM) µM is shown. This binding curve shows show the saturation point for the cold ligand (spiperone) is about 1.5 µM concentration.
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1. Ratnala, V. R. P.; Swarts, H. G. P.; VanOostrum, J.; Leurs, R.; DeGroot, H. J. M.; Bakker, R. A.; DeGrip, W. J., Large-scale overproduction, functional purification and ligand affinities of the His-tagged human histamine H1 receptor. Eur J Biochem 2004, 271 (13), 2636-2646. 2. Briggs, J.; Chung, H.; Caffrey, M., The temperature-composition phase diagram and mesophase structure characterization of the monoolein/water system. J Phy I 1996, 6 (5), 723-751. 3. Nollert, P., Lipidic cubic phases as matrices for membrane protein crystallization. Methods 2004, 34 (3), 348-353. 4. Conn, C. E.; Darmanin, C.; Sagnella, S. M.; Mulet, X.; Greaves, T. L.; Varghese, J. N.; Drummond, C. J., Incorporation of the dopamine D2L receptor and bacteriorhodopsin within bicontinuous cubic lipid phases. 1. Relevance to in meso crystallization of integral membrane proteins in monoolein systems. Soft Matter 2010, 6 (19), 4828-4837. 5. Andersson, S.; Hyde, S. T.; Larsson, K.; Lidin, S., Minimal-Surfaces and Structures - from Inorganic and Metal Crystals to Cell-Membranes and Bio-Polymers. Chem Rev 1988, 88 (1), 221-242.
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