Evidence for membrane thinning effect as the mechanism for peptide-induced pore formation

Antimicrobial peptides have two binding states in a lipid bilayer, a surface state S and a pore-forming state I. The transition from the S state to the I state has a sigmoidal peptide-concentration dependence indicating cooperativity in the peptide-membrane interactions. In a previous paper, we reported the transition of alamethicin measured in three bilayer conditions. The data were explained by a free energy that took into account the membrane thinning effect induced by the peptides. In this paper, the full implications of the free energy were tested by including another type of peptide, melittin, that forms toroidal pores, instead of barrel-stave pores as in the case of alamethicin. The S-to-I transitions were measured by oriented circular dichroism. The membrane thinning effect was measured by x-ray diffraction. All data were in good agreement with the theory, indicating that the membrane thinning effect is a plausible mechanism for the peptide-induced pore formations.     

Diffusion as a probe of peptide-induced membrane domain formation

Recently, we have shown that association with an antimicrobial peptide (AMP) can drastically alter the diffusion behavior of the constituent lipids in model membranes (Biochemistry 49, 4672-4678). In particular, we found that the diffusion time of a tracer fluorescent lipid through a confocal volume measured via fluorescence correlation spectroscopy (FCS) is distributed over a wide range of time scales, indicating the formation of stable and/or transient membrane species that have different mobilities. A simple estimate, however, suggested that the slow diffusing species are too large to be attributed to AMP oligomers or pores that are tightly bound to a small number of lipids. Thus, we tentatively ascribed them to membrane domains and/or clusters that possess distinctively different diffusion properties. In order to further substantiate our previous conjecture, herein we study the diffusion behavior of the membrane-bound peptide molecules using the same AMPs and model membranes. Our results show, in contrast to our previous findings, that the diffusion times of the membrane-bound peptides exhibit a much narrower distribution that is more similar to that of the lipids in peptide-free membranes. Thus, taken together, these results indicate that while AMP molecules prompt domain formation in membranes, they are not tightly associated with the lipid domains thus formed. Instead, they are likely located at the boundary regions separating various domains and acting as mobile fences.     

Behavioral and electrophysiological studies of peptide-induced emesis in dogs

The electrophysiological responses of neurons in the canine area postrema (AP) to ionophoretic application of neuropeptides and transmitters were studied and correlated with the presence or absence of an emetic response on systemic administration. Of 17 common neuropeptides 11 were emetic when applied systemically at doses of 0.03-0.35 mg/kg. The emesis was dose dependent and was no longer observed in animals with chronic ablation of the AP. The responses of 122 AP single units were recorded. Neurons were silent at rest, and most were excited by glutamate, apomorphine, and dopamine. Excitatory responses to each of eight emetic peptides were recorded in 22-65% of cells studied; no responses were found to two peptides that were not emetic. The response to glutamate was always a brief, high-frequency discharge; the responses to all 13 other excitatory substances were of long latency, low frequency, and long duration. With high ionophoretic current or multiple applications, units would frequently become spontaneously active for many minutes or longer. The similarity of response of so many substances on small neurons suggests a common ionic or metabolic mechanism underlying the response. The direct correlation between the occurrence of emesis on systemic administration and the presence of excitatory receptors on AP neurons provides strong support for the proposed role of the AP as the chemoreceptor trigger zone for emesis.     

Coarse-grained molecular simulation of self-assembly nanostructures of CTAB on nanoscale graphene

Coarse-grained molecular dynamics simulation has been performed to study the aggregated morphology of the cationic surfactant, cetyltrimethylammonium bromide (CTAB), adsorbed on nanoscale graphene surfaces. The CTAB surfactants can self-assemble on graphene to form various supramolecular morphologies and structures. The effect of packing density, thickness of graphene sheet and width of graphene nanoribbon on the CTAB–graphene self-assembly has been investigated. The buoyant densities of various graphene–CTAB assemblies were calculated, which increase with surfactant coverage and number of graphene layers. This result demonstrates that density gradient can be used to isolate graphenes with various layers. This simulation provides larger-scale microscopic insight into the supramolecular self-assembly nanostructures for the CTAB surfactants aggregated on graphene, which could be valuable to guide fabrication of graphene-based hybrid nanocomposites.     

Radiation-induced delay of nuclear pore formation

We have shown that radiation affects the nuclear envelope, a membrane structure-closely associated with DNA. The density of nuclear pores visible on freeze-etch surfaces decreased at a rate of 0.042 (pores/mum2)/100 rad with respect to unirradiated cells. This is interpreted as a radiation-induced delay in development of the nuclear envelope.     

Onset of anthrax toxin pore formation

Protective antigen (PA) is the anthrax toxin protein recognized by capillary morphogenesis gene 2 (CMG2), a transmembrane cellular receptor. Upon activation, seven ligand-receptor units self-assemble into a heptameric ring-like complex that becomes endocytozed by the host cell. A critical step in the subsequent intoxication process is the formation and insertion of a pore into the endosome membrane by PA. The pore conversion requires a change in binding between PA and its receptor in the acidified endosome environment. Molecular dynamics simulations totaling approximately 136 ns on systems of over 92,000 atoms were performed. The simulations revealed how the PA-CMG2 complex, stable at neutral conditions, becomes transformed at low pH upon protonation of His-121 and Glu-122, two conserved amino acids of the receptor. The protonation disrupts a salt bridge important for the binding stability and leads to the detachment of PA domain II, which weakens the stability of the PA-CMG2 complex significantly, and subsequently releases a PA segment needed for pore formation. The simulations also explain the great strength of the PA-CMG2 complex achieves through extraordinary coordination of a divalent cation.     

T cell studies in a peptide-induced model of systemic lupus erythematosus

We have previously reported that immunization with a peptide mimetope of dsDNA on a branched polylysine backbone (DWEYSVWLSN-MAP) induces a systemic lupus erythematosus-like syndrome in the nonautoimmune BALB/c mouse strain. To understand the mechanism underlying this breakdown in self tolerance, we examined the role of T cells in the response. Our results show that the anti-foreign and anti-self response induced by immunization is T cell dependent and is mediated by I-E(d)-restricted CD4(+) T cells of the Th1 subset. In addition, generation of the critical T cell epitope requires processing by APCs and depends on the presence of both DWEYSVWLSN and the MAP backbone. The breakdown in self tolerance does not occur through cross-reactivity between the T cell epitope of DWEYSVWLSN-MAP and epitopes derived from nuclear Ags. In this induced-model of SLE, therefore, autoreactivity results from the activation of T cells specific for foreign Ag and of cross-reactive anti-foreign, anti-self B cells. Despite the fact that tissue injury is mediated by Ab, the critical initiating T cell response is Th1.     

Coarse-grained biomolecular simulation with REACH: realistic extension algorithm via covariance Hessian

Coarse-graining of protein interactions provides a means of simulating large biological systems. Here, a coarse-graining method, REACH, is introduced, in which the force constants of a residue-scale elastic network model are calculated from the variance-covariance matrix obtained from atomistic molecular dynamics (MD) simulation. In test calculations, the C(alpha)-atoms variance-covariance matrices are calculated from the ensembles of 1-ns atomistic MD trajectories in monomeric and dimeric myoglobin, and used to derive coarse-grained force constants for the local and nonbonded interactions. Construction of analytical model functions of the distance-dependence of the interresidue force constants allows rapid calculation of the REACH normal modes. The model force constants from monomeric and dimeric myoglobin are found to be similar in magnitude to each other. The MD intra- and intermolecular mean-square fluctuations and the vibrational density of states are well reproduced by the residue-scale REACH normal modes without requiring rescaling of the force constant parameters. The temperature-dependence of the myoglobin REACH force constants reveals that the dynamical transition in protein internal fluctuations arises principally from softening of the elasticity in the nonlocal interactions. The REACH method is found to be a reliable way of determining spatiotemporal protein motion without the need for expensive computations of long atomistic MD simulations.     

pH-dependent pore formation properties of pardaxin analogues

The interaction of pardaxin, a shark-repellent neurotoxin, and its charge-modified analogues with vesicles and human erythrocytes is described. The following six analogues and derivatives were synthesized by a solid phase method: [Glu8, Glu16]pardaxin, [N1-succinamido,Glu8,Glu16]pardaxin, [N1,Lys8,Lys16-triacetyl]pardaxin, des-[1----9]pardaxin (Shai, Y., Bach, D., and Yanovsky, A. (1990) J. Biol. Chem. 265, 20202-20209), and des-[1----9] [Glu16]pardaxin. The relative hydrophobic characteristics of the analogues were examined using reverse-phase high performance liquid chromatography. The pH-dependent spectroscopic and functional characteristics of the analogues were also investigated at either neutral or acidic pH. Spectroscopic characterization was achieved by measuring circular dichroism both before and after binding to vesicles, at either neutral or acidic pH. The ability of the peptides to dissipate a diffusion potential, to cause calcein release or the pH-dependent release of 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt/p-xylene-bis[pyridinium bromide] from sonicated unilamellar liposomes, as well as measurements of cytolytic activity on human erythrocytes, served to functionally characterize the peptides. We show a direct correlation between alpha-helical content, the analogues' hydrophobicity, and their pore-forming properties at the different pH values tested. We also demonstrate that the charge of the N terminus and of the peptide backbone, but not of the C terminus, affects the secondary structure as well as the activities of the analogues. Finally, we show that the cytolytic activity of pardaxin at neutral pH is not retained by any of the analogues.     

Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s_o) and liquid-disordered (l_d) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l_o) and l_d phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s_o-l_d and l_o-l_d phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s(o)) and liquid-disordered (l(d)) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l(o)) and l(d) phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s(o)-l(d) and l(o)-l(d) phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

Coarse-grained molecular simulation of diffusion and reaction kinetics in a crowded virtual cytoplasm

We present a general-purpose model for biomolecular simulations at the molecular level that incorporates stochasticity, spatial dependence, and volume exclusion, using diffusing and reacting particles with physical dimensions. To validate the model, we first established the formal relationship between the microscopic model parameters (timestep, move length, and reaction probabilities) and the macroscopic coefficients for diffusion and reaction rate. We then compared simulation results with Smoluchowski theory for diffusion-limited irreversible reactions and the best available approximation for diffusion-influenced reversible reactions. To simulate the volumetric effects of a crowded intracellular environment, we created a virtual cytoplasm composed of a heterogeneous population of particles diffusing at rates appropriate to their size. The particle-size distribution was estimated from the relative abundance, mass, and stoichiometries of protein complexes using an experimentally derived proteome catalog from Escherichia coli K12. Simulated diffusion constants exhibited anomalous behavior as a function of time and crowding. Although significant, the volumetric impact of crowding on diffusion cannot fully account for retarded protein mobility in vivo, suggesting that other biophysical factors are at play. The simulated effect of crowding on barnase-barstar dimerization, an experimentally characterized example of a bimolecular association reaction, reveals a biphasic time course, indicating that crowding exerts different effects over different timescales. These observations illustrate that quantitative realism in biosimulation will depend to some extent on mesoscale phenomena that are not currently well understood.     

A thermodynamic approach to alamethicin pore formation

The structure and energetics of alamethicin Rf30 monomer to nonamer in cylindrical pores of 5 to 11 Å radius are investigated using molecular dynamics simulations in an implicit membrane model that includes the free energy cost of acyl chain hydrophobic area exposure. Stable, low energy pores are obtained for certain combinations of radius and oligomeric number. The trimer and the tetramer formed 6 Å pores that appear closed while the larger oligomers formed open pores at their optimal radius. The hexamer in an 8 Å pore and the octamer in an 11 Å pore give the lowest effective energy per monomer. However, all oligomers beyond the pentamer have comparable energies, consistent with the observation of multiple conductance levels. The results are consistent with the widely accepted “barrel-stave” model. The N terminal portion of the molecule exhibits smaller tilt with respect to the membrane normal than the C terminal portion, resulting in a pore shape that is a hybrid between a funnel and an hourglass. Transmembrane voltage has little effect on the structure of the oligomers but enhances or decreases their stability depending on its orientation. Antiparallel bundles are lower in energy than the commonly accepted parallel ones and could be present under certain experimental conditions. Dry aggregates (without an aqueous pore) have lower average effective energy than the corresponding aggregates in a pore, suggesting that alamethicin pores may be excited states that are stabilized in part by voltage and in part by the ion flow itself.     

Alamethicin-induced pore formation in biological membranes.

The effects of alamethicin on the membrane barrier function of rabbit erythrocytes, human platelets and sarcoplasmic reticulum vesicles, as well as on that of brain microsomes and liver mitochondria of the rat were compared. An upset of the barrier function was observed for plasma membranes of brain microsomes as well as for erythrocyte and platelet membranes at alamethicin concentrations ranging between 25-80 micrograms/ml. The membrane barrier functions of sarcoplasmic reticulum vesicles, of endoplasmic reticulum vesicles of rat brain microsomes, and of liver mitochondria were disturbed at 3-7 micrograms/ml alamethicin. The different sensitivities of plasma and intracellular membranes to alamethicin were supposed to be due to the presence of considerable quantities of cholesterol in plasma membranes as well as to peculiarities of their protein compositions.     

Revisiting peptide amphiphilicity for membrane pore formation

It has previously been shown that an amphipathic de novo designed peptide made of 10 leucines and four phenylalanines substituted with crown ethers induces vesicle leakage without selectivity. To gain selectivity against negatively charged dimyristoylphosphatidylglycerol (DMPG) bilayers, one or two leucines of the peptide were substituted with positively charged residues at each position. All peptides induce significant calcein leakage of DMPG vesicles. However, some peptides do not induce significant leakage of zwitterionic dimyristoylphosphatidylcholine vesicles and are thus active against only bacterial model membranes. The intravesicular leakage is induced by pore formation instead of membrane micellization. Nonselective peptides are mostly helical, while selective peptides mainly adopt an intermolecular β-sheet structure. This study therefore demonstrates that the position of the lysine residues significantly influences the secondary structure and bilayer selectivity of an amphipathic 14-mer peptide, with β-sheet peptides being more selective than helical peptides.     

Coarse-grained molecular dynamics simulation of interactions between cyclic lipopeptide Bacillomycin D and cell membranes

According to experimental studies, Bacillomycin D has strong antimicrobial activities, but the antimicrobial mechanism is still unknown. In this paper, the interaction mechanisms between this cyclic lipopeptide and three different charged cell membranes are studied via Coarse-Grained Molecular Dynamics (CG MD) simulations. A specific CG model for the cyclic lipopeptide Bacillomycin D was developed. The insertion of cyclic lipopeptide Bacillomycin D into DOPC, DOPC/DPPA and DOPC/DOTAP cell membranes was investigated. The position distribution and stability of Bacillomycin D in the three different cell membranes were analysed and compared based on density profile calculations. Additionally, we focused on the Radial Distribution Function (RDF) curves between amino acid residues with negative charges or strong hydrophobic properties and the head groups of two different cell membranes. Based on changes in the curvature of the three membranes, the cyclic lipopeptide Bacillomycin D can cause localised surface protrusions in DOPC/DOTAP membranes, inward depressions in the surface of DOPC/DPPA membranes and inhibition deformation in the surface of DOPC membranes. This study will help to further understand the antibacterial mechanism of the cyclic lipopeptide Bacillomycin D and provide a basis for the development of new antibiotics.     

On the mechanism of pore formation by melittin

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.     

The mechanism of pore formation by bacterial toxins

A remarkable group of proteins challenge the notions that protein sequence determines a unique three-dimensional structure, and that membrane and soluble proteins are very distinct. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. Recent structural studies provide ideas about how these changes take place. The recently solved structures of the beta-pore-forming toxins LukS, varepsilon-toxin and intermedilysin confirm that the pore-forming regions are initially folded up on the surfaces of the soluble precursors. To create the transmembrane pores, these regions must extend and refold into membrane-inserted beta-barrels.