Small molecule permeation across membrane channels: Chemical modification to quantify transport across OmpF

Biological channels facilitate the exchange of molecules across membranes, but surprisingly the general tools to quantify transport is missing. Electrochemistry analysis is often the method to study functional properties of channels. However, analysing the current fluctuation of channels typically with their constriction region (CR) in the middle does not allow conclusion on successful transport, i.e. distinguishing translocation from binding, herein denoted as Scheme 1 and 2. To distinguish both processes, we added an additional barrier at the channel exit acting as a molecular counter. To identify permeation, we compare the molecule residence time in WT with a chemically modified one at the exit. Only permeable molecules would see the blocker at the channel exit and subsequently a modified residence time. As an example, we use the well‐studied outer membrane channel from E. coli, OmpF. Inspection of the structure suggests the position 181 which locates below the CR, and subsequently mutated into cysteine (E181C) in an otherwise cysteine‐free system then functionalized it by covalent binding with one of the two blockers MTSES or GLT which is larger in size. We measured the passage of model peptides, mono‐, tri‐, hepta‐arginine through the porin. Using the voltage dependence of the dwell time as the signal for transport, both mono‐ and tri‐arginine fitted with scheme 1 whilst hepta‐arginine fell into scheme 2. Both schemes determination meets well with previous studies, which in turn verified our simple approach with a simple molecular sensor at the exit. Later, we analysed norfloxacin, as an instance for antibiotic permeation. The modulated dwell time suggests such molecule falls into scheme 1 which demonstrates possible successful permeation of norfloxacin across OmpFwt. This approach may discriminate binding from translocation for a wide range of substrates. A potential application can be screening for scaffolds to improve the permeability of antibiotics.

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