Tools to Quantify Molecular Transport: Electroosmosis Dominates Electrophoresis of Fluoroquinolones Across the Outer Membrane Porin F (OmpF)

Abstract

We report that the dynamics of antibiotic capture and transport across a voltage-biased OmpF nanopore is dominated by the electroosmotic flow rather than the electrophoretic force. By reconstituting an OmpF porin in an artificial lipid bilayer and applying an electric field across, we are able to elucidate the permeation of molecules, and their mechanism of transport. This field gives rise to an electrophoretic force acting directly on a charged substrate, but also indirectly via coupling to all other mobile ions causing an electroosmotic flow. The directionality and magnitude of this flow depends on the selectivity of the channel. Modifying the charge state of three different substrates (Norfloxacin, Ciprofloxacin, and Enoxacin) by varying the pH between 6 and 9, while the charge and selectivity of OmpF is conserved, allows us to work under conditions where EOF and electrophoretic forces add or oppose. This configuration allows us to identify and distinguish the contributions of the electroosmotic flow and the electrophoretic force on translocation. Statistical analysis of the resolvable dwell-times reveals rich kinetic details regarding the direction and the stochastic movement of antibiotics inside the nanopore. We quantitatively describe the electroosmotic velocity component experienced by the substrates, and their diffusion coefficients inside the porin with an estimate of the energy barrier experienced by the molecules, caused by the interaction with the channel wall, slowing down the permeation by several orders of magnitude.