The permeation of most antibiotics through the outer membrane of Gram-negative bacteria occurs through porin channels. To design drugs with increased activity against Gram-negative bacteria in the face of the antibiotic resistance crisis, the strict constraints on the physicochemical properties of the permeants imposed by these channels must be better understood. Here we show that a combination of high-resolution electrophysiology, new noise-filtering analysis protocols and atomistic biomolecular simulations reveals weak binding events between the β-lactam antibiotic ampicillin and the porin PorB from the pathogenic bacterium Neisseria meningitidis. In particular, an asymmetry often seen in the electrophysiological characteristics of ligand-bound channels is utilised to characterise the binding site and molecular interactions in detail, based on the principles of electro-osmotic flow through the channel. Our results provide a rationale for the determinants that govern the binding and permeation of zwitterionic antibiotics in porin channels.

The actin cortex is a thin cross-linked network attached to the plasma membrane, which is responsible for the cell's shape during migration, division, and growth. In a reductionist approach, we created a minimal actin cortex (MAC) attached to a lipid membrane to correlate the filamentous actin architecture with its viscoelastic properties. The system is composed of a supported 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayer doped with the receptor lipid phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P) to which a constitutively active mutant of ezrin, which is a direct membrane-cytoskeleton linker, is bound. The formation of the MAC on the supported lipid bilayer is analyzed as a function of increasing PtdIns(4,5)P/ezrin pinning points, revealing an increase in the intersections between actin filaments, that is, the node density of the MAC. Bead tracking microrheology on the membrane-attached actin network provides information about its viscoelastic properties. The results show that ezrin serves as a dynamic cross-linker for the actin cortex attached to the lipid bilayer and that the stiffness of the network is influenced by the pinning point density, relating the plateau storage modulus G to the node density of the MAC.

Supported planar lipid bilayers (SLBs) prepared by spreading of unilamellar vesicles on hydrophilic substrates such as silicon dioxide are frequently used to investigate lipid−protein interactions by means of surface-sensitive methods. In recent years, the receptor lipid phosphatidylinositol-4,5-bisphosphate (PtdIns-[4,5]P 2 ) became particularly important as a significant number of proteins bind to this lipid at the inner leaflet of the plasma membrane. Here, we investigated how the lipid PtdIns[4,5]P 2 distributes between the two leaflets of an SLB on SiO 2 surfaces. We prepared SLBs on SiO 2 by spreading small unilamellar vesicles and quantified the adsorption of PtdIns[4,5]P 2 binding proteins providing information about the accessibility of PtdIns[4,5]P 2 . We compared protein binding to PtdIns[4,5]P 2 in SLBs with that in lipid monolayers on a 1,1,1-trimethyl-N-(trimethylsilyl)silanamine-functionalized SiO 2 surface using reflectometric interference spectroscopy and atomic force microscopy. Our results clearly demonstrate that the accessibility of PtdIns[4,5]P 2 for protein binding is reduced in SLBs compared to that in supported hybrid membranes, which is discussed in terms of PtdIns[4,5]P 2 distribution in the two leaflets of SLBs.

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