Engineering the Extracellular Loops of Outermembrane Protein G in Creating a Nanopore Sensing Platform

Abstract

Outermembrane protein G (OmpG) is a 14-stranded beta barrel proin naturally found in the outermembrane of E. coli. It contains seven flexible extracellular loops that create a dynamic behavior in single channel recording termed gating. OmpG gating and its 8 Angstrom diameter make it unsuitable for sensing protein analytes that cannot enter its lumen, a process called translocation. Thus, we have taken advantage of its extracellular loops to facilitate binding of target analytes. We have seen that upon binding to loop 6, proteins cans be detected via the changes in OmpG gating. The changes in gating can be mediated by electrostatic interactions between the OmpG loops and the binding pocket of the protein analyte. We therefore extended our study to create OmpG nanopores with altered charged properties to understand the mechanism of detection and to enhance the binding signal of a specific target in a mixture. We also found that detection of proteins using OmpG can be achieved not only by using loop 6 but also with four other loops. With this knowledge we can engineer multiple binding sites into one nanopore to lower the limit of detection creating a more sensitive nanopore sensor. By gaining a fundamental understanding of OmpG and how it can interact with analytes will help in the building of a robust OmpG library for a plethora of target analytes.