Current Fluctuation Analysis to Study Mg2+-Binding in the Bacterial Porin OmpF

Abstract

OmpF is a protein that forms water-filled, wide channels in the outer membrane of Escherichia coli. Previous studies have shown that the narrowest part of OmpF aqueous pore is largely responsible for its characteristic transport properties. To a large extent the pore eyelet controls the conductance and selectivity of the channel. In addition, the channel crystal structure obtained from a MgCl2 solution of the protein showed that Mg2+ ions bind between two acidic residues located at the channel constriction. Subsequent studies have demonstrated that this binding causes OmpF selectivity inversion. Here, we aim to get further insight into the effects of MgCl2 binding on OmpF ion conduction through the analysis of current fluctuations. We use planar lipid bilayer electrophysiology to measure time-resolved single-channel currents and analyze the associated fluctuations. We first evaluate the high-frequency regime for different concentrations of KCl and identify a contribution to the spectrum that seems to arise from surface charge fluctuations. We then compare these results with those obtained in MgCl2. In addition, we analyze the low-frequency current noise, which shows the characteristic Lorentzian profile, and study its dependence on electrolyte concentration in the presence of monovalent and divalent cations.