A pH-tunable nanofluidic diode: electrochemical rectification in a reconstituted single ion channel

We report pH-dependent electrochemical rectification in a protein ion channel (the bacterial porin OmpF) reconstituted on a planar phospholipid membrane. The measurements performed at single-channel level show that the electric current is controlled by the protein fixed charge and it can be tuned by adjusting the local pH. Under highly asymmetric pH conditions, the channel behaves like a liquid diode. Unlike other nanofluidic devices that display also asymmetric conductance, here the microscopic charge distribution of the system can be explored by using the available high-resolution (2.4 A) channel crystallographic structure. Continuum electrostatics calculations confirm the hypothesized bipolar structure of the system. The selective titration of the channel residues is identified as the underlying physicochemical mechanism responsible for current rectification.     

A size-modified poisson–boltzmann ion channel model in a solvent of multiple ionic species: Application to voltage-dependent anion channel

We present a new size-modified Poisson–Boltzmann ion channel (SMPBIC) model and use it to calculate the electrostatic potential, ionic concentrations, and electrostatic solvation free energy for a voltage-dependent anion channel (VDAC) on a biological membrane in a solution mixture of multiple ionic species. In particular, the new SMPBIC model adopts a membrane surface charge density and a natural Neumann boundary condition to reflect the charge effect of the membrane on the electrostatics of VDAC. To avoid the singularity difficulties caused by the atomic charges of VDAC, the new SMPBIC model is split into three submodels such that the solution of one of the submodels is obtained analytically and contains all the singularity points of the SMPBIC model. The other two submodels are then solved numerically much more efficiently than the original SMPBIC model. As an application of this SMPBIC submodel partitioning scheme, we derive a new formula for computing the electrostatic solvation free energy. Numerical results for a human VDAC isoform 1 (hVDAC1) in three different salt solutions, each with up to five different ionic species, confirm the significant effects of membrane surface charges on both the electrostatics and ionic concentrations. The results also show that the new SMPBIC model can describe well the anion selectivity property of hVDAC1, and that the new electrostatic solvation free energy formula can significantly improve the accuracy of the currently used formula. © 2019 Wiley Periodicals, Inc.     

Protein retention in ion-exchange chromatography: effect of net charge and charge distribution

The charge regulated slab model is used to evaluate the salt dependence of the retention of Staphylococcal nuclease A and its mutants in cation-exchange chromatography. An important feature of this work is that the net charge of the proteins is varied in two different ways: (a) by changing the eluent pH so that the charges are created by protonation and (b) by point mutation at position 116. Since the structure of Staphylococcal nuclease and the mutants are known, the pH dependence of retention data of the different mutants gives detailed insights into the retention mechanism. Experimental results show that the salt dependence of retention is affected more strongly by changes of the eluent pH than by point mutations. This implies that the amino acid in position 116 has only a moderately strong interaction with the stationary phase surface and that a patch on one side of the protein surface is mainly responsible for the electrostatic interaction with the surface.     

Charging Metal-Organic Framework Membranes by Incorporating Crown Ethers to Capture Cations for Ion Sieving

Protein channels on the biofilm conditionally manipulate ion transport via regulating the distribution of charge residues, making analogous processes on artificial membranes a hot spot and challenge. Here, we employ metal–organic frameworks (MOFs) membrane with charge-adjustable subnano-channel to selectively govern ion transport. Various valent ions are binded with crown ethers embedded in the MOF cavity, which act as charged guest to regulate the channels’ charge state from the negativity to positivity. Compared with the negatively charged channel, the positive counterpart obviously enhances Li⁺/Mg²⁺ selectivity, which benefit from the reinforcement of the electrostatic repulsion between ions and the channel. Meanwhile, theoretical calculations reveal that Mg²⁺ transport through the more positively charged channel needed to overcome higher entrance energy barrier than that of Li⁺. This work provides a subtle strategy for ion-selective transport upon regulating the charge state of insulating membrane, which paves the way for the application like seawater desalination and lithium extraction from salt lakes.     

Divalent cations reduce the pH sensitivity of OmpF channel inducing the pK(a) shift of key acidic residues

In contrast to the highly-selective channels of neurophysiology employing mostly the exclusion mechanism, different factors account for the selectivity of large channels. Elucidation of these factors is essential for understanding the permeation mechanisms in ion channels and their regulation in vivo. The interaction between divalent cations and a protein channel, the bacterial porin OmpF, has been investigated paying attention to the channel selectivity and its dependence on the solution pH. Unlike the experiments performed in salts of monovalent cations, the channel is now practically insensitive to pH, being anion selective all over the pH range considered. Electrostatic calculations based on the available structural data suggest that the binding of divalent cations has two main effects: (i) the pK(a) values of key ionizable groups differ significantly from those of the isolated groups in solution and (ii) the cation binding has a decisive impact on the effective electric charge regulating the channel selectivity. A simple molecular model based on statistical thermodynamics provides additional qualitative explanations to the experimental findings that could also be useful for other related systems like synthetic nanopores, ion exchange membranes, and polyelectrolyte multilayers.     

Study of the Selectivity of SnO2 Supported Membranes Prepared by a Sol-Gel Route

In this work the sol-gel process was used to prepare SnO2 supported membranes with an average pore size of 2.5 nm. The effects of salt concentration (NaCl or CaCl2) and of the pH of the aqueous solutions used on the flux and selectivity through the SnO2 membrane were analyzed by permeation experiments and the results interpreted taking account of the zeta potential values determined from the electrophoretic mobility of the SnO2 powder aqueous dispersion. The results show that the ion flux (Na+, Ca2+ and Cl–) throughout the membrane is determined by the electrostatic repulsion among these species and the surface charge at the tin oxide-solution interface.     

Molecular dynamics and brownian dynamics investigation of ion permeation and anesthetic halothane effects on a proton-gated ion channel

Bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC) is activated to cation permeation upon lowering the solution pH. Its function can be modulated by anesthetic halothane. In the present work, we integrate molecular dynamics (MD) and Brownian dynamics (BD) simulations to elucidate the ion conduction, charge selectivity, and halothane modulation mechanisms in GLIC, based on recently resolved X-ray crystal structures of the open-channel GLIC. MD calculations of the potential of mean force (PMF) for a Na(+) revealed two energy barriers in the extracellular domain (R109 and K38) and at the hydrophobic gate of transmembrane domain (I233), respectively. An energy well for Na(+) was near the intracellular entrance: the depth of this energy well was modulated strongly by the protonation state of E222. The energy barrier for Cl(-) was found to be 3-4 times higher than that for Na(+). Ion permeation characteristics were determined through BD simulations using a hybrid MD/continuum electrostatics approach to evaluate the energy profiles governing the ion movement. The resultant channel conductance and a near-zero permeability ratio (P(Cl)/P(Na)) were comparable to experimental data. On the basis of these calculations, we suggest that a ring of five E222 residues may act as an electrostatic gate. In addition, the hydrophobic gate region may play a role in charge selectivity due to a higher dehydration energy barrier for Cl(-) ions. The effect of halothane on the Na(+) PMF was also evaluated. Halothane was found to perturb salt bridges in GLIC that may be crucial for channel gating and open-channel stability, but had no significant impact on the single ion PMF profiles.     

Colloidal stability of aqueous nanofibrillated cellulose dispersions

Cellulose nanofibrils constitute an attractive raw material for carbon-neutral, biodegradable, nanostructured materials. Aqueous suspensions of these nanofibrils are stabilized by electrostatic repulsion arising from deprotonated carboxyl groups at the fibril surface. In the present work, a new model is developed for predicting colloidal stability by considering deprotonation and electrostatic screening. This model predicts the fibril-fibril interaction potential at a given pH in a given ionic strength environment. Experiments support the model predictions that aggregation is induced by decreasing the pH, thus reducing the surface charge, or by increasing the salt concentration. It is shown that the primary mechanism for aggregation upon the addition of salt is the surface charge reduction through specific interactions of counterions with the deprotonated carboxyl groups, and the screening effect of the salt is of secondary importance.     

Stabilization of Liposomes by Polyelectrolytes: Mechanism of Interaction and Role of Experimental Conditions

ζ-potential measurements on LUVs allow to evidence the influence of pH, ionic salt concentration, and polyelectrolyte charge on the interaction between polyelectrolyte (chitosan and hyaluronan) and zwitterionic lipid membrane. First, chitosan adsorption is studied: adsorption is independent on the chitosan molecular weight and corresponds to a maximum degree of decoration of 40% in surface coverage. From the dependence with pH and independence with MW, it is concluded that electrostatic interactions are responsible of chitosan adsorption which occurs flat on the external surface of the liposomes. The vesicles become positively charged in the presence of around two repeat units of chitosan added per lipid accessible polar head in acid medium down to pH = 7.2. Direct optical microscopy observations of GUVs shows a stabilization of the composite liposomes under different external stresses (pH and salt shocks) which confirms the strong electrostatic interaction between the chitosan and the lipid membrane. It is also demonstrated that the liposomes are stabilized by chitosan adsorption in a very wide range of pH (2.0 < pH < 12.0). Then, hyaluronan (HA), a negatively charged polyelectrolyte, is added to vesicles; the vesicles turn rapidly negatively charged in presence of adsorbed HA Finally, we demonstrated that hyaluronan adsorbs on positively charged chitosan-decorated liposomes at pH < 7.0 leading to charge inversion in the liposome decorated by the chitosan-hyaluronan bilayer. Our results demonstrate the adsorption of positive and/or negative polyelectrolyte at the surface of lipidic vesicles as well as their role on vesicle stabilization and charge control.     

Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes

Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and observed the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin containing lipid bilayers is of potential for the efficient electrophysiological characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.     

Asymmetry of lipid bilayers induced by monovalent salt: atomistic molecular-dynamics study

Interactions between salt ions and lipid components of biological membranes are essential for the structure, stability, and functions of the membranes. The specific ionic composition of aqueous buffers inside and outside of the cell is known to differ considerably. To model such a situation we perform atomistic molecular-dynamics (MD) simulations of a single-component phosphatidylcholine lipid bilayer which separates two aqueous reservoirs with and without NaCl salt. To implement the difference in electrolyte composition near two membrane sides, a double bilayer setup (i.e., two bilayers in a simulation box) is employed. It turns out that monovalent salt, being in contact with one leaflet only, induces a pronounced asymmetry in the structural, electrostatic, and dynamical properties of bilayer leaflets after 50 ns of MD simulations. Binding of sodium ions to the carbonyl region of the leaflet which is in contact with salt results in the formation of "Na-lipids" complexes and, correspondingly, reduces mobility of lipids of this leaflet. In turn, attractive interactions of chloride ions (mainly located in the aqueous phase close to the water-lipid interface) with choline lipid groups lead to a substantial (more vertical) reorientation of postphatidylcholine headgroups of the leaflet adjoined to salt. The difference in headgroup orientation on two sides of a bilayer, being coupled with salt-induced reorientation of water dipoles, leads to a notable asymmetry in the charge-density profiles and electrostatic potentials of bilayer constitutes of the two leaflets. Although the overall charge density of the bilayer is found to be almost insensitive to the presence of salt, a slight asymmetry in the charge distribution between the two bilayer leaflets results in a nonzero potential difference of about 85 mV between the two water phases. Thus, a transmembrane potential of the order of the membrane potential in a cell can arise without ionic charge imbalance between two aqueous compartments.     

How protonation modulates the interaction between proteins and pH-responsive hydrogel films

Hydrogels of pH-responsive polymers are promising candidates for the design of functional biomaterials. In this context, understanding the complexity of the interaction between these materials and proteins is essential. A recently developed molecular-level equilibrium theory for protein adsorption on hydrogels of cross-linked polyacid chains allows for modeling size, shape, charge distribution, protonation state and conformational degrees of freedom of all chemical species in the system; proteins are described using a coarse-grained model of their crystallographic structure. This review summarizes our recent studies, which have focused on understanding how the interaction between proteins and pH-responsive hydrogel films depends on the pH and salt concentration, both in single protein solutions and mixtures. In particular, we discuss the key role that protonation plays in mediating the polymer-protein electrostatic attractions that drive adsorption. Deprotonation of the polyacid network modifies the nano-environment inside the hydrogel; the local pH drops inside the film. In single protein solutions, protonation of amino acid residues in this lower-pH environment favors adsorption to the hydrogel. Upon adsorption, the net charge of the protein can be several units more positive than in the solution. The various amino acids protonate differently, in a non-trivial way, which gives flexibility to the protein to enhance its positive charge and favor adsorption under a wide range of conditions. In binary and ternary protein solutions, amino acid protonation is the decisive factor for selective adsorption under certain conditions. We show that the polymer network composition and the solution pH can be used to separate and localize proteins within nanometer-sized regions.     

Synthesis,characterization and electrochemical properties of cation selective ion exchange composite membranes

In this work polystyrene based strontium phosphate membranes (SPMs) were prepared by applying different pressures. The membrane potential is measured with uni-univalent electrolytes (KCl, NaCl, and LiCl) solutions using saturated calomel electrodes (SCEs). The effective fixed charge density of these membranes is determined by the Torell, Meyer and Sievers method and it showed the dependence of membrane potential on the porosity, the charge on the membrane matrix, charge and size of permeating ions. The membranes are characterized by X-ray diffraction, scanning electron microscopy and IR spectroscopy. The order of surface charge density for electrolytes is KCl > NaCl > LiCl. Other parameters such as transport number, distribution coefficient, charge effectiveness and related parameters are calculated. The membrane was found to be mechanically stable, and can be operated over a wide pH range.     

Simulating the proton transfer in gramicidin A by a sequential dynamical Monte Carlo method

The large interest in long-range proton transfer in biomolecules is triggered by its importance for many biochemical processes such as biological energy transduction and drug detoxification. Since long-range proton transfer occurs on a microsecond time scale, simulating this process on a molecular level is still a challenging task and not possible with standard simulation methods. In general, the dynamics of a reactive system can be described by a master equation. A natural way to describe long-range charge transfer in biomolecules is to decompose the process into elementary steps which are transitions between microstates. Each microstate has a defined protonation pattern. Although such a master equation can in principle be solved analytically, it is often too demanding to solve this equation because of the large number of microstates. In this paper, we describe a new method which solves the master equation by a sequential dynamical Monte Carlo algorithm. Starting from one microstate, the evolution of the system is simulated as a stochastic process. The energetic parameters required for these simulations are determined by continuum electrostatic calculations. We apply this method to simulate the proton transfer through gramicidin A, a transmembrane proton channel, in dependence on the applied membrane potential and the pH value of the solution. As elementary steps in our reaction, we consider proton uptake and release, proton transfer along a hydrogen bond, and rotations of water molecules that constitute a proton wire through the channel. A simulation of 8 mus length took about 5 min on an Intel Pentium 4 CPU with 3.2 GHz. We obtained good agreement with experimental data for the proton flux through gramicidin A over a wide range of pH values and membrane potentials. We find that proton desolvation as well as water rotations are equally important for the proton transfer through gramicidin A at physiological membrane potentials. Our method allows to simulate long-range charge transfer in biological systems at time scales, which are not accessible by other methods.     

Adsorption of heterogeneously charged nanoparticles on a variably charged surface by the extended surface complexation approach: charge regulation, chemical heterogeneity, and surface complexation

Adsorption of randomly branched polyelectrolytes, "hairy" particles and internally structured macromolecules, collectively denoted as heterogeneously charged nanoparticles, on charged surfaces is important in many technological and natural processes. In this paper, we will focus on (1) the charge regulation of both the nanoparticle and the surface and (2) the surface complexation between the particle functional groups and the surface sites and will theoretically study the adsorption using the extended surface complexation approach. The model explicitly considers the electrochemical potential of a nanoparticle with an average (smeared-out) structure and charge both in bulk solution and on the surface to obtain the equilibrium adsorption. The chemical heterogeneity of the particle is described by a distribution of the protonation constant. Detailed analysis of the chemical potential of the adsorbed nanoparticle reveals that the pH and salt dependence of the adsorption can be largely explained by the balance between an energy gain resulting from the particle and surface charge regulation and the surface complexation and an energy loss from the unfavorable interparticle electrostatic repulsion close to the surface. This conclusion is also supported by the strong impacts that the chemical heterogeneity of the particle functional groups, the magnitude of the surface complexation, the number of the functional groups, and the size of the particle have on the adsorption.     

Behaviour of a cobalt spinel ultrafiltration membrane during salt filtration with different ionic strengths

The characterisation of the surface charge of a cobalt spinel ultrafiltration membrane was investigated in the presence of electrolyte solutions by means of streaming potential measurements. The selectivity of a membrane towards different salts depends on the electrostatic interactions between the species in solution and the charge of the membrane surface. Firstly, the values of the streaming potential coefficient were measured during the filtration of NaCl, CaCl₂, Na₂SO₄ and CaSO₄ as a function of pH and ionic strength. The iso-electric point was determined for each electrolyte. Secondly, the rejection rates of the electrolytes were measured by ionic chromatography. The rejection was explained by a correlation with the surface charge of the membrane.     

纳通道的物质传输特性及应用

近年来,随着材料科学、微纳加工技术和微纳尺度物质传输理论的发展,纳通道技术得到了越来越多的研究和关注。纳通道包括生物纳通道和人工纳通道,其孔径通常为1~100 nm。在这一尺度下,通道表面与通道内物质之间的作用概率大大增强,使得纳通道表现出许多与宏观体系不同的物质传输特性,例如通道表面电荷与通道内离子之间的静电作用产生了离子选择性,通道内电化学势的不对称分布产生了离子整流特性,物质传输过程中占据通道产生了阻塞脉冲特性等。纳通道中的这些物质传输特性在传感、分离、能源等领域具有广泛应用,例如通过对纳通道进行功能化修饰可以实现门控离子传输;利用亚纳米尺度的通道可以实现单分子传感;利用通道与传输物质之间的相互作用可以实现离子、分子、纳米粒子的分离;利用纳通道的离子选择性可以在通道内实现电荷分离,将不同形式的能量（如光、热、压力、盐差等）高效转化为电能。纳通道技术是化学、材料科学、纳米技术等多学科的交叉集合,在解决生物、环境、能源等基本问题方面具有良好的前景。该文综述了近10年来与纳通道物质传输理论以及纳通道技术应用相关的前沿研究,梳理了纳通道技术的发展过程,并对其在各个领域的应用进行了总结与展望。     

The Effects of Salt and pH on the Spermatozoa Agglutinating Activity of Carp Ovum Cystatin by Molecular Dynamics Simulations

In order to elucidate how the spermatozoa agglutinating activity of carp ovum cystatin is significantly affected by salt and pH in the atomic scale, an homology model of carp ovum cystatin was constructed based on the crystal structure of chicken egg white cystatin in this study. Although these two proteins exhibit only 36.11% of sequence identity, they share similar folds. Our homology model also shows that 16 positively charged residues are equally distributed on the surface of carp ovum cystatin, leading to agglutinating spermatozoa via electrostatic interaction. The structure of carp ovum cystatin is not significantly affected by salt and pH during 400 ps molecular dynamics simulations. However, the total accessible surface area (ASA) of the positively charged residues on the surface of carp ovum cystatin decreased with increasing salt concentration, leading to the abolishment of its spermatozoa agglutinating activity. Moreover, 13 Lys residues became deprotonated at pH 11, resulting in the decrease of the net positive charges on the surface of carp ovum cystatin and thus losing its spermatozoa agglutinating activity.     

Rapid separations of nile blue stained microorganisms as cationic charged species by chip-CE with LIF

Rapid detection of microorganisms by alternative methods is desirable. Electromigration separation methods have the capability to separate microorganisms according to their charge and size and laser-induced fluorescence (LIF) detection have single-cell detection capability. In this work, a new combined separation and detection scheme was introduced using chip-based capillary electrophoresis (chip-CE) platform with LIF detection. Three microorganisms Escherichia coli, Staphylococcus aureus, and Candida albicans were selected as representatives of Gram-positive bacteria, Gram-negative bacteria, and fungi. While their cells carry an overall negative charge in neutral to alkaline pH, staining them with nile blue (NB) provided highly sensitive LIF detection with excitation and emission wavelengths at 635 nm and 685 nm, respectively, and at the same time, the overall charge was converted to positive. Electrolyte pH and concentration of polyethylene oxide (PEO) significantly affected the resolution of the microorganisms. Their optimal separation in the 14 mm separation channel was achieved in less than 30 s (R(s) > 5.3) in an electrolyte consisting of 3.94 mM Tris, 0.56 mM boric acid, 0.013 mM ethylenediaminetetraacetic acid disodium salt dihydrate (pH 10.5), and 0.025% PEO, with injection/separation voltages of +1000/+1000 V. The separation mechanism is likely employing contributions to the overall cationic charge from both the prevalently anionic membrane proteins and the cationic NB. Importantly, the resulting cationic NB-stained cells exhibited excellent separation selectivity and efficiency of ～38000 theoretical plates for rapid separations within 30-40 s. The results indicate the potential of chip-CE for microbial analysis, which offers separations of a wide range of species with high efficiency, sensitivity, and throughput.     

Poisson-Boltzmann theory of the charge-induced adsorption of semi-flexible polyelectrolytes

A model is suggested for the structure of an adsorbed layer of a highly charged semi-flexible polyelectrolyte on a weakly charged surface of opposite charge sign. The adsorbed phase is thin, owing to the effective reversal of the charge sign of the surface upon adsorption, and ordered, owing to the high surface density of polyelectrolyte strands caused by the generally strong binding between polyelectrolyte and surface. The Poisson-Boltzmann equation for the electrostatic interaction between the array of adsorbed polyelectrolytes and the charged surface is solved for a cylindrical geometry, both numerically, using a finite element method, and analytically within the weak curvature limit under the assumption of excess monovalent salt. For small separations, repulsive surface polarization and counterion osmotic pressure effects dominate over the electrostatic attraction and the resulting electrostatic interaction curve shows a minimum at nonzero separations on the Angstrom scale. The equilibrium density of the adsorbed phase is obtained by minimizing the total free energy under the condition of equality of chemical potential and osmotic pressure of the polyelectrolyte in solution and in the adsorbed phase. For a wide range of ionic conditions and charge densities of the charged surface, the interstrand separation as predicted by the Poisson-Boltzmann model and the analytical theory closely agree. For low to moderate charge densities of the adsorbing surface, the interstrand spacing decreases as a function of the charge density of the charged surface. Above about 0.1 M excess monovalent salt, it is only weakly dependent on the ionic strength. At high charge densities of the adsorbing surface, the interstrand spacing increases with increasing ionic strength, in line with the experiments by Fang and Yang [J. Phys. Chem. B 101, 441 (1997)].