Antimicrobial peptides in toroidal and cylindrical pores

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize biological membranes. Their mechanism of action is still not well understood. Here we investigate the preference of alamethicin and melittin for pores of different shapes, using molecular dynamics (MD) simulations of the peptides in pre-formed toroidal and cylindrical pores. When an alamethicin hexamer is initially embedded in a cylindrical pore, at the end of the simulation the pore remains cylindrical or closes if glutamines in the N-termini are not located within the pore. On the other hand, when a melittin tetramer is embedded in toroidal pore or in a cylindrical pore, at the end of the simulation the pore is lined both with peptides and lipid headgroups, and, thus, can be classified as a toroidal pore. These observations agree with the prevailing views that alamethicin forms barrel-stave pores whereas melittin forms toroidal pores. Both alamethicin and melittin form amphiphilic helices in the presence of membranes, but their net charge differs; at pH ∼ 7, the net charge of alamethicin is − 1 whereas that of melittin is + 5. This gives rise to stronger electrostatic interactions of melittin with membranes than those of alamethicin. The melittin tetramer interacts more strongly with lipids in the toroidal pore than in the cylindrical one, due to more favorable electrostatic interactions.     

Retention of small charged impurities during ultrafiltration

Ultrafiltration is used to remove small impurities from a variety of processing streams. However, the clearance of small charged impurities may be inadequate due to electrostatic exclusion by the charged ultrafiltration membranes, an effect that has been largely unappreciated. Ultrafiltration experiments were performed to evaluate the transmission of several model impurities with different electrical charge through ultrafiltration membranes having different surface charge characteristics. Highly charged impurities are strongly rejected by charged cellulose and polyethersulfone membranes even though these solutes are much smaller than the membrane pore size. These effects could be eliminated by using high ionic strength solutions to shield the electrostatic interactions. The sieving data are in good agreement with model calculations based on the partitioning of charged spheres into charged cylindrical pores. Guidelines are developed for estimating conditions needed to obtain effective removal of small charged impurities through charged ultrafiltration membranes.     

A NUMERICAL TAXONOMIC STUDY OF NOSTOC AND ANABAENA1

Thirty characteristics of 14 Nostoc and 10 Anabaena species were analyzed from previously published data. Using standard numerical taxonomic methods, simple matching coefficients were calculated and a phenogram drawn. The analysis revealed that some of the central characteristics of Nostoc are: a punctiforme stage; motile reproductive stage; plant mass with a dull to shiny luster, non-veined surface, and nonfimbriate margin; some spherical vegetative cells; no cylindrical heterocysts; and some spherical, but no cylindrical akinetes. Some of the central characteristics of Anabaena that were revealed are: no punctiforme stage; a motile vegetative stage; plant mass with a shiny luster, veined surface, and fimbriate margin; no spherical vegetative cells; some cylindrical heterocysts; and some cylindrical, but no spherical, akinetes. In general, Anabaena has larger akinetes and vegetative cells than Nostoc. Based on 30 morphological characteristics and the clustering data of the phenogram, keys were constructed for the Nostoc and Anabaena species studied. The data clearly support two separate and distinct, though similar genera and, less sharply, the separation of the 24 species. The more useful characteristics for separation of the species are size and shape of akinetes, vegetative cells, and heterocysts; color and luster of plant mass; veined plant mass surface; margin fimbriate; and shape of plant mass in nature.     

How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

The human immune system can directly lyse invading micro‐organisms and aberrant host cells by generating pores in the cell envelope, called membrane attack complexes (MACs). Recent studies using single‐particle cryoelectron microscopy have revealed that the MAC is an asymmetric, flexible pore and have provided a structural basis on how the MAC ruptures single lipid membranes. Despite these insights, it remains unclear how the MAC ruptures the composite cell envelope of Gram‐negative bacteria. Recent functional studies on Gram‐negative bacteria elucidate that local assembly of MAC pores by surface‐bound C5 convertase enzymes is essential to stably insert these pores into the bacterial outer membrane (OM). These convertase‐generated MAC pores can subsequently efficiently damage the bacterial inner membrane (IM), which is essential for bacterial killing. This review summarizes these recent insights of MAC assembly and discusses how MAC pores kill Gram‐negative bacteria. Furthermore, this review elaborates on how MAC‐dependent OM damage could lead to IM destabilization, which is currently not well understood. A better understanding on how MAC pores kill bacteria could facilitate the future development of novel strategies to treat infections with Gram‐negative bacteria.     

Peptides released from reovirus outer capsid form membrane pores that recruit virus particles

Nonenveloped animal viruses must disrupt or perforate a cell membrane during entry. Recent work with reovirus has shown formation of size-selective pores in RBC membranes in concert with structural changes in capsid protein mu1. Here, we demonstrate that mu1 fragments released from reovirus particles are sufficient for pore formation. Both myristoylated N-terminal fragment mu1N and C-terminal fragment phi are released from particles. Both also associate with RBC membranes and contribute to pore formation in the absence of particles, but mu1N has the primary and sufficient role. Particles with a mutant form of mu1, unable to release mu1N or form pores, lack the ability to associate with membranes. They are, however, recruited by pores preformed with peptides released from wild-type particles or with synthetic mu1N. The results provide evidence that docking to membrane pores by virus particles may be a next step in membrane penetration after pore formation by released peptides.     

Charge formation in microporous membranes by single-acid site dissociation

The electrolyte concentration and pH dependence of the effective charge density of weak ion exchange membranes have been studied by combining solutions of the Poisson-Boltzmann equation in cylindrical pores with a simple dissociation equilibrium of weakly acid groups attached to the pore walls. Analytical expressions for the effective charge density and wall potential are presented which describe these quantities in terms of pH, electrolyte (1 : 1) concentration, acid constant, density of acid groups and the pore size. The concentration dependence of the effective fixed charge density experimentally observed for cellulose membranes and NaCl-solutions agrees quantitatively with the theoretical predictions. For track-etched mica membranes and KCl-solutions the influence of pH and electrolyte concentration on the effective charge density can be qualitatively explained. Also an interpretation of electro-osmotic findings obtained with an asymmetric cellulose acetate membrane and NaCl-solutions is given.     

Postsynaptic membranes in the electric tissue of Narcine: I. Organization and innervation of electric cells. Fine structure of nicotinic receptor-channel molecules revealed by transmission microscopy

The ventral, postsynaptic membranes of the electroplaques of Narcine were examined for structures corresponding in location, numbers, position and form with nicotinic acetylcholine receptor-channel molecules. Electron micrographs of conventionally fixed material showed that the thickened outer lamina of these membranes contained about 6100 stained structures/μm² ; and calculations from this packing density and the estimated area of postsynaptic membranes/g tissue yielded a total number of structures within a factor of two of the number of receptor molecules/g determined from the binding of alpha-bungarotoxin. Examination of tissue prepared with a tannic acid/glutaraldehyde fixative revealed that the structures were uniform in character, cylindrical in shape, transmembrane in position, and that each contained a central core. Their diameter matched that of isolated receptor molecules, and their size can account for most of the receptor protein. We conclude that these receptor molecules are largely intramembranous, cylindrical and exhibit transmembrane pores.     

Charge distribution and imperfect amphipathicity affect pore formation by antimicrobial peptides

Antimicrobial peptides often permeabilize biological membranes via a pore mechanism. Two pore types have been proposed: toroidal, where the pore is partly lined by lipid, and barrel-stave, where a cylindrical pore is completely lined by peptides. What drives the preference of antimicrobial peptides for a certain pore type is not yet fully understood. According to neutron scattering and oriented circular dichroism, melittin and MG-H2 induce toroidal pores whereas alamethicin forms barrel-stave pores. In previous work we found that indeed melittin seems to favor toroidal pores whereas alamethicin favors cylindrical pores. Here we designed mutants of these two peptides and the magainin analog MG-H2, aimed to probe how the distribution of charges along the helix and its imperfectly amphipathic structure influence pore formation. Molecular dynamics (MD) simulations of the peptides in a pre-formed cylindrical pore have been performed. The duration of the simulations was 136ns to 216ns. We found that a melittin mutant with lysine 7 neutralized favors cylindrical pores whereas a MG-H2 mutant with lysines in the N-terminal half of these peptides neutralized and an alamethicin mutant with a positive charge at the position 7 form semitoroidal pores. These results suggest that charged residues within the N-terminal half are important for toroidal pore formation. Toroidal pores produced by MG-H2 are more disordered than the melittin pores, likely because of the charged residues located in the middle of the MG-H2 helix (K11 and K14). Imperfect amphipathicity of melittin seems to play a role in its preference for toroidal pores since the substitutions of charged residues located within the nonpolar face by hydrophobic residues suppress evolution of a toroidal pore. The mutations change the position of lysine 7 near the N-terminus, relative to the lower leaflet headgroups. The MD simulations also show that the melittin P14A mutant forms a toroidal pore, but its configuration diverges from that of melittin and it is probably metastable.     

Antimicrobial peptides bind more strongly to membrane pores

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize bacterial membranes. Understanding their mechanism of action might help design better antibiotics. Using an implicit membrane model, modified to include pores of different shapes, we show that four AMPs (alamethicin, melittin, a magainin analogue, MG-H2, and piscidin 1) bind more strongly to membrane pores, consistent with the idea that they stabilize them. The effective energy of alamethicin in cylindrical pores is similar to that in toroidal pores, whereas the effective energy of the other three peptides is lower in toroidal pores. Only alamethicin intercalates into the membrane core; MG-H2, melittin and piscidin are located exclusively at the hydrophobic/hydrophilic interface. In toroidal pores, the latter three peptides often bind at the edge of the pore, and are in an oblique orientation. The calculated binding energies of the peptides are correlated with their hemolytic activities. We hypothesize that one distinguishing feature of AMPs may be the fact that they are imperfectly amphipathic which allows them to bind more strongly to toroidal pores. An initial test on a melittin-based mutant seems to support this hypothesis.     

Minimum Membrane Bending Energies of Fusion Pores

Membranes fuse by forming highly curved intermediates, culminating in structures described as fusion pores. These hourglass-like figures that join two fusing membranes have high bending energies, which can be estimated using continuum elasticity models. Fusion pore bending energies depend strongly on shape, and the present study developed a method for determining the shape that minimizes bending energy. This was first applied to a fusion pore modeled as a single surface and then extended to a more realistic model treating a bilayer as two monolayers. For the two-monolayer model, fusion pores were found to have metastable states with energy minima at particular values of the pore diameter and bilayer separation. Fusion pore energies were relatively insensitive to membrane thickness but highly sensitive to spontaneous curvature and membrane asymmetry. With symmetrical bilayers and monolayer spontaneous curvatures of ?0.1 nm^?1 (a typical value) separated by 6 nm (closest distance determined by repulsive hydration forces), fusion pore formation required 43–65 kT. The pore radius of ~2.25 nm fell within the range estimated from conductance measurements. With bilayer separation >6 nm, fusion pore formation required less energy, suggesting that protein scaffolds can promote fusion by bending membranes toward one another. With nonzero spontaneous monolayer curvature, the shape that minimized the energy change during fusion pore formation differed from the shape that minimized its energy after it formed. Thus, a nascent fusion pore will relax spontaneously to a new shape, consistent with the experimentally observed expansion of nascent fusion pores during viral fusion.     

Functional size of complement and perforin pores compared by confocal laser scanning microscopy and fluorescence microphotolysis

Confocal laser scanning microscopy and fluorescence microphotolysis (also referred to as fluorescence photobleaching recovery) were employed to study the transport of hydrophilic fluorescent tracers through complement and perforin pores. By optimizing the confocal effect it was possible to determine the exclusion limit of the pores in situ, i.e. without separation of cells and tracer solution. Single-cell flux measurements by fluorescence microphotolysis yielded information on the sample population distribution of flux rates. By these means a direct comparison of complement and perforin pores was made in sheep erythrocyte membranes. In accordance with previous studies employing a variety of different techniques complement pores were found to have a functional radius of approx. 50 A when generated at high complement concentrations. The flux rate distribution indicated that pore size heterogeneity was rather small under these conditions. Perforin pores, generated in sheep erythrocyte membranes at high perforin concentrations, were found to have a functional size very similar to complement pores. Furthermore, the functional size of the perforin pore seemed to be relatively independent of the dynamic properties of the target membrane since in two cell membranes which are very different in this regard, the human erythrocyte membrane and the plasma membrane of erythroleukemic cells, the functional radius of the perforin pore was also close to 50 A. A perforin-specific antibody reduced the functional radius of perforin pores to 45 A.     

Potential-dependent conductances in lipid membranes containing alamethicin.

This article is concerned primarily with the mechanism of the potential-dependent conductance induced in artificial lipid membranes by the cyclic polypeptide andibiotic alamethicin. It has already been shown from studies of the fluctuations that can be detected in very small membrane currents that alamethicin forms transient pores of some 0.6 nm in diameter and that, for small inorganic ions, these are poorly selective. The origin of these pores, their spatial distribution and interaction are discussed. It is demonstrated that the sensitivity of the membrane conductance to the applied potential arises only to a slight extent from the current-voltage relations for the individual pores, and that the main effect stems from the influence of the potential on the frequency of opening of the pores. From the properties of lipid membranes containing alamethicin in a wide variety of electrolytes, and from other evidence, it is concluded that the polypeptide reacts to the electric field more probably because it has dipole moment than because it binds ions. It is proposed that the conducting complex is capable of functioning in either of two orientations, and that it is these two possibilities that give rise to certain differences in the single channel characteristics for the two directions of the field.     

Arginine-Rich Peptides Destabilize the Plasma Membrane, Consistent with a Pore Formation Translocation Mechanism of Cell-Penetrating Peptides

Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes.     

Modeling of discrete currents of single ion channels of cell membranes using synthetic nanometer pores in polyethylene terephthalate films

Study of the conductivity of single supernarrow pores (1–15 nm in diameter) formed in thin membranes (10–12 μm in thickness) from polyethylene terephthalate (PETP) has revealed discrete changes in the currents passing through such pores when applied from an external source of potential difference of 200–1000 mV. Based on several characteristics, such discrete currents (discrete conductivity changes) appeared to be identical to the so-called currents of single ionic channels in cell membranes. The supernarrow pores whose properties are described in the present work were obtained by alkaline etching of tracks in thin PETP membranes (a variant of the so-called nuclear filters). On the walls of the pores, carboxyl groups, i.e., negative fixed charges, and their compensating counterion (cation) layer are formed. Upon setting the potential difference onto the PETP membrane, this cation layer is able to transfer current, through a process called surface conductance. In the case of nanometer-sized diameters of the pores, such surface conductance can turn out to be dominating. We have shown that these discrete changes of currents passing through the nanometer pores are associated with metastability of their surface conductance. In the highly cation-selective channels in the cell membranes, there should inevitably exist an area with dominating cation surface conductance and, hence, conductance metastability. Therefore, a new explanation is proposed of the characteristic discreteness of the currents of single cation-specific ionic channels in cell membranes. Such an explanation does not rule out the existence of any other traditional explanation of the discreteness of ion channel currents.     

Effect of extrusion pressure and lipid properties on the size and polydispersity of lipid vesicles.

The production of vesicles, spherical shells formed from lipid bilayers, is an important aspect of their recent application to drug delivery technologies. One popular production method involves pushing a lipid suspension through cylindrical pores in polycarbonate membranes. However, the actual mechanism by which the polydisperse, multilamellar lipid suspension breaks up into a relatively monodisperse population of vesicles is not well understood. To learn about factors influencing this process, we have characterized vesicles produced under different extrusion parameters and from different lipids. We find that extruded vesicles are only produced above a certain threshold extrusion pressure and have sizes that depend on the extrusion pressure. The minimum pressure appears to be associated with the lysis tension of the lipid bilayer rather than any bending modulus of the system. The flow rate of equal concentration lipid solutions through the pores, after being corrected for the viscosity of water, is independent of lipid properties.     

Electroporation of curved lipid membranes in ionic strength gradients

A thermodynamic theory for the membrane electroporation of curved membranes such as those of lipid vesicles and cylindrical membrane tubes has been developed. The theory covers in particular the observation that electric pore formation and shape deformation of vesicles and cells are dependent on the salt concentration of the suspending solvent. It is shown that transmembrane salt gradients can appreciably modify the electrostatic part of Helfrich's spontaneous curvature, elastic bending rigidity and Gaussian curvature modulus of charged membranes. The Gibbs reaction energy of membrane electroporation can be explicitely expressed in terms of salt gradient-dependent contributions of bending, the ionic double layers and electric surface potentials and dielectric polarisation of aqueous pores. In order to cover the various physical contribution to the chemical process of electroporation-resealing, we have introduced a generalised chemophysical potential covering all generalised forces and generalised displacements in terms of a transformed Gibbs energy formalism. Comparison with, and analysis of, the data of electrooptical relaxation kinetic studies show that the Gibbs reaction energy terms can be directly determined from turbidity dichroism (Planck's conservative dichroism). The approach also quantifies the electroporative cross-membrane material exchange such as electrolyte release, electrohaemolysis of red blood cells or uptake of drugs and dyes and finally gene DNA by membrane electroporation.     

Interactions of a charged nanoparticle with a lipid membrane: implications for gene delivery

We employ self-consistent field theory to study the thermodynamics of membrane-particle interactions in the context of gene delivery systems, with the aim to guide the design of dendrimers that can overcome the endosomal escape barrier by inserting into membranes and creating pores. We consider the interaction between a model polyamidoamine dendrimer and a membrane under controlled tension as a function of the separation between the dendrimer and the membrane. In all the cases we have studied, the lowest free energy state corresponds to the membrane partially wrapping the dendrimer. However, with moderate tension, we find that a G5 (or larger) generation dendrimer, through thermal fluctuation, can induce the formation of metastable pores. These metastable pores are subsequently shown to significantly lower the critical tension necessary for membrane rupture, thus possibly enhancing the release of the trapped genetic material from the endosome.     

Ca<Superscript>2+</Superscript>-Induced Phase Separation in the Membrane of Palmitate-Containing Liposomes and Its Possible Relation to Membrane Permeabilization

A Ca(2+)-induced phase separation of palmitic acid (PA) in the membrane of azolectin unilamellar liposomes has been demonstrated with the fluorescent membrane probe nonyl acridine orange (NAO). It has been shown that NAO, whose fluorescence in liposomal membranes is quenched in a concentration-dependent way, can be used to monitor changes in the volume of lipid phase. The incorporation of PA into NAO-labeled liposomes increased fluorescence corresponding to the expansion of membrane. After subsequent addition of Ca(2+), fluorescence decreased, which indicated separation of PA/Ca(2+) complexes into distinct membrane domains. The Ca(2+)-induced phase separation of PA was further studied in relation to membrane permeabilization caused by Ca(2+) in the PA-containing liposomes. A supposition was made that the mechanism of PA/Ca(2+)-induced membrane permeabilization relates to the initial stage of Ca(2+)-induced phase separation of PA and can be considered as formation of fast-tightening lipid pores due to chemotropic phase transition in the lipid bilayer.