Cherezov, V. & Caffrey, M. Membrane protein crystallization in lipidic mesophases. A mechanism study using X-ray microdiffraction. Faraday Discuss. 136, 195-212

Department of Molecular Biology, The Scripps Research Institute, La Jolla CA 92037, USA.
Faraday Discussions (Impact Factor: 4.61). 02/2007; 136:195-212; discussion 213-29. DOI: 10.1039/B618173B
Source: PubMed


The membrane structural biologist seeks to understand how membrane proteins function at a molecular level. One of the most direct ways of accomplishing this requires knowing the structure of the protein, ideally at atomic resolution. To date, this can only be done by the method of macromolecular crystallography. Integral to the method is the need for three-dimensional crystals of diffraction quality and their production represents a major rate-limiting step in the overall process of structure determination. The in meso method is a novel approach for crystallizing membrane proteins. It makes use of lipidic mesophases, the cubic phase in particular. A mechanism for how the method works has been proposed. In this study, we set out to test one aspect of the hypothesis which posits that the protein migrates from the bulk mesophase reservoir to the face of the crystal by way of a lamellar conduit. Using a sub-micrometer-sized X-ray beam the interface between a growing membrane protein crystal and the bulk cubic phase was interrogated with micrometer spatial resolution. Characteristic diffraction from the lamellar phase was observed at the interface as expected. This result supports the proposal that the protein uses a lamellar portal on its way from the bulk mesophase up and into the face of the crystal.

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    • "While the bulk monoolein cubic phase remained transparent and non-birefringent, the solution-exposed surface appeared shiny under microscopic inspection using crossed polarizers (not shown). We speculate that a thin section of the lipid material forms a portal lamellar phase at the solution exposed surface (Figure 6), allowing detergent and RC molecules to enter a bilayer structure that is connected to the curved lipid bilayer system within the LCP, a mechanism that is similar to the crystal growth hypothesis brought forward initially by [21] and later verified [22]. "
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    ABSTRACT: The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization parameters. Finally, we provide a model that explains the incorporation of the membrane protein from solution into the lipid phase via a portal lamellar phase.
    PLoS ONE 08/2011; 6(8):e24488. DOI:10.1371/journal.pone.0024488 · 3.23 Impact Factor
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  • Faraday Discussions 02/2007; 136:409-16. DOI:10.1039/b707653n · 4.61 Impact Factor
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