Article

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

Membrane Structural and Functional Biology Group, Schools of Biochemistry and Immunology and Medicine, Trinity College Dublin.
Journal of Visualized Experiments (Impact Factor: 1.33). 11/2010; DOI: 10.3791/1712
Source: PubMed

ABSTRACT

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile 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 alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

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    • "Purified native or SeMet-labeled 103His- TEV-Int 208–449 and 103His-TEV-Inv 147–390 in the size exclusion chromatography buffer were concentrated to 40 and 28 mg/ml, respectively, and then diluted with dH 2 O to 20 mg/ml. Monoolein (Nu-Chek Prep) was melted at 42 C and then 60 ml of molten monoolein was mixed with 40 ml of protein at 20 mg/ml in a coupled syringe apparatus as described previously by Caffrey and Cherezov (2009). The final concentration of protein in the lipidic mesophase was 8 mg/ml. "
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    • "Both crystal forms were remarkably reproducible: simple gradient screens described in Supplementary text produced crystals every time crystal trials were set up. This once again demonstrates that MO-based mesophase crystallogenesis can tolerate a broad range of conditions (Ai and Caffrey, 2000; Caffrey and Cherezov, 2009; Cherezov et al., 2001 "
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