Liu Wei, Cherezov Vadim
Molecular Biology, The Scripps Research Institute, USA.
J Vis Exp. 2011 Mar 28(49):2501. doi: 10.3791/2501.
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.
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，因此与多种功能障碍和疾病有关。然而，对膜蛋白的结构和功能的理解远远落后于其可溶性对应物，主要是由于其增溶以及生成具有衍射质量的晶体存在困难。在脂质中间相结晶（也称为在中间相或LCP结晶）是一种很有前景的技术，已成功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。与从去污剂溶液中进行传统结晶相比，在中间相结晶利用了类似天然的膜环境，通常会产生溶剂含量较低且有序性更好的晶体。该方法并不困难，但需要了解脂质相行为并具备处理粘性中间相材料的实践经验。在这里，我们展示了一种简单有效的方法来制备LCP，并使用注射器混合器在LCP的脂质双层中重构膜蛋白，然后将纳升量的LCP分配到检测或结晶板中，进行预结晶检测并从LCP基质中收获晶体。这些方案为进行中间相结晶试验提供了基本指导；然而，与任何结晶实验一样，需要进行广泛的筛选和优化，并且不一定能保证成功。
