Crystallizing membrane proteins for structure-function studies using lipidic mesophases.
Crystallizing membrane proteins for structure-function studies using lipidic mesophases.
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DOI：
10.1042/bst0390725
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发表时间：
2011-06
影响因子：
3.9
通讯作者：
Caffrey M
中科院分区：
文献类型：
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作者：
Caffrey M
近年来，用于膜蛋白结晶的脂质立方相法取得了一些令人瞩目的成功。在G蛋白偶联受体领域尤其如此，在过去的31年半里出现了6个新的晶体结构。慢慢地，它正在成为一种被接受的方法，具有经过验证的记录和令人信服的普遍性。然而，这并不是一种在每个膜结构生物学实验室都使用的方法，这是不幸的。不愿采用它的部分原因是，预计在处理晶体生长所在的粘稠立方体中间相时会遇到困难。用中间相生长的晶体采集和收集衍射数据也有些胆战心惊。人们承认，这种方法存在一些相关的挑战。然而，多年来，我们一直在努力使该方法变得用户友好。为此，已经开发了用于处理皮升至纳升体积范围内的中间相的工具，以便在手动和机器人模式下进行有效的结晶筛选。玻璃析晶板已经建成，为新生晶体提供无与伦比的光学质量和敏感度。脂类和沉淀剂筛选已被用于更合理的结晶形成方法，因此该方法现在可以应用于各种类型和大小的膜蛋白。在这篇文章中，概述了这些分类的进展，并总结了已产生的膜蛋白的方法。描述了进一步发展该方法所必须克服的挑战。
The lipidic cubic phase method for crystallizing membrane proteins has posted some high-profile successes recently. This is especially true in the area of G-protein-coupled receptors, with six new crystallographic structures emerging in the last 31/2 years. Slowly, it is becoming an accepted method with a proven record and convincing generality. However, it is not a method that is used in every membrane structural biology laboratory and that is unfortunate. The reluctance in adopting it is attributable, in part, to the anticipated difficulties associated with handling the sticky viscous cubic mesophase in which crystals grow. Harvesting and collecting diffraction data with the mesophase-grown crystals is also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. However, over the years, we have worked to make the method user-friendly. To this end, tools for handling the mesophase in the pico- to nano-litre volume range have been developed for efficient crystallization screening in manual and robotic modes. Glass crystallization plates have been built that provide unparalleled optical quality and sensitivity to nascent crystals. Lipid and precipitant screens have been implemented for a more rational approach to crystallogenesis, such that the method can now be applied to a wide variety of membrane protein types and sizes. In the present article, these assorted advances are outlined, along with a summary of the membrane proteins that have yielded to the method. The challenges that must be overcome to develop the method further are described.