Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores.

Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the POPG:POPC ratio. Vesicles (300 microM POPG) readily aggregated at HNP-2 concentrations above 1 microM, but no mixing of vesicle contents could be detected for concentrations as high as 2 microM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 microM POPG vesicles, 50% of entrapped solute is released by 0.7 microM HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (approximately 400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.     

Fluorescent probes for asymmetric lipid bilayers: Synthesis and properties in phosphatidyl choline liposomes and erythrocyte membranes

Summary We have synthesized three sets of fluorescent probes which we believe will be useful in studies of asymmetric membranes and have studied their interactions with model lipid bilayers and erythrocyte membranes. The probes were designed to partition preferentially into one face of a lipid bilayer with asymmetrically disposed phospholipids and to report lipid transitions in that monolayer.We synthesized more than twenty probes containing anthroyl-, dansyl-, or pyrene rings with acidic, basic, and neutral functional groups and alkyl spacers of various lengths. The interactions of these probes with liposomes of phosphatidyl choline and with erythrocyte membranes were characterized to determine whether probe insertion was asymmetric, how deeply the probe penetrated the bilayer, and whether the probe reflected thermotropic phase transitions in model membranes. The set of variously charged anthroyl esters, analogs of local anaesthetics, appears to be promising for studies of asymmetric membranes.Fluorescent probes have been used extensively to provide information on the lipid regions of biological membranes. Membrane fluidity, a composite of molecular packing and motion of acyl chains in lipid bilayers, has been assessed with a variety of fluorescent probes, the fluorescence of which undergoes some measurable change at the temperature of the membrane's thermotropic phase transition. A large number of fluorescent probes have been used for this purpose. Bashford, Morgan and Radda (Bashford, C.L., Morgan, C.G., Radda, G.K. 1976;Biochim. Biophys. Acta 426: 157) and Thulborn and Sawyer (Thulborn, K.R., Sawyer, W.H. 1978;Biochim. Biophys. Acta 511: 125) synthesized several fatty acid derivatives in which an anthracene group is attached (in ester linkage) along the acyl chain at various positions, and have shown that this set of probes may be useful in probing membrane fluidity at differentdepths within the bilayer.This report describes the synthesis and properties of several sets of amphipathic fluorescent probes, which may partition unequally into the two faces of an asymmetric lipid bilayer, and may therefore provide information about membranes complementary to that obtainable with existing probes.     

Pressure adaptation of teleost gill Na+/K+-adenosine triphosphatase: role of the lipid and protein moieties

Summary The effects of temperature and pressure on Na⁺/K⁺-adenosine triphosphatases (Na⁺/K⁺-ATPases) from gills of marine teleost fishes were examined over a range of temperatures (10–25°C) and pressures (1–680 atm). The relationship between gill membrane fluidity and Na⁺/K⁺-ATPase activity was studied using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The increase in temperature required to offset the membrane ordering effects of high pressure was 0.015–0.025°C·atm^-1, the same coefficient that applied to Na⁺/K⁺-ATPase activities. Thus, temperature-pressure combinations yielding the same Na⁺/K⁺-ATPase activity also gave similar estimates of membrane fluidity. Substituion of endogenous lipids with lipids of different composition altered the pressure responses of Na⁺/K⁺-ATPase. Na⁺/K⁺-adenosine triphosphatase became more sensitive to pressure in the presence of chicken egg phosphatidylcholine, but phospholipids isolated from fish gills reduced the inhibition by pressure of Na⁺/K⁺-ATPase. Cholesterol increased enzyme pressure sensitivity. Membrane fluidity and pressure sensitivity of Na⁺/K⁺-ATPase were correlated, but the effects of pressure also dependent on the source of the enzyme. Our results suggest that pressure adaptation of Na⁺/K⁺-ATPase is the result of both changes in the primary structure of the protein and homeoviscous adaptation of the lipid environment.Abbreviations EDTA; DPH 1,6-diphenyl-1,3,5-hexatriene - PC phosphatidylcholine - PL phospholipid - SDH succinate dehydrogenase     

Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic film model

Summary A simple viscoelastic film model is presented, which predicts a breakdown electric potential having a dependence on the electric pulse length which approximates the available experimental data for the electric breakdown of lipid bilayers and cell membranes (summarized in the reviews of U. Zimmermann and J. Vienken, 1982,J. Membrane Biol. 67:165 and U. Zimmermann, 1982,Biochim. Biophys. Acta 694:227). The basic result is a formula for the time of membrane breakdown (up to the formation of pores): =(/C)/( _m ² ₀ ² U ⁴/24Gh ³+T ²/Gh–1), where is a proportionality coefficient approximately equal to ln(h/2₀),h being the membrane thickness and ₀ the amplitude of the initial membrane surface shape fluctuation ( is usually of the order of unity), represents the membrane shear viscosity,G the membranes shear elasticity modules, _m the membrane relative permittivity, ₀=8.85×10^–12 Fm,U the electric potential across the membrane, the membrane surface tension andT the membrane tension. This formula predicts a critical potentialU _c ;U _c =(24Gh ³/ _m ² ₀ ² )^1/4 (for = andT=0). It is proposed that the time course of the electric field-induced membrane breakdown can be divided into three stages: (i) growth of the membrane surface fluctuations, (ii) molecular rearrangements leading to membrane discontinuities, and (iii) expansion of the pores, resulting in the mechanical breakdown of the membrane.     

Experimental and observational evidence reveals that predators in natural environments do not regulate their prey: They are passengers,not drivers

Among both ecologists and the wider community there is a tacit assumption that predators regulate populations of their prey. But there is evidence from a wide taxonomic and geographic range of studies that predators that are adapted to co-evolved prey generally do not regulate their prey. This is because predators either cannot reproduce as fast as their prey and/or are inefficient hunters unable to catch enough prey to sustain maximum reproduction. The greater capacity of herbivores to breed successfully is, however, normally restricted by a lack of enough food of sufficient quality to support reproduction. But whenever this shortage is alleviated by a large pulse of food, herbivores increase their numbers to outbreak levels. Their predators are unable to contain this increase, but their numbers, too, surge in response to this increase in food. Eventually both their populations will crash once the food supply runs out, first for the herbivores and then for the predators. Then an “over-run” of predators will further depress the already declining prey population, appearing to be controlling its abundance. This latter phenomenon has led many ecologists to conclude that predators are regulating the numbers of their prey. However, it is the same process that is revealed during outbreaks that limits populations of both predator and prey in “normal” times, although this is usually not readily apparent. Nevertheless, as all the diverse cases discussed here attest, the abundance of predators and their co-evolved prey are both limited by their food: the predators are passengers, not drivers.     

The structure of polyunsaturated lipid bilayers important for rhodopsin function: a neutron diffraction study

The structure of oriented 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine bilayers with perdeuterated stearoyl- or docosahexaenoyl hydrocarbon chains was investigated by neutron diffraction. Experiments were conducted at two different relative humidities, 66 and 86%. At both humidities we observed that the polyunsaturated docosahexaenoyl chain has a preference to reside near the lipid water interface. That leaves voids in the bilayer center that are occupied by saturated stearoyl chain segments. This uneven distribution of saturated- and polyunsaturated chain densities is likely to result in membrane elastic stress that modulates function of integral receptor proteins like rhodopsin.     

Responses to reversed NH3 and NH4+ gradients in a teleost (Ictalurus punctatus), an elasmobranch (Raja erinacea), and a crustacean (Callinectes sapidus):evidence for NH4+/H+ exchange in the teleost and the elasmobranch

Ammonia excretion rates of channel catfish, Ictalurus punctatus, little skate (Raja erinacea), and blue crab (Callinectes sapidus) were measured in experimental regimes which permitted simultaneous assessment of the partial pressure gradients for nonionized NH3 and the chemical concentration gradients of NH4+. Under conditions of low external ammonia, the average ammonia excretion was +295 microM kg-1 h-1 for catfish, +149 microM kg-1 h-1 for blue crabs, and +59 microM kg-1 h1 for skates with partial pressure gradients of +72.5 mu Torr, +413 mu Torr, and +24.4 mu Torr, respectively; and [NH4+] gradients of +189 microM l-1, +643 microM l-1, and +107 microM l-1 (positive indicating greater from inside to medium). When the external ammonia was increased to 1.15 mM l-1, both gradients were reversed, and the net ammonia movement was initially from the external water into all three species. In the catfish the inward movement ceased, however, and ammonia excretion eventually resumed in the face of reversed gradients of both NH3 partial pressure and [NH4+]. Unidirectional Na+ influx, indicative of a Na+/NH4+ exchange, did not increase. The ammonia data, changes in titratable acidity, and net apparent H+ efflux were all consistent with a linked extrusion of internal NH4+ for external H+. Incorporation of such an exchange into a computer simulation model of the ammonia equilibrium and exchange system duplicated the experimental data. Other hypotheses failed to match experimental data, or failed to predict internal ammonia levels lower than outside. In the crab, internal ammonia levels rose rapidly to concentrations and partial pressures above the external medium until excretion was reestablished, with no evidence of maintenance of a reversed gradient. In the skate, internal concentrations rose appreciably in the first hour and continued to rise for 6-8 h, with no resumption of ammonia excretion. The interspecies differences appear to be due at least partly to differences in ammonia permeability of the gills.     

Further evidence for the importance of lipid bilayers in the interaction between lactate dehydrogenase and phosphatidylserine

Lactate dehydrogenase (LDH) is one of the glycolytic enzymes, which have been proved to have the capability to reverse non-specific adsorption on cellular membranous structures in vitro, as well as on the structural proteins of the contractile system of muscle cells. It has been suggested that this binding may play a physiological role, as it alters the enzyme's kinetic properties. Our previous studies on this enzyme showed that its interaction with some anionic phospholipids reveals similar characteristics and similar effect on the activity of the enzyme to those which had been observed for the interaction with membranous structures. Disruption of the lipid bilayers by nonionic detergent (Tween 20) restored the enzyme activity inhibited by the presence of phosphatidylserine (PS) liposomes. In this study, we used the measurement of enzyme tryptophanyl fluorescence spectra to monitor the interaction and possible changes in the enzyme conformation. The investigation provided further evidence of the importance of the bilayer structure in this interaction. Similarly to the effect on the activity of the enzyme, the addition of Tween 20 diminishes the quenching of the LDH tryptophanyl fluorescence, and finally completely restores the fluorescence.     

Characterization of the fluorophore 4-heptadecyl-7-hydroxycoumarin: a probe for the head-group region of lipid bilayers and biological membranes

The fluorophore 4-heptadecyl-7-hydroxycoumarin was used as a probe to study the properties of phospholipid bilayers at the lipid-water interface. To this end, the steady-state fluorescence anisotropy, the differential polarized phase fluorometry, and the emission lifetime of the fluorophore were measured in isotropic viscous medium, in lipid vesicles, and in the membrane of vesicular stomatitis virus. In the isotropic medium (glycerol), the probe showed an increase in the steady-state fluorescence anisotropy with a decrease in temperature, but the emission lifetime was unaffected by the change in temperature. In glycerol, the observed and predicted values for maximum differential tangents of the probe were identical, indicating that in isotropic medium 4-heptadecyl-7-hydroxycoumarin is a free rotator. Nuclear magnetic resonance and differential scanning calorimetric studies with lipid vesicles containing 1-2 mol % of the fluorophore indicated that the packaging density of the choline head groups was affected in the presence of the probe with almost no effect on the fatty acyl chains. The fluorophore partitioned equally well in the gel and liquid-crystalline phase of the lipids in the membrane, and the phase transition of the bilayer lipids was reflected in the steady-state fluorescence anisotropy of the probe. The presence of cholesterol in the lipid vesicles had a relatively small effect on the dynamics of lipids in the liquid-crystalline state, but a significant disordering effect was noted in the gel state. One of the most favorable properties of the probe is that its emission lifetime was unaffected by the physical state of the lipids or by the temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of tannin on cellular membranes: Novel insights from concerted studies on lipid bilayers and native cells

Hydrolyzable tannin (3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl-β-d-glucose) has a dual effect on the cell membrane: (1) it binds to a plasmalemmal protein of the Chara corallina cell (C₅₀ = 2.7 ± 0.3 μM) and (2) it forms ionic channels in the lipid membrane. Based on these facts, a molecular model for the interaction of tannins with the cell membrane is proposed. The model suggests that the molecules of hydrolyzable tannin bind electrostatically to the outer groups of the membrane protein responsible for the Ca²⁺-dependent chloride current and blocks it. Some tannin molecules penetrate into the hydrophobic region of the membrane, and when a particular concentration is reached, they form ion-conducting structures selective toward Cl^?.     

Mechanical properties of lipid bilayers and regulation of mechanosensitive function: From biological to biomimetic channels

Material properties of lipid bilayers, including thickness, intrinsic curvature and compressibility regulate the function of mechanosensitive (MS) channels. This regulation is dependent on phospholipid composition, lateral packing and organization within the membrane. Therefore, a more complete framework to understand the functioning of MS channels requires insights into bilayer structure, thermodynamics and phospholipid structure, as well as lipid-protein interactions. Phospholipids and MS channels interact with each other mainly through electrostatic forces and hydrophobic matching, which are also crucial for antimicrobial peptides. They are excellent models for studying the formation and stabilization of membrane pores. Importantly, they perform equivalent responses as MS channels: (1) tilting in response to tension and (2) dissipation of osmotic gradients. Lessons learned from pore forming peptides could enrich our knowledge of mechanisms of action and evolution of these channels. Here, the current state of the art is presented and general principles of membrane regulation of mechanosensitive function are discussed.     

Simulations of skin barrier function: free energies of hydrophobic and hydrophilic transmembrane pores in ceramide bilayers

Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion, and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO) weaken the barrier function of the skin. We have used the potential of mean constraint force (PMCF) method to calculate the free energy of pore formation in ceramide bilayers in both the innate gel phase and in the DMSO-induced fluidized state. Our simulations show that the fluid phase bilayers form archetypal water-filled hydrophilic pores similar to those observed in phospholipid bilayers. In contrast, the rigid gel-phase bilayers develop hydrophobic pores. At the relatively small pore diameters studied here, the hydrophobic pores are empty rather than filled with bulk water, suggesting that they do not compromise the barrier function of ceramide membranes. A phenomenological analysis suggests that these vapor pores are stable, below a critical radius, because the penalty of creating water-vapor and tail-vapor interfaces is lower than that of directly exposing the strongly hydrophobic tails to water. The PMCF free energy profile of the vapor pore supports this analysis. The simulations indicate that high DMSO concentrations drastically impair the barrier function of the skin by strongly reducing the free energy required for pore opening.     

Site-directed mutagenesis and phylogenetic comparisons of the Escherichia coli Tus protein: DNA-protein interactions alone can not account for Tus activity

The Tus protein of Escherichia coli is capable of arresting DNA replication in an orientation-dependent manner when bound to specific sequences in the bacterial chromosome called Ter sites. Arrest of DNA replication has been postulated to occur either by a barrier mechanism, where Tus acts as a physical block to replication fork progression, or through protein-protein interactions between Tus and some component of the replication fork. A previous mutational analysis of Tus suggested that the amino acids in the L1 loop might play a role in replication arrest. Site-directed mutagenesis of amino acids in the L1 loop and other amino acid residues on the "non-permissive" face of Tus was performed to identify residues that affected Tus function. One mutant, E47Q, gave results that are inconsistent with the barrier model, showing a greater affinity for the Ter site (with a t 1/2 of 348 min versus 150 min for wild-type Tus) but a reduced ability to arrest DNA replication in vivo. In addition to the site-directed mutagenesis studies, the tus genes of Salmonella, Klebsiella, and Yersinia were sequenced and the proteins expressed in E. coli to assess their ability to arrest DNA replication. The results presented here support a role for protein-protein interactions in Tus function, and suggest that residues E47 and E49 participate in replication fork arrest.     

Calcium binding to a human factor IXa derivative lacking gamma-carboxyglutamic acid: evidence for two high-affinity sites that do not involve beta-hydroxyaspartic acid

A derivative of human blood clotting factor IXa beta lacking gamma-carboxyglutamic acid (Gla) residues was prepared by limited proteolysis with chymotrypsin, and subsequently examined for its ability to bind calcium ions. By amino acid analysis, Gla-domainless human factor IXa beta contained 0.3-0.4 moles of beta-hydroxyaspartic acid per mole of protein. Equilibrium dialysis experiments demonstrated that Gla-domainless human factor IXa beta retained two high-affinity calcium binding sites (Kd=52 microM), a finding essentially identical to that observed for Gla-domainless bovine factor IX that contains 0.8-0.9 moles of beta-hydroxyaspartic acid per mole of protein. These data strongly suggest that the beta-hydroxyaspartic acid residue in these proteins does not participate in their high affinity calcium sites.