Small Molecule Permeation Across Membrane Channels: Chemical Modification to Quantify Transport Across OmpF
2018-09-04T14:20:43Z (GMT) by
Biological channels facilitate the exchange of small molecules across membranes, but surprisingly there is a lack of general tools for the identification and quantification of transport (i.e., translocation and binding). Analyzing the ion current fluctuation of a typical channel with its constriction region in the middle does not allow a direct conclusion on successful transport. For this, we created an additional barrier acting as a molecular counter at the exit of the channel. To identify permeation, we mainly read the molecule residence time in the channel lumen as the indicator whether the molecule reached the exit of the channel. As an example, here we use the well-studied porin, OmpF, an outer membrane channel from <i>E. coli</i>. Inspection of the channel structure suggests that aspartic acid at position 181 is located below the constriction region (CR) and we subsequently mutated this residue to cysteine, where else cysteine free and functionalized it by covalent binding with 2-sulfonatoethyl methanethiosulfonate (MTSES) or the larger glutathione (GLT) blockers. Using the dwell time as the signal for transport, we found that both mono-arginine and tri-arginine permeation process is prolonged by 20% and 50% respectively through OmpF<sub>E181C</sub>MTSES, while the larger sized blocker modification OmpF<sub>E181C</sub>GLT drastically decreased the permeation of mono-arginine by 9-fold and even blocked the pathway of the tri-arginine. In case of the hepta-arginine as substrate, both chemical modifications led to an identical ‘blocked’ pattern observed by the dwell time of ion current fluctuation of the OmpF<sub>wt</sub>. As an instance for antibiotic permeation, we analyzed norfloxacin, a fluoroquinolone antimicrobial agent. The modulation of the interaction dwell time suggests possible successful permeation of norfloxacin across OmpF<sub>wt</sub>. This approach may discriminate blockages from translocation events for a wide range of substrates. A potential application could be screening for scaffolds to improve the permeability of antibiotics.