Changes in Salt Concentration Modify the Translocation of Neutral Molecules through a ΔCymA Nanopore in a Non-monotonic Manner.

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.

Computational Modeling of Ion Transport in Bulk and through a Nanopore Using the Drude Polarizable Force Field.

In the past two decades, molecular dynamics simulations have become the method of choice for elucidating the transport mechanisms of ions through various membrane channels. Often, these simulations heavily rely on classical nonpolarizable force fields (FFs), which lack electronic polarizability in the treatment of the electrostatics. The recent advancements in the Drude polarizable FF lead to a complete set of parameters for water, ions, protein, and lipids, allowing for a more realistic modeling of membrane proteins. However, the quality of these Drude FFs remains untested for such systems. Here, we examine the quality of this FF set in two ways, i.e., (i) in simple ionic aqueous solution simulations and (ii) in more complex membrane channel simulations. First, the aqueous solutions of KCl, NaCl, MgCl2, and CaCl2 salts are simulated using the polarizable Drude and the nonpolarizable CHARMM36 FFs. The bulk conductivity has been estimated for both FF sets using applied-field simulations for several concentrations and temperatures in the case of all investigated salts and compared to experimental findings. An excellent improvement in the ability of the Drude FF to reproduce the experimental bulk conductivities for KCl, NaCl, and MgCl2 solutions can be observed but not in the case of CaCl2. Moreover, the outer membrane channel OmpC from the bacterium Escherichia coli has been employed to examine the ability of the polarizable and nonpolarizable FFs to reproduce ion transport-related quantities known from experiment. Unbiased and applied-field simulations have been performed in the presence of KCl using both FF sets. Unlike for the bulk systems of aqueous salt solutions, it has been found that the Drude FF is not accurate in modeling KCl transport properties across the OmpC porin.

Structural insights into the main S-layer unit of Deinococcus radiodurans reveal a massive protein complex with porin-like features.

In the extremophile bacterium Deinococcus radiodurans, the outermost surface layer is tightly connected with the rest of the cell wall. This integrated organization provides a compact structure that shields the bacterium against environmental stresses. The fundamental unit of this surface layer (S-layer) is the S-layer deinoxanthin-binding complex (SDBC), which binds the carotenoid deinoxanthin and provides both, thermostability and UV radiation resistance. However, the structural organization of the SDBC awaits elucidation. Here, we report the isolation of the SDBC with a gentle procedure consisting of lysozyme treatment and solubilization with the nonionic detergent n-dodecyl-ß-d-maltoside, which preserved both hydrophilic and hydrophobic components of the SDBC and allows the retention of several minor subunits. As observed by low-resolution single-particle analysis, we show that the complex possesses a porin-like structural organization, but is larger than other known porins. We also noted that the main SDBC component, the protein DR_2577, shares regions of similarity with known porins. Moreover, results from electrophysiological assays with membrane-reconstituted SDBC disclosed that it is a nonselective channel that has some peculiar gating properties, but also exhibits behavior typically observed in pore-forming proteins, such as porins and ionic transporters. The functional properties of this system and its porin-like organization provide information critical for understanding ion permeability through the outer cell surface of S-layer-carrying bacterial species.