Department of Biosystems Science and Engineering, ETH Zurich , CH-4058 Basel, Switzerland.
Nano Lett. 2013;13(11):5585-93. doi: 10.1021/nl403232z. Epub 2013 Oct 3.
Elucidating the mechanisms by which proteins translocate small molecules and ions through transmembrane pores and channels is of great interest in biology, medicine, and nanotechnology. However, the characterization of pore forming proteins in their native state lacks suitable methods that are capable of high-resolution imaging (~1 nm) while simultaneously mapping physical and chemical properties. Here we report how force-distance (FD) curve-based atomic force microscopy (AFM) imaging can be applied to image the native pore forming outer membrane protein F (OmpF) at subnanometer resolution and to quantify the electrostatic field and potential generated by the transmembrane pore. We further observe the electrostatic field and potential of the OmpF pore switching "on" and "off" in dependence of the electrolyte concentration. Because electrostatic field and potential select for charged molecules and ions and guide them to the transmembrane pore the insights are of fundamental importance to understand the pore function. These experimental results establish FD-based AFM as a unique tool to image biological systems to subnanometer resolution and to quantify their electrostatic properties.
阐明蛋白质通过跨膜孔道和通道转运小分子和离子的机制，在生物学、医学和纳米技术领域具有重要意义。然而，在天然状态下对孔形成蛋白进行表征缺乏合适的方法，这些方法能够进行高分辨率成像（约 1nm），同时还能绘制物理和化学性质的图谱。在这里，我们报告了如何将基于力-距离（FD）曲线的原子力显微镜（AFM）成像应用于亚纳米分辨率下对天然孔形成的外膜蛋白 F（OmpF）进行成像，并定量测量跨膜孔产生的静电场和电势。我们进一步观察到 OmpF 孔在依赖于电解质浓度时“开启”和“关闭”的静电场和电势。由于静电场和电势会选择带电荷的分子和离子，并引导它们进入跨膜孔，因此这些见解对于理解孔的功能具有重要的基础意义。这些实验结果确立了基于 FD 的 AFM 作为一种独特的工具，可以对生物系统进行亚纳米分辨率的成像，并对其静电特性进行定量分析。
