Highlights d Projection map of a native S-layer at 4.5-Å resolution by cryo-EC d SDBC structure at 11-Å resolution by cryo-EM single-particle analysis d SDBC in situ localization and orientation d S-layer as a stabilizing component of cell-wall status

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 it, 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 in which electroosmotic flow 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, which slows down the permeation by several orders of magnitude.

Quantifying the passage of the large peptide protamine (Ptm) across CymA, apassive channel for cyclodextrin uptake,isinthe focus of this study.Using areporter-pair-based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation through CymA is the rate-limiting factor.F urthermore,w e reconstituted single CymA channelsi nto planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage-dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1-2 events per second and long dwell times,i na greement with the liposome study.A pplied-field and steered molecular dynamics simulations added an atomistic view of the permeation events.Itcan be concluded that ac oncentration gradient of 1 mm Ptm leads to at ranslocation rate of about one molecule per second and per channel.

The voltage-dependent transport through biological and artificial nanopores is being used in many applications such as DNA or protein sequencing and sensing. The primary approach to determine the transport has been to measure the temporal ion current fluctuations caused by solutes when applying external voltages. Crossing the nanoscale confinement in the presence of an applied electric field primarily relies on two factors, i.e., the electrophoretic drag and electroosmosis. The electroosmotic flow (EOF) is a voltage-dependent ion-associated flow of solvent molecules, i.e., usually water, and depends on many factors, such as pH, temperature, pore diameter, and also the concentration of ions. The exact interplay between these factors is so far poorly understood. In this joint experimental and computational study, we have investigated the dependence of the EOF on the concentration of the buffer salt by probing the transport of α-cyclodextrin molecules through the ΔCymA channel. For five different KCl concentrations in the range between 0.125 and 2 M, we performed applied-field molecular dynamics simulations and analyzed the ionic flow and the EOF across the ΔCymA pore. To our surprise, the concentration-dependent net ionic flux changes non-monotonically and nonlinearly and the EOF is seen to follow the same pattern. On the basis of these findings, we were able to correlate the concentration-dependent EOF with experimental kinetic constants for the translocation of α-cyclodextrin through the ΔCymA nanopore. Overall, the results further improve our understanding of the EOF-mediated transport through nanopores and show that the EOF needs to seriously be taken into consideration when analyzing the permeation of (neutral) substrates through nanopores.

Cobalt bisdicarbollides (COSANs) are inorganic boron-based anions that have been previously reported to permeate by themselves through lipid bilayer membranes, a propensity that is related to their superchaotropic character. We now introduce their use as selective and efficient molecular carriers of otherwise impermeable hydrophilic oligopeptides through both artificial and cellular membranes, without causing membrane lysis or poration at low micromolar carrier concentrations. COSANs transport not only arginine-rich but also lysine-rich peptides, whereas low-molecular-weight analytes such as amino acids as well as neutral and anionic cargos (phalloidin and BSA) are not transported. In addition to the unsubstituted isomers (known as ortho- and meta-COSAN), four derivatives bearing organic substituents or halogen atoms have been evaluated, and all six of them surpass established carriers such as pyrenebutyrate in terms of activity. U-tube experiments and black lipid membrane conductance measurements establish that the transport across model membranes is mediated by a molecular carrier mechanism. Transport experiments in living cells showed that a fluorescent peptide cargo, FITC-Arg8, is delivered into the cytosol.