Quantitative Imaging of the Electrostatic Field of a Transmembrane Protein at Subnanometer Resolution by the use of Atomic Force Microscopy

Abstract

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. These translocation mechanisms are mainly based on electrostatic interactions. 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 sub-nanometer 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 unique tool to image biological systems to sub-nanometer resolution and to quantify their electrostatic properties.View Large Image | View Hi-Res Image | Download PowerPoint Slide