Equilibrium Partitioning of Binary Polymer Mixtures into Biological Nanopores

The cell interior, enclosed by membrane barriers, is a condensed solution of inorganic ions, polymers, carbohydrates, polynucleotides, and a large number of other organic molecules. Within cells, transport of metabolites and biopolymers, such as polynucleotides and proteins, occurs partly through specific transmembrane pores (mesoscopic ion channels) spanning cellular compartments. Examples of such functions are translocation of matrix RNA molecules from cell nucleus through nuclear pore complexes, ejection of viral genome from bacterial virus capsids into host bacterial cells, and translocation of protein factors across toxin channels in biological membranes. All these processes, that occur in the cellular milieu, are mediated by complex membrane structures and must be affected by molecular crowding. However, the effects of crowding are insufficiently addressed. Particular effects of certain types of molecular ``crowders\u27\u27 have only begun to be understood. Partially they stem from the dramatic complexity of the cellular translocation machinery, which makes direct observation of crowding phenomena extremely challenging. In addressing pore-assisted metabolite transport, a simplified experimental system with isolated protein channels in artificial membranes has been a useful model to probe and to assess crowding effects of such transport. In the experimental scheme employed here, a single pore is spontaneously assembled into an artificial bilayer separating two voltage-clamped electrolyte compartments. As the electric field is applied across the pore, the resulting ion current can be detected with high precision; interference of channel-passing or channel-excluded polymers with the ion flow gives a sensitive report on the studied phenomena of molecular crowding. In the absence of a field, polymers partition ``passively\u27\u27 into the pores, a direct result of the ``osmotic stress\u27\u27 induced by the polymers (crowders) themselves. Here, we study partitioning of polymers from a non-ideal binary mixture composed of polymers of different molecular weights going into structurally different ion channels. This is based on the assumption that in a two-component polymer mixture, one component that is preferentially excluded from the channel cavity will actively force the other component into the channel cavity. In order to assess the extent to which our results are useful in understanding concrete examples of ion-conducting aqueous pores and size-dependent forced partitioning into these pores. We describe the equation of state of a polymer mixture by its osmotic pressure, study the effects of polymer crowding on electrolyte solutions, investigate the partitioning of polymers from such mixtures into structurally different ion channels

Tools to Quantify Molecular Transport: Electroosmosis Dominates Electrophoresis of Fluoroquinolones Across the Outer Membrane Porin F (OmpF)

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, 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 where EOF 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, slowing down the permeation by several orders of magnitude

A general approach to protein folding using thermostable exoshells

10.1038/s41467-021-25996-4NATURE COMMUNICATIONS12