Crystallization of Membrane Proteins in Lipidic Mesophases
Crystallization of Membrane Proteins in Lipidic Mesophases
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DOI：
10.3791/2501
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发表时间：
2011-03-01
影响因子：
1.2
通讯作者：
Cherezov, Vadim
中科院分区：
文献类型：
--
作者：
Liu, Wei;Cherezov, Vadim
膜蛋白在活细胞中执行与信号转导、运输和能量转换相关的关键功能，并且因此与多种功能障碍和疾病有关。然而，膜蛋白的结构和功能的理解是强烈滞后于其可溶性合作伙伴，主要是由于与其溶解和衍射质量晶体的产生相关的困难。介晶相结晶（也称为介晶或LCP结晶）是一种很有前途的技术，其被成功地应用于获得微生物视紫红质、光合蛋白、外膜β桶和G蛋白偶联受体的高分辨率结构。在介观结晶中，利用天然的膜环境，并且与从洗涤剂溶液的传统结晶相比，通常产生具有较低溶剂含量和更好有序性的晶体。该方法并不难，但需要了解脂相行为和处理粘性中间相材料的实践。在这里，我们展示了一个简单而有效的方法，使LCP和重构膜蛋白的LCP的脂质双层使用注射器混合器，然后分配到一个测定或结晶板的LCP纳升部分，进行预结晶测定和收获晶体从LCP矩阵。这些协议提供了一个基本的指导，在介孔结晶试验的方法，然而，与任何结晶实验，广泛的筛选和优化是必需的，并不一定保证成功的结果。
Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.