An approach to prepare membrane proteins for single-molecule imaging
An approach to prepare membrane proteins for single-molecule imaging
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DOI：
10.1002/anie.200504506
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Lakey, Jeremy H.
中科院分区：
文献类型：
--
作者：
Cisneros, David A.;Muller, Daniel J.;Lakey, Jeremy H.
膜蛋白在从X射线晶体学和NMR光谱方法获得的高分辨率结构数据库中的代表性不足。[1]此外，它们经常形成大的，有时是短暂的超分子复合物。替代的方法，如冷冻电子显微镜（EM）和原子力显微镜（AFM）是必不可少的工具，能够提供有关嵌入在脂质膜中的膜蛋白的结构-功能关系的补充信息。两种方法提高了这些方法的分辨率，二维结晶和单粒子重建。在EM中，晶体提供电子衍射数据，可以提高分辨率，而单粒子平均可以用于> 200 kDa的蛋白质。高分辨率的AFM拓扑图可以揭示单个天然膜蛋白的结构细节，但作为一个先决条件，蛋白质必须吸附到原子级平坦的云母和密集包装在膜中，以限制其横向流动性。[2]虽然单个粒子的平均值显示了它们的共同结构，但选定的例子可用于表征结构灵活性、可变性和构象变化。[3]已经发表了许多在金上自组装单层的AFM分析的例子，[4]并且已经开发了硫脂质来在金上创建膜模拟表面。[5-7]然而，这些还没有被结合起来作为一种手段来成像膜蛋白。在这里，我们展示了一个共价组装膜蛋白成像的方法，避免结晶。原子平面金，工程蛋白质和化学修饰的脂质相结合，以快速组装固定和完全定向的样品（图1）。由此产生的单膜蛋白质的双折射模式AFM形貌图，
Membrane proteins are under represented in the database of high-resolution structures obtained from X-ray crystallographic and NMR spectroscopic methods.[1] Furthermore, they often form large and sometimes transient supramolecular complexes. Alternative approaches such as cryo-electron microscopy (EM) and Atomic force microscopy (AFM) are essential tools that are able to provide complementary information on the structure–function relationship of membrane proteins embedded in the lipid membrane. Two approaches increase the resolution of these methods, 2D crystallization and single-particle reconstruction. In EM, crystals provide electron diffraction data which can increase resolution, whereas single-particle averaging can be used for proteins> 200kDa. High-resolution AFM topographs can reveal structural details of single native membrane proteins but, as a prerequisite, the proteins must be adsorbed to atomically flat mica and densely packed in a membrane to restrict their lateral mobility.[2] Although averaging of single particles shows their common structures, selected examples can be used to characterize structural flexibility, variability, and conformational changes.[3] Many examples of AFM analysis of self-assembled monolayers on gold have been published,[4] and thiolipids have been developed to create membrane mimetic surfaces on gold.[5–7] However, these have not yet been combined as a means to image membrane proteins.Herein we demonstrate a covalent assembly approach to membrane protein imaging that avoids crystallization. Atomically flat gold, engineered proteins, and chemically modified lipids are combined to rapidly assemble immobile and fully oriented samples (Figure1). The resulting tapping-mode AFM topographs of single membrane proteins, set against a