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The cubicon method for concentrating membrane proteins in the cubic mesophase

Nature Protocols volume 12, pages 1745–1762 (2017)Cite this article

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Abstract

The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible—using traditional methods—to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution. Using several rounds of reconstitution, the protein concentration in the bilayer of the cubic mesophase can be ramped up stepwise from less than a milligram per milliliter to tens of milligrams per milliliter for crystallogenesis. The general applicability of the method is demonstrated with five integral membrane proteins: the β2-adrenergic G protein-coupled receptor (β2AR), the peptide transporter (PepTSt), diacylglycerol kinase (DgkA), the alginate transporter (AlgE) and the cystic fibrosis transmembrane conductance regulator (CFTR). In the cases of β2AR, PepTSt, DgkA and AlgE, an effective 20- to 45-fold concentration was realized, resulting in a protein-laden mesophase that allowed the formation of crystals using the in meso method and structure determination to resolutions ranging from 2.4 Å to 3.2 Å. In addition to opening up in meso crystallization to a broader range of integral membrane protein targets, the cubicon method should find application in situations that require membrane protein reconstitution in a lipid bilayer at high concentrations. These applications include functional and biophysical characterization studies for ligand screening, drug delivery, antibody production and protein complex formation. A typical cubicon experiment can be completed in 3–5 h.

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Figure 1: Membrane protein types yielding structures based on in meso crystallization and the corresponding protein concentrations used for crystallization.
Figure 2: Mesophase identity and microstructure of hydrated lipids as affected by detergent at 20 °C, determined using SAXS.
Figure 3: Appearance of the lipid, aqueous solution and mesophase at various stages in the cubicon process.
Figure 4: The cubicon method for raising membrane protein concentration in the bilayer of the cubic mesophase by sequential rounds of reconstitution.
Figure 5: Structures solved based on crystals grown using protein-laden mesophase generated by the cubicon method.

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Acknowledgements

The authors thank past and current members of the Membrane Structural and Functional Biology Group for their many helpful contributions. The work was funded by Science Foundation Ireland (12/IA/1255) and the Cystic Fibrosis Foundation. P.M. was supported by a grant (WELBIO CR-2012S-04) to C. Govaerts (Université libre de Bruxelles) from the Belgian National Funds for Scientific Research. The support from beamline scientists at the Swiss Light Source, Villigen, Switzerland and the Diamond Light Source, Didcot, UK, is gratefully acknowledged.

Author information

Author notes

  1. Pikyee Ma and Dietmar Weichert: These authors contributed equally to this work.

Authors and Affiliations

  1. Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland

    Pikyee Ma, Dietmar Weichert & Martin Caffrey

  2. Department of Biochemistry and Biophysics and Cystic Fibrosis Treatment and Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Luba A Aleksandrov, Timothy J Jensen & John R Riordan

  3. Beijing Advanced Innovation Center for Structural Biology, School of Medicine,

    Xiangyu Liu

  4. , Tsinghua University, Beijing, China

    Xiangyu Liu

  5. Department of Molecular and Cellular Physiology, School of Medicine,

    Brian K Kobilka

  6. Stanford University, Stanford, California, USA

    Brian K Kobilka

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Contributions

P.M. concentrated CFTR; produced and purified DgkA and PepTSt; concentrated, analyzed, crystallized, collected and processed diffraction data for DgkA, PepTSt and β2AR, and SAXS data for DDM, LMNG and DMNG; and solved, refined and analyzed the structures of DgkA, PepTSt and β2AR. D.W. produced and purified AlgE and concentrated, analyzed, crystallized and solved its structure; refined and analyzed the structure of AlgE; and prepared samples and analyzed SAXS data for LDAO and for the mesophase samples of the reference proteins that were used for crystallization. L.A.A. and T.J.J. produced and purified CFTR. X.L. produced and purified β2AR. M.C. designed the research and J.R.R., B.K.K. and M.C. provided project supervision. M.C. wrote the manuscript with input from D.W. and P.M.

Corresponding author

Correspondence to Martin Caffrey.

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The authors declare no competing financial interests.

Integrated supplementary information

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/2θ 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 Lα (L) phase are shown. Actual 2D SAXS diffraction patterns, upon which the I/2θ plots are based, are included as insets.

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.

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.

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.

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.

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 β2AR 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.

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 μL) of excess aqueous solution (numbered by reconstitution round from 1 - 7 in a, from 8 - 14 in b and from 15 - 17 in c) with a loading series that includes the indicated amounts of BSA (lanes 9 - 11 in a and 10 - 12 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.

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 μL) 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.

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 PepTSt.

Quantitation was carried out by comparing the level of Coomassie Blue staining observed with aliquots (5 μL) 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 PepTSt (lanes 2 - 4 in a and 2-3 in b). In this case, no protein was detected indicating complete partitioning of PepTSt into the 9.7 MAG mesophase at each round of reconstitution. Molecular weight markers were included for reference.

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 β2AR.

Quantitation was carried out by comparing the level of Coomassie Blue staining observed with aliquots (5 μL) 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 β2AR (lanes 2 - 5 in a and b). In this case, no protein was detected indicating complete partitioning of β2AR into the 9.9 MAG/cholesterol mesophase at each round of reconstitution. Molecular weight markers were included for reference.

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 μL) 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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 1–3, and the Supplementary Methods. (PDF 2810 kb)

Materials and procedures used to implement the cubicon method.

The protein used in this demonstration is CFTR and the lipid is 9.9 MAG containing 10% (wt/wt) cholesterol. The video was recorded in staged sequences to optimize viewing quality. Accordingly, the volumes of protein solution and lipid shown in the video differ slightly from those in the text of this Protocol. However, the volume ratios and the procedures followed are exactly as described in the Protocol. (MP4 23295 kb)

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Ma, P., Weichert, D., Aleksandrov, L. et al. The cubicon method for concentrating membrane proteins in the cubic mesophase. Nat Protoc 12, 1745–1762 (2017). https://doi.org/10.1038/nprot.2017.057

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