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 (PepT_St), diacylglycerol kinase (DgkA), the alginate transporter (AlgE) and the cystic fibrosis transmembrane conductance regulator (CFTR). In the cases of β₂AR, PepT_St, 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.

中文翻译：

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在立方中间相中浓缩膜蛋白的立方方法。

脂质立方相（内消旋）方法是生成晶体和完整膜蛋白的高分辨率X射线结构的重要方法。但是，由于不稳定，使用传统方法将某些膜蛋白和复合物浓缩到适合于介观结晶和结构确定的值可能是不可能的。本文描述的立方方法利用了膜蛋白的两亲性质及其从水溶液中优先分配到脂质双层中的自然趋势。使用几轮重构，立方中间相双层中的蛋白质浓度可以逐步增加，从少于每毫升毫克增加到每毫升数十毫克，以进行结晶生成。₂ -肾上腺素能G蛋白偶联受体（β ₂ AR），肽转运体（PEPT_圣），二酰基甘油激酶（DgkA），藻酸盐转运体（ALGE）和囊性纤维化跨膜传导调节蛋白（CFTR）。在β的情况下₂ AR，PEPT_圣DgkA和AlgE的有效浓度为20到45倍，产生了一个蛋白质负载的中间相，该中间相允许使用in in meso方法和结构测定法形成晶体，分辨率范围为2.4到3.2Å。除了在介观结晶中开放到更广泛的完整膜蛋白靶标范围外，cubicon方法还应运用于需要在脂双层中以高浓度重建膜蛋白的情况。这些应用包括功能和生物物理表征研究，用于配体筛选，药物递送，抗体生产和蛋白质复合物形成。一个典型的立方​​实验可以在3-5小时内完成。