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Crystallizing membrane proteins using lipidic mesophases

Nature Protocols volume 4, pages 706–731 (2009)Cite this article

Abstract

A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and α-helical and β-barrel proteins. Its most recent successes are the human-engineered β2-adrenergic and adenosine A2A G protein–coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase and for setting up crystallizations in manual mode. Methods for harvesting microcrystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about 1 h.

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Figure 1: Temperature-composition phase diagram of the monoolein/water system determined under 'conditions of use' in the heating and cooling directions from 20 °C.
Figure 2: Cartoon representation of the events proposed to take place during the crystallization of an integral membrane protein from the lipidic cubic mesophase.
Figure 3: The in meso crystallization robot.
Figure 4: A flowchart summarizing the steps involved in and time required for setting up an in meso membrane protein crystallization trial.
Figure 5: The glass sandwich crystallization plate.
Figure 6: The lipid-mixing device.
Figure 7: Ways to set up in meso crystallization trials using commercial plates.
Figure 8: Microsyringe-repeating dispenser.
Figure 9: Crystals of membrane proteins growing in the lipidic mesophase.
Figure 10: Images of birefringent solid and liquid crystalline lipidic phases recorded using polarized light microscopy.
Figure 11: Nonbirefringent 'objects' with sharp edges and corners that appear in lipid mesophases under in meso crystallization conditions.
Figure 12: Distinguishing protein from salt crystals.
Figure 13: A key for scoring the outcome of in meso crystallization trials.
Figure 14: Small-angle X-ray diffraction patterns of hydrated monoolein in the cubic and sponge phases.
Figure 15: X-ray diffraction patterns of the various phases that can occur in the monoolein/screen solution/water system.
Figure 16: Spectrophotometry/fluorescence cuvette and accessories.
Figure 17: Spectrophotometric and visual properties of BtuB in detergent solution and in the cubic phase.
Figure 18: Initial BtuB microcrystal hits.
Figure 19: Absorption spectrum of LH2 in detergent solution and in the lipidic cubic phase.
Figure 20: Initial β2AR-T4L microcrystal hits.

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Acknowledgements

We thank members and associates of the Caffrey group, past and present, for their assorted contributions over the years to this work. In particular, we acknowledge the contributions of D. Aragao, A-c Cheng, J. Clogston, D. Hart, N. Hoefer, B. Hummel, D. Li, W. Liu, J. Lyons, Y. Misquitta, L. Muthusubramaniam, A. Peddi, B. Sun, J. Tan and Y. Zheng. This work was supported in part by grants from Science Foundation Ireland (02-IN1-B266), the National Institutes of Health (RO1 program: GM61070 and GM75915; the NIH Roadmap Initiative: P50 GM073197; and the Protein Structure Initiative: U54 GM074961) and the National Science Foundation (IIS-0308078).

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  1. Membrane Structural and Functional Biology Group, University of Limerick, Limerick, Ireland

    Martin Caffrey

  2. Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA

    Vadim Cherezov

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Correspondence to Martin Caffrey.

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Caffrey, M., Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat Protoc 4, 706–731 (2009). https://doi.org/10.1038/nprot.2009.31

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