Crystallizing membrane proteins for structure determination using lipidic mesophases.
Crystallizing membrane proteins for structure determination using lipidic mesophases.
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DOI：
10.3791/1712
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发表时间：
2010-11-21
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影响因子：
--
通讯作者：
Porter, Christopher
中科院分区：
文献类型：
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作者：
Caffrey, Martin;Porter, Christopher
详细的协议结晶膜蛋白通过使用介电中间相描述。这种方法被称为介观立方相或介观方法。该方法已被证明是非常通用的，因为它已被用于解决原核和真核蛋白质，蛋白质是单体，同源和异源多聚体，含发色团和无发色团，和α-螺旋和β-桶蛋白质的X射线晶体结构。最近成功用于介晶化的是人工程化的β 2-肾上腺素能和腺苷A2 a G蛋白偶联受体。提出了用于将膜蛋白重组成基于单油酸甘油酯的中间相的协议，以及用于在手动模式下设置结晶。整个过程中的其他步骤，如晶体收获，将在未来的视频文章中解决。制备负载蛋白质的中间相和手动设置结晶板所需的时间为约1小时。
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.