Membrane protein structure determination using crystallography and lipidic mesophases: recent advances and successes.
Membrane protein structure determination using crystallography and lipidic mesophases: recent advances and successes.
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DOI：
10.1021/bi300010w
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发表时间：
2012-08-14
期刊：
影响因子：
2.9
通讯作者：
Dukkipati A
中科院分区：
文献类型：
--
作者：
Caffrey M;Li D;Dukkipati A
β2-肾上腺素能受体与激动剂及其同源G蛋白复合物的晶体结构最近才得到解决。现在有可能在分子细节上探索这种典型的跨膜受体结合激动剂的方式，跨膜传递冲动或信号事件，并启动一系列G蛋白导向的细胞内反应。使用在合理设计的介晶中间相中生长的三元复合物的晶体通过所谓的介晶方法确定结构。该方法在G蛋白偶联受体领域被证明是特别有用的，在过去的五年中，已经解决了十三种不同受体类型的结构。除了受体之外，该方法已被证明可用于各种各样的整合膜蛋白类别，包括细菌和真核视紫红质、光捕获复合物II（LHII）、光合反应中心、细胞色素氧化酶、β-桶、交换剂和整合膜肽。这证明了该方法的多功能性和范围，并支持了这样的观点，即在介观方法应包括在严重的膜结构生物学家的武器库。然而，要实现这一点，必须克服不愿意采用它的部分原因是，必须克服与处理晶体生长的粘性、粘稠的立方中间相相关的预期困难。收获和收集中间相生长晶体的衍射数据也有些令人不安。人们承认，该方法存在一些挑战。多年来，我们一直致力于建立该方法在分子水平上的工作原理，并使其用户友好。为此，已经开发了用于在皮科至纳升体积范围内处理中间相的工具，用于手动和机器人模式下的高效结晶筛选。已经实施了用于评价作为晶体发生的前奏的重构成立方相的双层的膜蛋白的功能活性的方法。玻璃结晶板已经建成，提供无与伦比的光学质量和灵敏度的新生晶体。脂质和沉淀剂筛选已被设计用于更合理的结晶形成方法，使得该方法现在可以应用于更广泛的膜蛋白类型。在这篇当前主题的文章中，这些分类的进展沿着概述了膜蛋白的方法。的前景和必须克服的挑战，以进一步发展的方法进行了说明。
The crystal structure of the β2-adrenergic receptor in complex with an agonist and its cognate G protein has just recently been solved. It is now possible to explore in molecular detail the means by which this paradigmatic transmembrane receptor binds agonist, communicates the impulse or signalling event across the membrane and sets in motion a series of G protein-directed intracellular responses. The structure was determined using crystals of the ternary complex grown in a rationally designed lipidic mesophase by the so-called in meso method. The method is proving to be particularly useful in the G protein-coupled receptor field where the structures of thirteen distinct receptor types have been solved in the past five years. In addition to receptors, the method has proven useful with a wide variety of integral membrane protein classes that include bacterial and eukaryotic rhodopsins, a light harvesting complex II (LHII), photosynthetic reaction centers, cytochrome oxidases, β-barrels, an exchanger, and an integral membrane peptide. This attests to the versatility and range of the method and supports the view that the in meso method should be included in the arsenal of the serious membrane structural biologist. For this to happen however, the reluctance in adopting it attributable, in part, to the anticipated difficulties associated with handling the sticky, viscous cubic mesophase in which crystals grow must be overcome. 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. Over the years, we have endeavored to establish how the method works at a molecular level and to make it user-friendly. To these ends, tools for handling the mesophase in the pico- to nano-liter volume range have been developed for highly efficient crystallization screening in manual and robotic modes. Methods have been implemented for evaluating the functional activity of membrane proteins reconstituted into the bilayer of the cubic phase as a prelude to crystallogenesis. Glass crystallization plates have been built that provide unparalleled optical quality and sensitivity to nascent crystals. Lipid and precipitant screens have been designed for a more rational approach to crystallogenesis such that the method can now be applied to an even wider variety of membrane protein types. In this Current Topics article, these assorted advances are outlined along with a summary of the membrane proteins that have yielded to the method. The prospects for and the challenges that must be overcome to further develop the method are described.