Probing the Orientation of Reconstituted Maltoporin Channels at the Single-protein Level

Abstract

Recently we have shown that maltoporin channels reconstituted into black lipid membranes have pronounced asymmetric properties in both ion conduction and sugar binding. This asymmetry revealed also that maltoporin insertion is directional. However, the orientation in the lipid bilayer remained an open question. To elucidate the orientation, we performed point mutations at each side of the channel and analyzed the ion current fluctuation caused by an asymmetric maltohexaose addition. In a second series we used a chemically modified maltohexaose sugar molecule with inhibited entry possibility from the periplasmic side. In contrast to the natural outer cell wall of bacteria, we found that the maltoporin inserts in artificial lipid bilayer in such a way that the long extracellular loops are exposed to the same side of the membrane than protein addition. Based on this orientation, the directional properties of sugar binding were correlated to physiological conditions. We found that nature has optimized maltoporin channels by lowering the activation barriers at each extremity of the pore to trap sugar molecules from the external medium and eject them most efficiently to the periplasmic side.

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

“…Inspection of the porin structure suggests that the permeation of molecules through the channel is driven by molecular interactions with the surface rather than by free diffusion 68,69 . The first evidence for facilitated diffusion through a binding site was found in maltoporin, a maltose-specific channel from the outer membrane in E. coli [70][71][72][73] . a closer look at the OmpF structure revealed that it possesses a possible affinity site for ampicillin molecules (FIG.…”

“…Addition of antibiotics or sugar molecules on one (or both) side of the bilayer causes them to diffuse towards the channel. High-resolution ion-current fluctuation analysis allows detection of the passage of single molecules and thus characterization of facilitated permeation of sugars and antibiotics [68][69][70] . Addition of millimolar concentrations of ampicillin to the system can cause fluctuations in the ion current.…”

“…Increasing the ampicillin concentration causes a corresponding increase in the number of fluctuations observed, whereas the average time of channel blockage by antibiotic molecules is independent of the antibiotic concentration. Analysing the frequency of such fluctuations allows us to obtain the kinetic rates for interaction with the affinity site in the channel 68,70 .…”

“…The average through maltoporin (lamb) led to the development of an asymmetric single-binding-site model [70][71][72][73] The symbols P and aP indicate a channel state that is unoccupied or occupied by a bound antibiotic molecule, respectively; a and a′′ indicate free extracellular and periplasmic substrate, respectively.…”

“…However, for simplicity, we only describe the symmetric model only. Following the previous derivation we can calculate the flux 70,74 with Equation 3. Nature Reviews | Microbiology…”