Characterizing the mesophase behavior of hydrated 9.9 MAG at 20 C and at increasing concentrations of DDM by SAXS.

Transcription

1 Supplementary Figure 1 Characterizing the mesophase behavior of hydrated 9.9 MAG at 20 C and at increasing concentrations of DDM by SAXS. Individual panels show I/2 SAXS profiles at the specified concentration of detergent in the mesophase. The concentration of detergent in the solution used to make the mesophase is shown in brackets. Miller indices for individual reflections associated with the cubic-pn3m (P), cubic-ia3d (I) and L (L) phase are shown. Actual 2D SAXS diffraction patterns, upon which the I/2 plots are based, are included as insets.

2 Supplementary Figure 2 Characterizing the mesophase behavior of hydrated 9.7 MAG at 20 C and at increasing concentrations of DDM by SAXS. Additional details are provided in the legend to Supplementary Figure 1.

3 Supplementary Figure 3 Characterizing the mesophase behavior of hydrated 9.9 MAG with 10 %(wt/wt) cholesterol at 20 C and at increasing concentrations of LMNG by SAXS. Additional details are provided in the legend to Supplementary Figure 1.

4 Supplementary Figure 4 Characterizing the mesophase behavior of hydrated 9.9 MAG at 20 C and at increasing concentrations of LDAO by SAXS. Additional details are provided in the legend to Supplementary Figure 1.

5 Supplementary Figure 5 Characterizing the mesophase behavior of hydrated 9.9 MAG with 10 %(wt/wt) cholesterol at 20 C and at increasing concentrations of DMNG by SAXS. Additional details are provided in the legend to Supplementary Figure 1.

6 Supplementary Figure 6 Characterizing the phase behavior and microstructure of protein-laden mesophase of the type used for crystallization following rounds of reconstitution by the Cubicon method at 20 C. In all cases, the cubic mesophase prevails. Average lattice parameters for the cubic-ia3d (I) and Pn3m (P) phases are indicated (box inserts). The slight broadening of peaks in the 2AR SAXS pattern may originate from a smaller mesophase domain size that, in turn, may derive from the protein, the LMNG detergent and cholesterol present in the hosting 9.9 MAG lipid. Broadening does not compromise the reliability of the mesophase identification and characterization. Additional details are provided in the legend to Supplementary Figure 1.

7 Supplementary Figure 7 SDS-PAGE analysis of the protein content of excess aqueous solution formed following individual rounds of reconstitution during application of the Cubicon method with CFTR. Quantitation was carried out by comparing the level of Coomassie Blue staining observed with aliquots (5 L) of excess aqueous solution (numbered by reconstitution round from 1-7 in a, from 8-14 in b and from in c) with a loading series that includes the indicated amounts of BSA (lanes 9-11 in a and in b). In this case, no protein was detected indicating complete partitioning of CFTR into the 9.9 MAG/cholesterol mesophase at each round of reconstitution. BSA was used as a reference for quantitation because CFTR cannot be concentrated enough for such use. Molecular weight markers were included for reference.

8 Supplementary Figure 8 SDS-PAGE analysis of the protein content of excess aqueous solution formed following individual rounds of reconstitution during application of the Cubicon method with DgkA. Quantitation was carried out by comparing the level of Coomassie Blue staining observed with aliquots (5 L) of excess aqueous solution (numbered by reconstitution round from 1-6 in a and from 7-12 in b) with a loading series that includes the indicated amounts of DgkA (lanes 2-5 in a and b). In this case, no protein was detected indicating complete partitioning of DgkA into the 9.9 MAG mesophase at each round of reconstitution. Molecular weight markers were included for reference.

9 Supplementary Figure 9 SDS-PAGE analysis of the protein content of excess aqueous solution formed following individual rounds of reconstitution during application of the Cubicon method with PepT St. Quantitation was carried out by comparing the level of Coomassie Blue staining observed with aliquots (5 L) of excess aqueous solution (numbered by reconstitution round from 1-6 in a and from 7-12 in b) with a loading series that includes the indicated amounts of PepT St (lanes 2-4 in a and 2-3 in b). In this case, no protein was detected indicating complete partitioning of PepT St into the 9.7 MAG mesophase at each round of reconstitution. Molecular weight markers were included for reference.

10 Supplementary Figure 10 SDS-PAGE analysis of the protein content of excess aqueous solution formed following individual rounds of reconstitution during application of the Cubicon method with 2AR. Quantitation was carried out by comparing the level of Coomassie Blue staining observed with aliquots (5 L) of excess aqueous solution (numbered by reconstitution round from 1-6 in a and from 7-12 in b) with a loading series that includes the indicated amounts of 2AR (lanes 2-5 in a and b). In this case, no protein was detected indicating complete partitioning of 2AR into the 9.9 MAG/cholesterol mesophase at each round of reconstitution. Molecular weight markers were included for reference.

11 Supplementary Figure 11 SDS-PAGE analysis of the protein content of excess aqueous solution formed following individual rounds of reconstitution during application of the Cubicon method with AlgE. Quantitation was carried out by comparing the level of Coomassie Blue staining observed with aliquots (5 L) of excess aqueous solution (numbered by reconstitution round from 1-5 in a and from 6-10 in b) with a loading series that includes the indicated amounts of AlgE (lanes 2-4 in a and b). In this case, trace amounts of protein were detected following several of the rounds of reconstitution. This was particularly noticeable in rounds 3, 4, 5 and 9. We attribute the loss of protein not to a failure to partition into the bilayer of the mesophase, rather to the LDAO detergent creating a finer, more dispersed mesophase some of which is lost irreproducibly in the excess aqueous solution removed after each reconstitution round. Comparing staining levels in the reference AlgE and solution lanes we estimate that 0.15% of the total protein was lost during the Cubicon process. Increasing the mixing time from 3 to 7 min had no effect on the results. Molecular weight markers were included for reference.

12 P a g e 1 Supplementary Methods Protein production. The thermostable DgkA Δ4 mutant (Ile53Cys, Ile70Leu, Met96Leu, Val107Asp) was overexpressed and purified as described previously 1, with the exception that DDM was used in place of n-decyl -D-maltopyranoside (DM) (Buffer A) from the detergent exchange step onwards. PepT St 2, 2 AR-T4L 3, AlgE 4, and CFTR 5 were expressed and purified following established protocols. The proteins were stored and used for rounds of reconstitution in Buffers B, C, D and E, respectively. Buffer compositions are listed in Table 2. Mesophase characterization. The effect of detergent concentration on the phase behavior and microstructure of the hydrated lipids used in this study was established by SAXS at 20 C as described 6-8. Samples for phase determination at or close to full hydration were prepared at 3:2 (for 9.9 MAG and 9.9 MAG with 10% (wt/wt) cholesterol) or 1:1 (for 9.7 MAG) lipid-to-detergent solution volume ratio at room temperature (20 22 C) in a coupled syringe mixer 9. Stock solutions ranging from 0 to 1.25 M detergent, corresponding to a detergent concentration in the final mesophase ranging from 0 to 0.5 M, were added to one syringe of the coupled mixer. The requisite amount of lipid to produce a sample with 3:2 or 1:1 lipid-todetergent solution volume ratio was added to the second syringe. The two syringes were coupled and the components homogenized by mechanical mixing 9. In the case of LMNG, which has low solubility, a known weight of detergent powder was placed in one syringe of the mixer and MilliQ-water and lipid were added in appropriate amounts to achieve the desired sample composition to the other. The contents were mixed as described 8. Homogenized mesophase samples were transferred to 1 mm diameter glass capillaries, and flame and 5-min epoxy sealed. SAXS measurements were made at the PX I beamline (Swiss Light Source) using a 10 x 20 or 10 x 30 m 2 beam at to 1 Å (1.24 x 10 4 to 1.27 x 10 4 ev) and 1.87 x to 2.79 x photons/s and an exposure time of 0.1 to 0.15 s. Powder diffraction patterns were recorded with a Pilatus 6M detector at a sample-to-detector distance of 120 cm. SAXS patterns were circularly averaged using ALBULA (DECTRIS Ltd.). The data are reported as scattered intensity (I) versus scattering angle (2θ) plots. Peaks in the I/2θ plots were indexed manually into the different mesophase types and space groups were identified as described previously 7. The nature of the mesophase in the presence of a 3-fold volume excess of buffer solution was investigated, as above, by SAXS with samples prepared by mixing 30 L of Buffers A E (Table 2) with 10 L of the appropriate lipid or lipid mixture. The behavior of the protein-laden mesophases of the type used for crystallization following rounds of reconstitution by the Cubicon method was also investigated, as above, by SAXS (Supplementary Figure 6).

13 P a g e 2 Crystallization and structure determination. DgkA 1, 2 AR 3, PepT St 2 and AlgE 4 have all been crystallized by the in meso method. These same successful protocols or slight variations thereof were used in the current study to generate crystals for structure determination. The mesophase used to set up crystallization trials was that generated by the Cubicon method, just described. The final protein concentrations in the mesophase entering crystallization were estimated at 4.8 mg DgkA/mL, 18 mg 2AR/mL, 8 mg AlgE/mL and 5 mg PepT St /ml based on the volume of dilute protein solution of known protein concentration and lipid used in the rounds of reconstitution. The assumption made is that all of the protein in the starting protein solution ends up in the mesophase. As evidenced by the lack of protein in the excess aqueous solution following rounds of reconstitution this assumption holds true for DgkA, 2 AR and PepT St. For AlgE, ~ 0.15% of the protein was lost in the separated excess aqueous solution and has been accounted for in the above calculations. Trials were set up following well-established methods that employed 50 nl mesophase and 800 nl precipitant solution per well in 96-well glass sandwich plates 10. Crystals of DgkA were grown at 4 C whilst 2 AR, PepT St and AlgE were crystallized at 20 C. DgkA in 9.9 MAG as host lipid was crystallized using a precipitant containing % (vol/vol) MPD, 3% (vol/vol) 1,4-butanediol, 100 mm LiNO 3, 100 mm NaCl and 100 mm sodium citrate ph 5.6. PepT St was crystallized in 9.7 MAG using a solution containing % (vol/vol) PEG 400, mm NH 4 H 2 PO 4 and 100 mm Hepes ph 7.0 as precipitant. β 2 AR in 9.9 MAG doped with 10% (wt/wt) cholesterol was crystallized with a precipitant solution containing 30 35% (vol/vol) PEG 400, 5 10% (vol/vol) 1,4-butanediol, mm Na 2 SO 4 and 100 mm Bis-Tris ph 7.0. Crystals of AlgE were grown in 9.9 MAG using a precipitant solution containing 38% (vol/vol) PEG 400, 0.1 M sodium citrate ph 5.6 and 0.1 M Bis-Tris propane ph 5.0. Crystals were harvested with m micromounts (MiTeGen) and were snapcooled without added cryo-protectant, as described 11. X-ray diffraction data were collected on beamlines I04 and I24 at the Diamond Light Source (Didcot, UK) and beamlines PXI and PXII at the Swiss Light Source (Villigen, Switzerland). Data were acquired using a 10 x 10 m 2 or 10 x 18 m 2 microfocus X- ray beam. DgkA: 100 % transmission at 1.2 x photons/s, 0.2 oscillation, 0.05 s exposure, 10 x 10 m 2 beam, a detector distance of 600 mm, and a wavelength of Å. β 2 AR-T4L: 100 % transmission at 1.2 x photons/s, 0.2 o oscillation, 0.06 s exposure, 10 x 10 m 2 beam, a detector distance of 600 mm and a wavelength of Å. PepT St : 100 % transmission at 2.32 x photons/s, 0.2 oscillation, 0.1 s exposure, 10 x 10 m 2 beam and a detector distance of 350 mm and a wavelength of AlgE: 100 % transmission at 2.08 x photons/s, 0.2 oscillation, 0.1 s exposure, 10 x 10 m 2 beam and a detector distance of 450 mm and a wavelength of Å.

14 P a g e 3 For all four proteins, complete data sets were collected with single crystals. Data were processed using the XIA2 12 pipeline to XDS 13 and scaled with AIMLESS 14 or XSCALE 13. Initial phases for DgkA, PepT St, β 2 AR and AlgE were obtained by molecular replacement using Phaser 15 with the protein component of published structures (3ZE5, 4D2B, 2RH1 and 4AFK, respectively) as search models. Iterative rounds of model refinement were performed in PHENIX 16, using Coot 17 for model building. Refinement statistics are reported in Supplementary Table 3. The structures are similar to their molecular replacement models with root mean square deviation (RMSD) values of 0.23, 0.14, 0.07 and 0.27 Å, respectively. Favored and allowed percentages in the Ramachandran plot were % and 2.00 % for DgkA, 98.42% and 1.59% for PepT St, 98.39% and 1.61% for β 2 AR and 96.5% and 3.5% for AlgE. No outliers were found. Coordinates and structure factors have been deposited in the Protein Data Bank ( under record identifier 5D6I for DgkA, 5D6K for PepT St, 5D6L for β 2 AR and 5IYU for AlgE. Structures were visualized and figures drawn with PyMol (PyMOL Molecular Graphics System, Schrödinger, LLC).

15 P a g e 4 Supplementary Table 1 Effect of excess aqueous solution on the mesophase behavior of different MAGs with and without cholesterol at 20 C. For each lipid/detergent combination the lattice parameter of the cubic-pn3m phase that formed at or close to full hydration, as reported in Fig. 2, and in an excess of aqueous solution is shown. Excess aqueous solution refers to mesophase prepared as a 3:1 by volume ratio of buffer to molten lipid. Buffers used with the different lipids and their compositions are listed in Table 2. In all cases, the only mesophase present was the cubic-pn3m phase. Lipid + Buffer Lattice parameter (Å) at or close to full hydration Lattice parameter (Å) in excess aqueous solution 9.9 MAG + Buffer A MAG + Buffer B MAG + 10% (wt/wt) cholesterol + Buffer C MAG + Buffer D MAG + 10 %(wt/wt) cholesterol + Buffer E

16 P a g e 5 Supplementary Table 2 Values used in calculating fold concentration for the proteins used in this Cubicon protocol.* CFTR DgkA AlgE 2AR PepT Protein concentration in the final mesophase post- Cubicon 7.2 mg/ml 4.8 mg/ml 8 mg/ml 18 mg/ml 5 mg/ml Protein concentration of the dilute starting solution 0.5 mg/ml 0.5 mg/ml 1 mg/ml 1 mg/ml 0.5 mg/ml Protein concentration in the equivalent stock solution 7.2 mg/0.4 ml = 18 mg/ml 4.8 mg/0.4 ml = 12 mg/ml 8 mg/0.4 ml = 20 mg/ml 18 mg/0.4 ml = 45 mg/ml 5 mg/0.5 ml = 10 mg/ml Fold concentration * A step-by-step calculation using CFTR as an example is provided in Step 26 of the Protocol.

17 P a g e 6 Supplementary Table 3 Data collection and refinement statistics. β 2 AR PepT St DgkA AlgE Data collection Space group C121 C222 1 P C121 Cell dimensions a, b, c (Å) , , , , , 72.77, , 73.39, α, β, γ ( ) 90.00, , 90.00, 90.00, 90.00, 90.00, 90.00, , Resolution (Å) ( )* ( )* ( )* ( )* R merge (0.814) (0.964) (1.33) (0.598) R pim (0.554) (0.462) (0.504) (0.381) I / σi 7.3 (1.5) 12.4 (1.8) 16.6 (1.4) 4.7 (1.7) Completeness (%) 94.4 (96.7) 99.8 (99.4) 99.7 (97.9) 99.2 (99.3) Redundancy 2.8 (2.8) 5.2 (5.3) 7.9 (7.9) 3.3 (3.4) Refinement Resolution (Å) No. of reflections 10,858 25,142 11,778 12,694 R work / R free 22.18/ / / /28.22 No. of atoms Protein 3,543 3,742 2,569 3,515 Ligand/ion Water B-factors Protein Ligand/ion N/A Water N/A N/A R.m.s. deviations Bond lengths (Å) Bond angles ( ) *Numbers in parentheses indicate values for highest-resolution shell All structures were solved using data collected from a single crystal

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