Ionic partition and transport in multi-ionic channels: a molecular dynamics simulation study of the OmpF bacterial porin

We performed all-atom molecular dynamics simulations studying the partition of ions and the ionic current through the bacterial porin OmpF and two selected mutants. The study is motivated by new, interesting experimental findings concerning their selectivity and conductance behavior at neutral pH. The mutations considered here are designed to study the effect of removal of negative charges present in the constriction zone of the wild-type OmpF channel (which contains, on one side, a cluster with three positive residues, and on the other side, two negatively charged residues). Our results show that these mutations induce an exclusion of cations from the constriction zone of the channel, substantially reducing the flow of cations. In fact, the partition of ions inside the mutant channels is strongly inhomogeneous, with regions containing an excess of cations and regions containing an excess of anions. Interestingly, the overall number of cations inside the channel is larger than the number of anions, this excess being different for each protein channel. We found that the differences in ionic charge inside these channels are justified by the differences in electric charge between the wild-type OmpF and the mutants, following an electroneutral balance.     

Brownian dynamics simulation of ion flow through porin channels

Bacterial porins, which allow the passage of solutes across the outer bacterial membrane, are structurally well characterized. They therefore lend themselves to detailed studies of the determinants of ion flow through transmembraneous channels. In a comparative study, we have performed Brownian dynamics simulations to obtain statistically significant transfer efficiencies for cations and anions through matrix porin OmpF, osmoporin OmpK36, phosphoporin PhoE and two OmpF charge mutants.The simulations show that the electrostatic potential at the highly charged channel constriction serves to enhance ion permeability of either cations or anions, dependent on the type of porin. At the same time translocation of counterions is not severely impeded. At the constriction, cations and anions follow distinct trajectories, due to the segregation of basic and acidic protein residues.Simulated ion selectivity and relative conductance agree well with experimental values, and are dependent crucially on the charge constellation at the pore constriction. The experimentally observed decrease in ion selectivity and single channel conductance with increasing ionic strength is well reproduced and can be attributed to electrostatic shielding of the pore lining.     

How pore mouth charge distributions alter the permeability of transmembrane ionic channels.

This paper investigates the effects that surface dipole layers and surface charge layers along the pore mouth-water interface can have on the electrical properties of a transmembrane channel. Three specific molecular sources are considered: dipole layers formed by membrane phospholipids, dipole layers lining the mouth of a channel-forming protein, and charged groups in the mouth of a channel-forming protein. We find, consistent with previous work, that changing the lipid-water potential difference only influences channel conduction if the rate-limiting step takes place well inside the channel constriction. We find that either mouth dipoles or mouth charges can act as powerful ion attractors increasing either cation or anion concentration near the channel entrance to many times its bulk value, especially at low ionic strengths. The effects are sufficient to reconcile the apparently contradictory properties of high selectivity and high conductivity, observed for a number of K+ channel systems. We find that localizing the electrical sources closer to the constriction entrance substantially increases their effectiveness as ion attractors; this phenomenon is especially marked for dipolar distributions. An approximate treatment of electrolyte shielding is used to discriminate between the various mechanisms for increasing ionic concentration near the constriction entrance. Dipolar potentials are far less sensitive to ionic strength variation than potentials due to fixed charges. We suggest that the K+ channel from sarcoplasmic reticulum does not have a fixed negative charge near the constriction entrance; we suggest further that the Ca+2-activated K+ channel from transverse tubule does have such a charge.     

Backbone dipoles generate positive potentials in all proteins: origins and implications of the effect

Asymmetry in packing the peptide amide dipole results in larger positive than negative regions in proteins of all folding motifs. The average side chain potential in 305 proteins is 109 +/- 30 mV (2. 5 +/- 0.7 kcal/mol/e). Because the backbone has zero net charge, the non-zero potential is unexpected. The larger oxygen at the negative and smaller proton at the positive end of the amide dipole yield positive potentials because: 1) at allowed phi and psi angles residues come off the backbone into the positive end of their own amide dipole, avoiding the large oxygen; and 2) amide dipoles with their carbonyl oxygen surface exposed and amine proton buried make the protein interior more positive. Twice as many amides have their oxygens exposed than their amine protons. The distribution of acidic and basic residues shows the importance of the bias toward positive backbone potentials. Thirty percent of the Asp, Glu, Lys, and Arg are buried. Sixty percent of buried residues are acids, only 40% bases. The positive backbone potential stabilizes ionization of 20% of the acids by >3 pH units (-4.1 kcal/mol). Only 6.5% of the bases are equivalently stabilized by negative regions. The backbone stabilizes bound anions such as phosphates and rarely stabilizes bound cations.     

Contributions of counter-charge in a potassium channel voltage-sensor domain

Voltage-sensor domains couple membrane potential to conformational changes in voltage-gated ion channels and phosphatases. Highly coevolved acidic and aromatic side chains assist the transfer of cationic side chains across the transmembrane electric field during voltage sensing. We investigated the functional contribution of negative electrostatic potentials from these residues to channel gating and voltage sensing with unnatural amino acid mutagenesis, electrophysiology, voltage-clamp fluorometry and ab initio calculations. The data show that neutralization of two conserved acidic side chains in transmembrane segments S2 and S3, namely Glu293 and Asp316 in Shaker potassium channels, has little functional effect on conductance-voltage relationships, although Glu293 appears to catalyze S4 movement. Our results suggest that neither Glu293 nor Asp316 engages in electrostatic state-dependent charge-charge interactions with S4, likely because they occupy, and possibly help create, a water-filled vestibule.     

Crystal structure of osmoporin OmpC from E. coli at 2.0 A

Porins form transmembrane pores in the outer membrane of Gram-negative bacteria with matrix porin OmpF and osmoporin OmpC from Escherichia coli being differentially expressed depending on environmental conditions. The three-dimensional structure of OmpC has been determined to 2.0 A resolution by X-ray crystallography. As expected from the high sequence similarity, OmpC adopts the OmpF-like 16-stranded hollow beta-barrel fold with three beta-barrels associated to form a tight trimer. Unlike in OmpF, the extracellular loops form a continuous wall at the perimeter of the vestibule common to the three pores, due to a 14-residues insertion in loop L4. The pore constriction and the periplasmic outlet are very similar to OmpF with 74% of the pore lining residues being conserved. Overall, only few ionizable residues are exchanged at the pore lining. The OmpC structure suggests that not pore size, but electrostatic pore potential and particular atomic details of the pore linings are the critical parameters that physiologically distinguish OmpC from OmpF.     

Cation-selective Pathway of OmpF Porin Revealed by Anomalous X-ray Diffraction

The OmpF porin from the Escherichia coli outer membrane folds into a trimer of β-barrels, each forming a wide aqueous pore allowing the passage of ions and small solutes. A long loop (L3) carrying multiple acidic residues folds into the β-barrel pore to form a narrow “constriction zone”. A strong and highly conserved charge asymmetry is observed at the constriction zone, with multiple basic residues attached to the wall of the β-barrel (Lys16, Arg42, Arg82 and Arg132) on one side, and multiple acidic residues of L3 (Asp107, Asp113, Glu117, Asp121, Asp126, Asp127) on the other side. Several computational studies have suggested that a strong transverse electric field could exist at the constriction zone as a result of such charge asymmetry, giving rise to separate permeation pathways for cations and anions. To examine this question, OmpF was expressed, purified and crystallized in the P6₃ space group and two different data sets were obtained at 2.6 Å and 3.0 Å resolution with K⁺ and Rb⁺, respectively. The Rb⁺-soaked crystals were collected at the rubidium anomalous wavelength of 0.8149 Å and cation positions were determined. A PEG molecule was observed in the pore region for both the K⁺ and Rb⁺-soaked crystals, where it interacts with loop L3. The results reveal the separate pathways of anions and cations across the constriction zone of the OmpF pore.     

Ionization states of residues in OmpF and mutants: effects of dielectric constant and interactions between residues

To understand ion permeation, one must assign correct ionization states to titratable amino acid residues in protein channels. We report on the effects of physical and methodological assumptions in calculating the protonation states at neutral bulk pH of titratable residues lining the lumen of the native Escherichia coli OmpF channel, and five mutants. We systematically considered a wide range of assumed protein dielectric constants and all plausible combinations of protonation states for electrostatically interacting side chains, and three different levels of accounting for solute shielding: 1), full nonlinear Poisson-Boltzmann; 2), linearized Poisson-Boltzmann; and 3), neglect of solute shielding. For this system we found it acceptable to neglect solute shielding, a result we postulate to be generalizable to narrow lumens of other protein channels. For the large majority of residues, the protonation state at neutral bulk pH was found to be independent of the assumed dielectric constant of the protein, and unambiguously determined by the calculation; for native OmpF only Asp-127 has a protonation state that is sensitive to the assumed protein dielectric constant. Our results are significant for understanding two published experimental observations: the structure of the narrow part of the channel, and the ionic selectivity of OmpF mutants.     

Energization-induced redistribution of charge carriers near membranes

The electric field arising from proton pumping across a topologically closed biological membrane causes accumulation close to the membrane of ionic charges equivalent to the charge of the pumped protons, positive on the side towards which protons are pumped, negative on the other side. We shall call this the 'active surface charge'. We here use the Poisson-Boltzmann equation to evaluate the effects of zwitterionic buffer molecules and uncharged proteins in the aqueous phase bordering the membrane on the magnitude and ionic composition of the active surface charge. For the positive side of the membrane, the main results are: (1) If the membrane is freely accessible to bulk phase ions, pumped protons exchange with these ions, such that the active surface charge consists of salt cations. (2) If a significant fraction of the ions in bulk solution consists of buffer molecules, then some of the pumped protons will remain close to the membrane and constitute a major fraction of the active surface charge. (3) If a protein layer borders the membrane, a significant part of the transmembrane electric potential difference exists within that protein layer and protons inside this layer dominate the active surface charge. (4) On the negative side of the membrane the corresponding phenomena would occur. (5) All these effects are strictly dependent on the transmembrane electric potential difference arising from proton pumping and would come in addition to the well known effects of buffers and electrically charged proteins on the retention of scalar protons. (6) No additional proton diffusion barrier may be required to account for a deficit in number of protons observed in the aqueous bulk phase upon aeration-induced proton pumping.     

Role of charged residues at the OmpF porin channel constriction probed by mutagenesis and simulation

The channel constriction of OmpF porin, a pore protein in the bacterial outer membrane, is highly charged due to the presence of three arginines (R42, R82, and R132) and two acidic residues (D113 and E117). The influence of these charges on ion conductance, ion selectivity, and voltage gating has been studied with mutants D113N/E117Q, R42A/R82A/R132A/D113N/E117Q, and V18K/G131K, which were designed to remove or add protein charge at the channel constriction. The crystal structures revealed no or only local changes compared to wild-type OmpF, thus allowing a comparative study. The single-channel conductance of the isosteric D113N/E117Q variant was found to be 2-fold reduced, and that of the pentuple mutant was 70% of the wild-type value, despite a considerably larger pore cross section. Ion selectivity was drastically altered by the mutations with cation/anion permeability ratios ranging from 1 to 12. Ion flow through these and eight other mutants, which have been characterized previously, was simulated by Brownian dynamics based on the detailed crystal structures. The calculated ion selectivity and relative channel conductance values agree well with the experimental data. This demonstrates that ion translocation through porin is mainly governed by pore geometry and charge, the two factors that are properly represented in the simulations.     

Residue ionization and ion transport through OmpF channels

Single trimeric channels of the general bacterial porin, OmpF, were reconstituted into planar lipid membranes and their conductance, selectivity, and open-channel noise were studied over a wide range of proton concentrations. From pH 1 to pH 12, channel transport properties displayed three characteristic regimes. First, in acidic solutions, channel conductance is a strong function of pH; it increases by approximately threefold as the proton concentration decreases from pH 1 to pH 5. This rise in conductance is accompanied by a sharp increase in cation transport number and by pronounced open-channel low-frequency current noise with a peak at ~pH 2.5. Random stepwise transients with amplitudes at ~1/5 of the monomer conductance are major contributors to this noise. Second, over the middle range (pH 5 ÷ pH 9), channel conductance and selectivity stay virtually constant; open channel noise is at its minimum. Third, over the basic range (pH 9 ÷ pH 12), channel conductance and cation selectivity start to grow again with an onset of a higher frequency open-channel noise. We attribute these effects to the reversible protonation of channel residues whose pH-dependent charge influences transport by direct interactions with ions passing through the channel.     

Membrane dipole potential modulates proton conductance through gramicidin channel: movement of negative ionic defects inside the channel.

The effect of membrane dipole potential on gramicidin channel activity in bilayer lipid membranes (BLMs) was studied. Remarkably, it appeared that proton conductance of gramicidin A (gA) channels responded to modulation of the dipole potential oppositely as compared with gA alkali metal cation conductance. In particular, the addition of phloretin, known to reduce the membrane dipole potential, resulted in a decrease in gA proton conductance, on one hand, and an increase in gA alkali metal conductance, on the other hand, whereas 6-ketocholestanol, the agent raising the membrane dipole potential, provoked an increase in gA proton conductance as opposed to a decrease in the alkali metal cation conductance. The peculiarity of the 6-ketocholestanol effect consisted in its dependence on the H(+) concentration. The experiments with the impermeant dipolar compound, phloridzin, showed that the response of proton transport through gramicidin channels to varying the membrane dipole potential did not change qualitatively if the dipole potential of only one monolayer or both monolayers of the BLM was altered. In contrast to gA proton conductance, the single-channel lifetime changed similarly with varying the membrane dipole potential, regardless of the kind of permeant cations (protons or potassium ions). The results of this study could be tentatively accounted for by an assumption that one of the rate-limiting steps of proton conduction through gramicidin channels represents, in fact, movement of negatively charged species (negative ionic defects) across a membrane.     

Histidine scanning mutagenesis of basic residues of the S4 segment of the shaker k+ channel

The voltage sensor of the Shaker potassium channel is comprised mostly of positively charged residues in the putative fourth transmembrane segment, S4 (Aggarwal, S.K., and R. MacKinnon. 1996. Neuron. 16:1169-1177; Seoh, S.-A., D. Sigg, D.M. Papazian, and F. Bezanilla. 1996. Neuron. 16:1159-1167). Movement of the voltage sensor in response to a change in the membrane potential was examined indirectly by measuring how the accessibilities of residues in and around the sensor change with voltage. Each basic residue in the S4 segment was individually replaced with a histidine. If the histidine tag is part of the voltage sensor, then the gating charge displaced by the voltage sensor will include the histidine charge. Accessibility of the histidine to the bulk solution was therefore monitored as pH-dependent changes in the gating currents evoked by membrane potential pulses. Histidine scanning mutagenesis has several advantages over other similar techniques. Since histidine accessibility is detected by labeling with solution protons, very confined local environments can be resolved and labeling introduces minimal interference of voltage sensor motion. After histidine replacement of either residue K374 or R377, there was no titration of the gating currents with internal or external pH, indicating that these residues do not move in the transmembrane electric field or that they are always inaccessible. Histidine replacement of residues R365, R368, and R371, on the other hand, showed that each of these residues traverses entirely from internal exposure at hyperpolarized potentials to external exposure at depolarized potentials. This translocation enables the histidine to transport protons across the membrane in the presence of a pH gradient. In the case of 371H, depolarization drives the histidine to a position that forms a proton pore. Kinetic models of titrateable voltage sensors that account for proton transport and conduction are presented. Finally, the results presented here are incorporated into existing information to propose a model of voltage sensor movement and structure.     

Imaging the electrostatic potential of transmembrane channels: atomic probe microscopy of OmpF porin

The atomic force microscope (AFM) was used to image native OmpF porin and to detect the electrostatic potential generated by the protein. To this end the OmpF porin trimers from Escherichia coli was reproducibly imaged at a lateral resolution of approximately 0.5 nm and a vertical resolution of approximately 0.1 nm at variable electrolyte concentrations of the buffer solution. At low electrolyte concentrations the charged AFM probe not only contoured structural details of the membrane protein surface but also interacted with local electrostatic potentials. Differences measured between topographs recorded at variable ionic strength allowed mapping of the electrostatic potential of OmpF porin. The potential map acquired by AFM showed qualitative agreement with continuum electrostatic calculations based on the atomic OmpF porin embedded in a lipid bilayer at the same electrolyte concentrations. Numerical simulations of the experimental conditions showed the measurements to be reproduced quantitatively when the AFM probe was included in the calculations. This method opens a novel avenue to determine the electrostatic potential of native protein surfaces at a lateral resolution better than 1 nm and a vertical resolution of approximately 0.1 nm.     

The protonation state of the Glu-71/Asp-80 residues in the KcsA potassium channel: a first-principles QM/MM molecular dynamics study

Although a few x-ray structures of the KcsA K(+) channel have been crystallized several issues concerning the mechanisms of the ionic permeation and the protonation state of the selectivity filter ionizable side chains are still open. Using a first-principles quantum mechanical/molecular mechanical simulation approach, we have investigated the protonation state of Glu-71 and Asp-80, two important residues located in the vicinity of the selectivity filter. Results from the dynamics show that a proton is shared between the two residues, with a slight preference for Glu-71. The proton is found to exchange on the picosecond timescale, an interesting phenomenon that cannot be observed in classical molecular dynamics. Simulations of different ionic loading states of the filter show that the probability for the proton transfer is correlated with the filter occupancy. In addition, the Glu-71/Asp-80 pair is able to modulate the potential energy profile experienced by a K(+) ion as it translates along the pore axis. These theoretical predictions, along with recent experimental results, suggest that changes of the filter structure could be associated with a shift in the Glu-Asp protonation state, which in turn would influence the ion translocation.     

Mechanism of type‐III protein secretion: Regulation of FlhA conformation by a functionally critical charged‐residue cluster

The bacterial flagellum contains a specialized secretion apparatus in its base that pumps certain protein subunits through the growing structure to their sites of installation beyond the membrane. A related apparatus functions in the injectisomes of gram‐negative pathogens to export virulence factors into host cells. This mode of protein export is termed type‐III secretion (T3S). Details of the T3S mechanism are unclear. It is energized by the proton gradient; here, a mutational approach was used to identify proton‐binding groups that might function in transport. Conserved proton‐binding residues in all the membrane components were tested. The results identify residues R147, R154 and D158 of FlhA as most critical. These lie in a small, well‐conserved cytoplasmic domain of FlhA, located between transmembrane segments 4 and 5. Two‐hybrid experiments demonstrate self‐interaction of the domain, and targeted cross‐linking indicates that it forms a multimeric array. A mutation that mimics protonation of the key acidic residue (D158N) was shown to trigger a global conformational change that affects the other, larger cytoplasmic domain that interacts with the export cargo. The results are discussed in the framework of a transport model based on proton‐actuated movements in the cytoplasmic domains of FlhA.     

The electron-transfer site of spinach plastocyanin

Two sites for electron transfer have been proposed for plastocyanin: one near the copper ion and the other close to the acid patch formed by residues 42-45. Calculations of electrostatic properties of spinach plastocyanin and ionic strength dependences of electron-transfer reactions of this protein have been used to distinguish between these two sites. Calculations show that the electric potential field of spinach plastocyanin is highly asymmetric and that the protein has a dipole moment of 360 D. The negative end of the dipole axis emerges between the negative patches formed by residues 42-45, which is though to be the cation binding site, and residues 59-61. The angles between the dipole vector and vectors from the center of mass to the copper ion and to the acid patch are 90 degrees and 30 degrees, respectively. The angle between the dipole vector and a line from the center of mass to the site of electron transfer is evaluated from the ionic strength dependence of electron-transfer rates at pH 7.8 with the help of equations developed by Van Leeuwen et al. [van Leeuwen, J.W., Mofers, F.J.M., & Veerman, E.C.I. (1981) Biochim. Biophys. Acta 635, 434] and Van Leeuwen [van Leeuwen, J.W. (1983) Biochim. Biophys. Acta 743, 408]. The angles found are 85 degrees, 110 degrees, and 75 +/- 15 degrees for reactions with tris(1,10-phenanthroline)cobalt(III), hexacyanoferrate(III), and ferrocytochrome c, respectively. The electric potential field calculations suggest that the hexacyanoferrate(III) interaction angle corresponds to a unique site of minimum repulsion at the hydrophobic region of the protein surface, close to the copper ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

FTIR spectroscopic characterization of the cytochrome aa3 from Acidianus ambivalens: evidence for the involvement of acidic residues in redox coupled proton translocation

The aa(3)-type quinol oxidase from Acidianus ambivalens is a divergent member of the heme-copper oxidases superfamily, namely, concerning the putative channels for intraprotein proton conduction. In this study, we used electrochemically induced FTIR difference spectroscopy to identify residues involved in redox-coupled protonation changes. In the spectral region characteristic for the nu(C=O) mode from protonated aspartic or glutamic acid side chains, a number of prominent features can be observed between 1790 and 1710 cm(-)(1), clearly indicating the reorganization or protonation of more than four protonatable residues upon electron transfer. A direct comparison of the Fourier-transform infrared difference spectra at different pH values reveals the noteworthy high pK of >8 for some of these residues, and the protonation of two of them. These acidic residues may play a role in the proton transport to the oxygen reducing site, in proton pumping pathways, or in protonation reactions concomitant with quinone reduction. Whereas the residues contributing between 1790 and 1750 cm(-)(1) have the typical position of an aspartic/glutamic acid side chain buried in the protein, a position closer to the surface is suggested for the residues contributing below approximately 1730 cm(-)(1). The possible involvement of residues contributing between 1750 and 1720 cm(-)(1) in the quinone binding site is demonstrated on the basis of experiments in the presence and absence of ubiquinone-2 and of the native electron carrier of the A. ambivalens respiratory chain, caldariella quinone. Most signals seen here are not observable in comparable spectra of typical members of the heme copper oxidase superfamily and thus reflect unique features of the enzyme from the hyperthermoacidophilic archaeon A. ambivalens.     

Focusing of the electrostatic potential at EF-hands of calbindin D(9k): titration of acidic residues

Biological functions for a large class of calmodulin-related proteins, such as target protein activation and Ca(2+) buffering, are based on fine-tuned binding and release of Ca(2+) ions by pairs of coupled EF-hand metal binding sites. These are abundantly filled with acidic residues of so far unknown ionization characteristics, but assumed to be essential for protein function in their ionized forms. Here we describe the measurement and modeling of pK(a) values for all aspartic and glutamic acid residues in apo calbindin D(9k), a representative of calmodulin-related proteins. We point out that while all the acidic residues are ionized predominantly at neutral pH, the onset of proton uptake by Ca(2+) ligands with high pK(a) under these conditions may have functional implications. We also show that the negative electrostatic potential is focused at the bidental Ca(2+) ligand of each site, and that the potential is significantly more negative at the N-terminal binding site.     

The Role of Electrostatic Interactions for Cytochrome c Oxidase Function

In recent years, the enormous increase in high-resolution three-dimensional structures of proteins together with the development of powerful theoretical techniques have provided the basis for a more detailed examination of the role of electrostatics in determining the midpoint potentials of redox-active metal centers and in influencing the protonation behavior of titratable groups in proteins. Based on the coordinates of the Paracoccus denitrificans cytochrome c oxidase, we have determined the electrostatic potential in and around the protein, calculated the titration curves for all ionizable residues in the protein, and analyzed the response of the protein environment to redox changes at the metal centers. The results of this study provide insight into how charged groups can be stabilized within a low-dielectric environment and how the range of their electrostatic effects can be modulated by the protein. A cluster of 18 titratable groups around the heme a ₃–Cu_B binuclear center, including a hydroxide ion bound to the copper, was identified that accounts for most of the proton uptake associated with redox changes at the binuclear site. Predicted changes in net protonation were in reasonable agreement with experimentally determined values. The relevance of these findings in the light of possible mechanisms of redox-coupled proton movement is discussed.