A spectroscopic study of the membrane interaction of the antimicrobial peptide Pleurocidin

The cationic amphipathic alpha-helical antibiotic peptide, pleurocidin, from the winter flounder Pleuronectes americanus associates strongly with anionic membranes where it is able to translocate across the membrane, cause dye leakage from vesicles and induce pore like channel conductance. To investigate the mechanism of pleurocidin antibiotic activity in more detail we have applied a variety of spectroscopic techniques to study the interaction of pleurocidin with model membranes. At neutral pH the peptide inserts into membranes containing anionic lipids and, as shown by proton-decoupled 15N solid-state NMR spectroscopy of macroscopically oriented samples, is aligned parallel to the membrane surface. 2H solid-state NMR spectroscopy of chain deuterated phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) lipids in mixed membranes shows that pleurocidin interacts with both the zwitterionic PE and anionic PG but disrupts the lipid acyl chain order of the anionic PG lipids more effectively. At acidic pH the three histidine residues of pleurocidin become protonated and positively charged which does not alter the membrane disrupting effect nor the location of the peptide in the membrane. The results are interpreted in terms of a structural model for pleurocidin inserted into anionic lipid membranes and the implications of our data are discussed in terms of a general mechanism for the antibiotic activity.     

Comparison of salt effects on the reactions of acetylcholinesterase with cationic and anionic inhibitors

The influence of inorganic salts on the inhibition of acetylcholinesterase by charged organophosphorous inhibitors has been studied. It has been shown that the salt effect on the reaction of acetylcholinesterase with anionic bis(p-nitrophenyl) phosphate is determined by the influence of added salts on the activity coefficient of the inhibitor. In contrast to the salt effects on the reaction of acetylcholinesterase with cationic compounds, it does not include contribution from the enzyme charges. The smaller salt effect in the case of anionic inhibitor can be explained assuming that the anionic inhibitor does not form a non-covalent complex with the enzyme before the phosphorylation step of the reaction. Comparison of salt effects on the substrate turnover showed that in the case of cholinesterases from natural sources they are larger than in the case of enzymes expressed in recombinant cell clones. The enhanced salt effects may result from post-translational modification of the enzyme.     

Effect of salt concentration on membrane lysis pressure

Cell membranes are capable of withstanding significant osmotic stress, the exact amount of which varies with the lipid composition. In this paper, we examine the effect that salt concentration has on the lysis pressure of membranes containing anionic lipids. Vesicles containing varying amounts of phosphatidylcholine and phosphatidylglycerol were osmotically stressed using NaCl as the osmolyte. The lysis pressure was observed to vary linearly with the Debye screening length and the extent of the variation was linear with anionic lipid content. The implications these results have for cells that frequently encounter low solute environments are discussed.     

Effect of salt concentration on membrane lysis pressure

Cell membranes are capable of withstanding significant osmotic stress, the exact amount of which varies with the lipid composition. In this paper, we examine the effect that salt concentration has on the lysis pressure of membranes containing anionic lipids. Vesicles containing varying amounts of phosphatidylcholine and phosphatidylglycerol were osmotically stressed using NaCl as the osmolyte. The lysis pressure was observed to vary linearly with the Debye screening length and the extent of the variation was linear with anionic lipid content. The implications these results have for cells that frequently encounter low solute environments are discussed.     

The effects of some cations and anions on spin labeled cytoplasmic membranes of bacillus subtilis

The effects of CaCl₂, MgCl₂, LaCl₃ and some alkali halides on $\text{Bacillus subtilis}$ cytoplasmic membranes were studied using stearic acid spin labels. The results indicate that two mechanisms are operating when these ions interact with $\text{B. subtilis}$ membranes. At low ionic concentrations (0 to 0.1 M) there is direct cation binding to the anionic membranes which in the case of Ca²⁺, Mg²⁺ and La³⁺, confers rigidity on the membranes and reaches saturation when the number of cations present equals the number of anionic sites on the lipids. At high concentrations there is a further effect on the membranes that parallels the known organizing/disorganizing effects that the ions studied have on water structure.     

Membrane composition and successful bioaugmentation. Studies of the interactions of model thylakoid and plasma cyanobacterial and bacterial membranes with fungal membrane-lytic enzyme Lecitase ultra

Cyanobacterial/bacterial consortia are frequently inoculated to soils to increase the soil fertility and to accelerate the biodegradation of organic pollutants. Moreover, such consortia can also be successfully applied in landfills especially for the biodegradation of plastic wastes. However, the bioaugmentation techniques turn out frequently inefficient due to the competition of the indigenous microorganisms attacking directly these inoculated or secreting to their surroundings cell wall and membrane-lytic enzymes. It can be hypothesized that the resistance of the microbial membrane to the enzymatic degradation is correlated with its lipid composition. To verify this hypothesis glycolipid and phospholipid Langmuir monolayers were applied as models of thylakoid and plasma cyanobacterial and bacterial membranes. Hybrid fungal enzyme Lecitase ultra joining the activity of lipase and phospholipase A1 was applied as the model of fungal membrane-lytic enzyme. It turned out that anionic thylakoid lipids sulfoquinovosyldiacylglycerol and phosphatidylglycerols were the main targets of Lecitase ultra in the model multicomponent thylakoid membranes. The resistance of the model plasma bacterial membranes to enzymatic degradation depended significantly to their composition. The resistance increased generally when the unsaturated lipids were exchanged to their saturated counterparts. However, most resistant turned out the membranes composed of unsaturated phosphatidylamine and saturated anionic phospholipids.     

Helix formation in methylated copolymers of lysine and alanine.

Random copolymers of lysine and alanine, 2:1 and 1:1, were trimethylated on the lysine amino groups to quaternary ammonium groups. Methylated and unmethylated polymers were prepared with Cl- or ClO4- as the counterion. CD spectra were measured for increasing concentration of peptide without added salt, and at constant peptide concentration in increasing NaCl or NaClO4. Unmethylated peptides, as the chloride, form alpha-helix more readily than do the methylated peptides. The opposite occurs with ClO4- as counterion. The helix-promoting effect of methylated lysine residues (ClO4- counterion) is diminished by the presence of alanine, as compared with effects when lysine is the only type of residue. The effect of methylation of proteins on helix formation may depend on the types of anionic groups with which the protein may be involved.     

Calcium-Lipid Interactions Observed with Isotope-Edited Infrared Spectroscopy

Calcium ions bind to lipid membranes containing anionic lipids; however, characterizing the specific ion-lipid interactions in multicomponent membranes has remained challenging because it requires nonperturbative lipid-specific probes. Here, using a combination of isotope-edited infrared spectroscopy and molecular dynamics simulations, we characterize the effects of a physiologically relevant (2 mM) Ca²⁺ concentration on zwitterionic phosphatidylcholine and anionic phosphatidylserine lipids in mixed lipid membranes. We show that Ca²⁺ alters hydrogen bonding between water and lipid headgroups by forming a coordination complex involving the lipid headgroups and water. These interactions distort interfacial water orientations and prevent hydrogen bonding with lipid ester carbonyls. We demonstrate, experimentally, that these effects are more pronounced for the anionic phosphatidylserine lipids than for zwitterionic phosphatidylcholine lipids in the same membrane.     

The distribution of anionic sites on the surfaces of mitochondrial membranes. Visual probing with polycationic ferritin

Polycationic ferritin, a multivalent ligand, was used as a visual probe to determine the distribution and density of anionic sites on the surfaces of rat liver mitochondrial membranes. Both the distribution of bound polycationic ferritin and the topography of the outer surface of the inner mitochondrial membrane were studied in depth by utilizing thin sections and critical-point dried, whole mount preparations for transmission electron microscopy and by scanning electron microscopy. Based on its relative affinity for polycationic ferritin, the surface of the inner membrane contains discrete regions of high density and low density anionic sites. Whereas the surface of the cristal membrane contains a low density of anionic sites, the surface of the inner boundary membrane contains patches of high density anionic sites. The high density anionic sites on the inner boundary membrane were found to persist as stable patches and did not dissociate or randomize freely when the membrane was converted osmotically to a spherical configuration. The observations suggest that the inner mitochondrial membrane is composed of two major regions of anionic macromolecular distinction. It is well-known that an intermembrane space exists between the two membranes of the intact mitochondrion; however, a number of contact sites occur between the two membranes. We determined that the outer membrane, partially disrupted by treatment with digitonin, remains attached to the inner membrane at these contact sites as inverted vesicles. Such attached vesicles show that the inner surface of the outer membrane contains anionic sites, but of decreased density, surrounding the contact sites. Thus, the intermembrane space in the intact mitochondrion may be maintained by electronegative surfaces of the two mitochondrial membranes. The distribution of anionic sites on the outer surface of the outer membrane is random. The nature and function of fixed anionic surface charges and membrane contact sites are discussed with regard to recent reports relating to calcium transport, protein assembly into mitochondrial membranes, and membrane fluidity.     

Electrical properties and molecular architecture of the channel formed by Escherichia coli hemolysin in planar lipid membranes

A 107 kDa hemolysin from Escherichia coli is able to open pores in lipid membranes. By studying its interaction with planar phospholipid bilayers we have derived some structural information on the organization of the pore. We measured the current-voltage characteristic and the ion selectivity of the channel both in neutral membranes, made of egg phosphatidylcholine (PC) and in negatively charged membranes, made of a 1:1 mixture of PC with phosphatidylserine (PS). Experiments were performed varying both the pH and the salt concentration of the bathing KCl solution. In neutral membranes the pore is ohmic and its conductance increases almost linearly with the salt concentration. The channel is cation-selective at high pH but nearly unselective at low pH. We interpret these results in terms of a minimal model based on classical electro-diffusional theories assuming that the pore is wide and bears a negative charge at its entrances. In membranes containing the acidic lipid the current-voltage curve is non-linear in such a way to suggest that the trans (but not the cis) entrance of the pore is affected by the surface potential of the membrane. Applying our model we find that the trans and cis entrances are located, respectively, about 0.5 nm and more than 5 nm apart from the plane of the membrane. We confirmed the asymmetric disposition of the channel by enzymatic digestion of preformed pores. This was effective only when the enzyme was applied on the cis side.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an alpha-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Functional and conformational modulation of human cytochrome P450 1B1 by anionic phospholipids

We investigated the interaction of human P450 1B1 (CYP1B1) with various phospholipid bilayers using the N-terminally deleted (Δ2-4)CYP1B1 and (Δ2-26)CYP1B1 enzymes. Among anionic phospholipids, phosphatidic acid (PA) and cardiolipin specifically increased the catalytic activities, membrane binding affinities, and thermal stabilities of both CYP1B1 proteins when phosphatidylcholine matrix was gradually replaced with these anionic phospholipids. PA- or cardiolipin-dependent changes of CYP1B1 conformation were revealed by altered Trp fluorescence and CD spectra. However, both PA and cardiolipin exerted more significant effects with the (Δ2-4)CYP1B1 than the (Δ2-26)CYP1B1 implying the functional importance of N-terminal region for the interaction with the phospholipid membranes. In contrast, other anionic phospholipids such as phosphatidylserine and the neutral phospholipid phosphatidylethanolamine had no apparent effects on the catalytic activity or conformation of CYP1B1. These data suggest that the chemical and physical properties of membranes influenced by PA or cardiolipin composition are critical for the functional roles of CYP1B1.     

Biophysical Investigation of the Membrane-Disrupting Mechanism of the Antimicrobial and Amyloid-Like Peptide Dermaseptin S9

Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with ²H- and ³¹P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Some effects of salts on staining: Use of the Donnan Equilibrium to describe staining of tissue sections with acid and basic dyes

Summary Using a wide variety of acid and basic dyes, and dyebaths of various pH’s and salt contents, it was shown that various effects of neutral inorganic salts on the staining of tissue sections could be explained by the Donnan Membrane Equilibrium. Thus the simultaneous increases in staining of some tissue components and decreases in staining of others, which occur on adding salt to the dyebaths, are often explicable in terms of the Donnan Equilibrium. The variation in staining intensity with dye charge, seen with a number of acid dyes, also agreed with the predictions of the Donnan Equilibrium. The Donnan Equilibrium was applied to understanding the mode of action of various biological staining methods requiring salt in the dyebaths; namely the Best’s Carmine glycogen stain, the Alcian Blue and Congo Red methods for amyloid, and the salty periodic acid-Schiff procedure for anionic mucosubstances.     

Effect of pH, salt, and coupling state on the interaction of ferredoxin with the chloroplast membrane

To determine if the interaction between ferredoxin and ferredoxin:NADP reductase is similar to the interaction between the purified proteins when the ferrodoxin:NADP reductase is membrane bound, the effect of pH, salt, and coupling state on the Km for ferredoxin in NADP reduction by chloroplast membranes has been examined. Increasing pH and salt concentrations as well as uncouplers all resulted in increases in the Km for ferredoxin. The pH and salt effects on the Km are similar to effects observed by others (C. Batie and H. Kamin (1981) J. Biol. Chem. 256, 7756-7763) on the dissociation constant for a complex between the two purified proteins, although the salt effect on the Km appears to be affected by the surface potential of the chloroplast membrane. These results suggest that the interaction between ferredoxin and the membrane-bound ferredoxin:NADP reductase is not greatly different from the interaction which has been characterized between the two purified proteins.     

A study of the membrane association and regulatory effect of the phospholemman cytoplasmic domain

Phospholemman (PLM) is a single-span transmembrane protein belonging to the FXYD family of proteins. PLM (or FXYD1) regulates the Na,K-ATPase (NKA) ion pump by altering its affinity for K(+) and Na(+) and by reducing its hydrolytic activity. Structural studies of PLM in anionic detergent micelles have suggested that the cytoplasmic domain, which alone can regulate NKA, forms a partial helix which is stabilized by interactions with the charged membrane surface. This work examines the membrane affinity and regulatory function of a 35-amino acid peptide (PLM(38-72)) representing the PLM cytoplasmic domain. Isothermal titration calorimetry and solid-state NMR measurements confirm that PLM(38-72) associates strongly with highly anionic phospholipid membranes, but the association is weakened substantially when the negative surface charge is reduced to a more physiologically relevant environment. Membrane interactions are also weakened when the peptide is phosphorylated at S68, one of the substrate sites for protein kinases. PLM(38-72) also lowers the maximal velocity of ATP hydrolysis (V(max)) by NKA, and phosphorylation of the peptide at S68 gives rise to a partial recovery of V(max). These results suggest that the PLM cytoplasmic domain populates NKA-associated and membrane-associated states in dynamic equilibrium and that phosphorylation may alter the position of the equilibrium. Interestingly, peptides representing the cytoplasmic domains of two other FXYD proteins, Mat-8 (FXYD3) and CHIF (FXYD4), have little or no interaction with highly anionic phospholipid membranes and have no effect on NKA function. This suggests that the functional and physical properties of PLM are not conserved across the entire FXYD family.     

Membrane binding kinetics of protein kinase C betaII mediated by the C2 domain.

Conventional isoforms of protein kinase C (PKC) are activated when their two membrane-targeting modules, the C1 and C2 domains, bind the second messengers diacylglycerol (DG) and Ca2+, respectively. This study investigates the mechanism of Ca2+-induced binding of PKC betaII to anionic membranes mediated by the C2 domain. Stopped-flow fluorescence spectroscopy reveals that Ca2+-induced binding of the isolated C2 domain to anionic vesicles proceeds via at least two steps: (1) rapid binding of two or more Ca2+ ions to the free domain with relatively low affinity and (2) diffusion-controlled association of the Ca2+-occupied domain with vesicles. Ca2+ increases the affinity of the C2 domain for anionic membranes by both decreasing the dissociation rate constant (k(off)) and increasing the association rate constant (k(on)) for membrane binding. For binding to vesicles containing 40 mol % anionic lipid in the presence of 200 microM Ca2+, k(off) and k(on) are 8.9 s(-1) and 1.2 x 10(10) M(-1) x s(-1), respectively. The k(off) value increases to 150 s(-1) when free Ca2+ levels are rapidly reduced, decreasing the average lifetime of the membrane-bound C2 domain (tau = k(off)(-1)) from 110 ms in the presence of Ca2+ to 6.7 ms when Ca2+ is rapidly removed. Experiments addressing the role of electrostatic interactions reveal that they stabilize either the initial C2 domain-membrane encounter complex or the high-affinity membrane-bound complex. Specifically, lowering the phosphatidylserine mole fraction or including MgCl2 in the binding reaction decreases the affinity of the C2 domain for anionic vesicles by both reducing k(on) and increasing k(off) measured in the presence of 200 microM Ca2+. These species do not affect the k(off) value when Ca2+ is rapidly removed. Studies with PKC betaII reveal that Ca2+-induced binding to membranes by the full-length protein proceeds minimally via two kinetically resolvable steps: (1) a rapid bimolecular association of the enzyme with vesicles near the diffusion-controlled limit and, most likely, (2) subsequent conformational changes of the membrane-bound enzyme. As is the case for the C2 domain, k(off) for full-length PKC betaII increases when Ca2+ is rapidly removed, reducing tau from 11 s in the presence of Ca2+ to 48 ms in its absence. Thus, both the C2 domain and the slow conformational change prolong the lifetime of the PKC betaII-membrane ternary complex in the presence of Ca2+, with rapid membrane release triggered by removal of Ca2+. These results provide a molecular basis for cofactor regulation of PKC whereby the C2 domain searches three-dimensional space at the diffusion-controlled limit to target PKC to relatively common anionic phospholipids, whereupon a two-dimensional search is initiated by the C1 domain for the more rare, membrane-partitioned DG.     

Role of membrane lipids in the mechanism of bacterial species selective toxicity by two alpha/beta-antimicrobial peptides

We have previously shown that two synthetic antimicrobial peptides with alternating alpha- and beta-amino acid residues, designated simply as alpha/beta-peptide I and alpha/beta-peptide II, had toxicity toward bacteria and affected the morphology of bacterial membranes in a manner that correlated with their effects on liposomes with lipid composition similar to those of the bacteria. In the present study we account for the weak effects of alpha/beta-peptide I on liposomes or bacteria whose membranes are enriched in phosphatidylethanolamine (PE) and why such membranes are particularly susceptible to damage by alpha/beta-peptide II. The alpha/beta-peptide II has marked effects on unilamellar vesicles enriched in PE causing vesicle aggregation and loss of their internal aqueous contents. The molecular basis of these effects is the ability of alpha/beta-peptide II to induce phase segregation of anionic and zwitterionic lipids as shown by fluorescence and differential scanning calorimetry. This phase separation could result in the formation of defects through which polar materials could pass across the membrane as well as form a PE-rich membrane domain that would not be a stable bilayer. alpha/beta-Peptide II is more effective in this regard because, unlike alpha/beta-peptide I, it has a string of two or three adjacent cationic residues that can interact with anionic lipids. Although alpha/beta-peptide I can destroy membrane barriers by converting lamellar to non-lamellar structures, it does so only weakly with unilamellar vesicles or with bacteria because it is not as efficient in the aggregation of these membranes leading to the bilayer-bilayer contacts required for this phase conversion. This study provides further understanding of why alpha/beta-peptide II is more toxic to micro-organisms with a high PE content in their membrane as well as for the lack of toxicity of alpha/beta-peptide I with these cells, emphasizing the potential importance of the lipid composition of the cell surface in determining selective toxicity of anti-microbial agents.     

Rectification properties and pH-dependent selectivity of meningococcal class 1 porin

We studied the current rectification properties and selectivity of class 1 porin (PorA) from Neisseria meningitidis (strain H44/76 Δ3Δ4) reconstituted in planar lipid membranes varying salt concentrations and pH. PorA channel shows voltage gating with a characteristic time remarkably longer than other porins. Its current-voltage asymmetry, evaluated as the current rectification ratio, changes nonmonotonically with salt concentration. Interestingly, it reaches its maximum value at physiological concentration. Porin selectivity, quantified by reversal potential measurements, is also significantly asymmetric. Depending on the direction of the salt gradient, the channel becomes more or less selective (10:1 vs. 5:1 Na⁺/Cl⁻). Besides, the reversal potential measurements suggest that porin inserts directionally following the concentration gradient. Measurements over a wide range of pH show that although PorA is strongly cation selective at pH >6, its selectivity gradually changes to anionic in an acidic medium (pH < 4). We show that a continuum electrodiffusion model quantitatively accounts for conductance and reversal potential measurements at positive and negative applied voltages.