Membrane-protein structure determination by solid-state NMR spectroscopy of microcrystals
Membrane-protein structure determination by solid-state NMR spectroscopy of microcrystals
复制标题
DOI：
10.1038/nmeth.2248
复制
发表时间：
2012-12-01
期刊：
影响因子：
48
通讯作者：
Linke, Dirk
中科院分区：
文献类型：
--
作者：
Shahid, Shakeel Ahmad;Bardiaux, Benjamin;Linke, Dirk
在现有的原子分辨率结构中，膜蛋白在很大程度上代表性不足。在蛋白质纯化过程中使用去污剂阻碍了X射线晶体学中有序晶体的形成，并导致分子翻滚较慢，阻碍了溶液状态NMR的应用。固态魔角旋转核磁共振波谱是一种新兴的膜蛋白结构生物学方法，可以克服这些技术问题。在这里，我们提出了固态NMR结构的小肠结肠炎耶尔森氏菌粘附素A（YadA）的跨膜结构域。该样品来自结晶试验，仅产生衍射差的微晶。我们使用单一的、均匀的C-13-和N-15-标记的样品解决了结构。此外，固态NMR使我们能够获得有关结构部分的灵活性和流动性的信息，这些信息与进化保守信息相结合，为YadA的自动转运机制提供了新的见解。
Membrane proteins are largely underrepresented among available atomic-resolution structures. The use of detergents in protein purification procedures hinders the formation of well-ordered crystals for X-ray crystallography and leads to slower molecular tumbling, impeding the application of solution-state NMR. Solid-state magic-angle spinning NMR spectroscopy is an emerging method for membrane-protein structural biology that can overcome these technical problems. Here we present the solid-state NMR structure of the transmembrane domain of the Yersinia enterocolitica adhesin A (YadA). The sample was derived from crystallization trials that yielded only poorly diffracting microcrystals. We solved the structure using a single, uniformly C-13- and N-15-labeled sample. In addition, solid-state NMR allowed us to acquire information on the flexibility and mobility of parts of the structure, which, in combination with evolutionary conservation information, presents new insights into the autotransport mechanism of YadA.