Sumino Ayumi, Uchihashi Takayuki, Oiki Shigetoshi
PRESTO, Japan Science and Technology Agency (JST) , 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012, Japan.
Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of Fukui , 23-3 Matsuokashimoaizuki, Yoshida-gun, Fukui 910-1193, Japan.
J Phys Chem Lett. 2017 Feb 16;8(4):785-793. doi: 10.1021/acs.jpclett.6b03058. Epub 2017 Feb 1.
Here, we have developed a method of oriented reconstitution of the KcsA potassium channel amenable to high-resolution AFM imaging. The solubilized full-length KcsA channels with histidine-tagged (His-tag) C-terminal ends were attached to a Ni-coated mica surface, and then detergent-destabilized liposomes were added to fill the interchannel space. AFM revealed that the membrane-embedded KcsA channels were oriented with their extracellular faces upward, seen as a tetrameric square shape. This orientation was corroborated by the visible binding of a peptide scorpion toxin, agitoxin-2. To observe the cytoplasmic side of the channel, a His-tag was inserted into the extracellular loop, and the oppositely oriented channels provided wholly different images. In either orientation, the channels were individually dispersed at acidic pH, whereas they were self-assembled at neutral pH, indicating that the oriented channels are allowed to diffuse in the membrane. This method is readily applicable to membrane proteins in general for AFM imaging.
在此，我们开发了一种适用于高分辨率原子力显微镜（AFM）成像的KcsA钾通道定向重组方法。将带有组氨酸标签（His标签）C末端的可溶性全长KcsA通道附着在镀镍云母表面，然后添加去污剂使其不稳定的脂质体以填充通道间空间。原子力显微镜显示，嵌入膜中的KcsA通道以其细胞外表面向上的方向排列，呈四聚体方形。一种肽蝎毒素agitoxin-2的可见结合证实了这种方向。为了观察通道的细胞质侧，将His标签插入细胞外环，而方向相反的通道提供了完全不同的图像。在任一方向上，通道在酸性pH下单独分散，而在中性pH下它们会自组装，这表明定向通道能够在膜中扩散。这种方法通常很容易应用于膜蛋白的原子力显微镜成像。
