Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy
Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy
复制标题
DOI：
10.3791/55148
复制
发表时间：
2017-03-01
影响因子：
1.2
通讯作者：
Jegerschold, Caroline
中科院分区：
文献类型：
--
作者：
Kumar, Ramakrishnan B.;Zhu, Lin;Jegerschold, Caroline
当连接到膜表面时，单配体蛋白发挥其功能，并且这种相互作用取决于特定的脂质组成和足够区域的可用性来执行功能。纳米盘用于提供具有受控尺寸和脂质含量的膜表面。在没有结合的外在蛋白质，磷钨酸钠染色的纳米盘出现硬币堆叠时，从侧面观察透射电子显微镜（TEM）。因此，该方案旨在有意促进堆叠;因此，防止堆叠可以解释为膜结合蛋白与纳米盘的结合。在进一步的步骤中，蛋白质-纳米盘复合物的TEM图像可以用标准的单颗粒方法处理，以产生低分辨率结构作为更高分辨率cryoEM工作的基础。此外，纳米盘提供适合于TEM或非变性凝胶电泳的样品。为了说明该方法，提出了纳米盘上的5-脂氧合酶的Ca 2+诱导的结合。
Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.