Defensins promote fusion and lysis of negatively charged membranes.

Defensins, a family of cationic peptides isolated from mammalian granulocytes and believed to permeabilize membranes, were tested for their ability to cause fusion and lysis of liposomes. Unlike alpha-helical peptides whose lytic effects have been extensively studied, the defensins consist primarily of beta-sheet. Defensins fuse and lyse negatively charged liposomes but display reduced activity with neutral liposomes. These and other experiments suggest that fusion and lysis is mediated primarily by electrostatic forces and to a lesser extent, by hydrophobic interactions. Circular dichroism and fluorescence spectroscopy of native defensins indicate that the amphiphilic beta-sheet structure is maintained throughout the fusion process. Taken together, these results support the idea that protein-mediated membrane fusion depends not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form a three-dimensional amphiphilic structure, which promotes the efficient mixing of the lipids between membranes. A molecular model for membrane fusion by defensins is presented, which takes into account the contributions of electrostatic forces, hydrophobic interactions, and structural amphiphilicity.     

Folding amphipathic helices into membranes: amphiphilicity trumps hydrophobicity

High amphiphilicity is a hallmark of interfacial helices in membrane proteins and membrane-active peptides, such as toxins and antimicrobial peptides. Although there is general agreement that amphiphilicity is important for membrane-interface binding, an unanswered question is its importance relative to simple hydrophobicity-driven partitioning. We have examined this fundamental question using measurements of the interfacial partitioning of a family of 17-residue amidated-acetylated peptides into both neutral and anionic lipid vesicles. Composed only of Ala, Leu, and Gln residues, the amino acid sequences of the peptides were varied to change peptide amphiphilicity without changing total hydrophobicity. We found that peptide helicity in water and interface increased linearly with hydrophobic moment, as did the favorable peptide partitioning free energy. This observation provides simple tools for designing amphipathic helical peptides. Finally, our results show that helical amphiphilicity is far more important for interfacial binding than simple hydrophobicity.     

Structure-activity relationships of diastereomeric lysine ring size analogs of the antimicrobial peptide gramicidin S: mechanism of action and discrimination between bacterial and animal cell membranes

Structure-activity relationships were examined in seven gramicidin S analogs in which the ring-expanded analog GS14 [cyclo-(VKLKVdYPLKVKLdYP)] is modified by enantiomeric inversions of its lysine residues. The conformation, amphiphilicity, and self-association propensity of these peptides were investigated by circular dichroism spectroscopy and reversed phase high performance liquid chromatography. (31)P nuclear magnetic resonance spectroscopic and dye leakage experiments were performed to evaluate the capacity of these peptides to induce inverse nonlamellar phases in, and to permeabilize phospholipid bilayers; their growth inhibitory activity against the cell wall-less mollicute Acholeplasma laidlawii B was also examined. The amount and stability of beta-sheet structure, effective hydrophobicity, propensity for self-association in water, ability to disrupt the organization of phospholipid bilayers, and ability to inhibit A. laidlawii B growth are strongly correlated with the facial amphiphilicity of these GS14 analogs. Also, the magnitude of the parameters segregate these peptides into three groups, consisting of GS14, the four single inversion analogs, and the two multiple inversion analogs. The capacity of these peptides to differentiate between bacterial and animal cell membranes exhibits a biphasic relationship with peptide amphiphilicity, suggesting that there may only be a narrow range of peptide amphiphilicity within which it is possible to achieve the dual therapeutic requirements of high antibiotic effectiveness and low hemolytic activity. These results were rationalized by considering how the physiochemical properties of these GS14 analogs are likely to be reflected in their partitioning into lipid bilayer membranes.     

Novel polymerizable surfactants with pH-sensitive amphiphilicity and cell membrane disruption for efficient siRNA delivery

Small interfering RNA (siRNA) is a promising new therapeutic modality that can specifically silence disease-related genes. The main challenge for successful clinical development of therapeutic siRNA is the lack of efficient delivery systems. In this study, we have designed and synthesized a small library of novel multifunctional siRNA carriers, polymerizable surfactants with pH-sensitive amphiphilicity based on the hypothesis that pH-sensitive amphiphilicity and environmentally sensitive siRNA release can result in efficient siRNA delivery. The polymerizable surfactants comprise a protonatable amino head group, two cysteine residues, and two lipophilic tails. The surfactants demonstrated pH-sensitive amphiphilic hemolytic activity or cell membrane disruption with rat red blood cells. Most of the surfactants resulted in low hemolysis at pH 7.4 and high hemolysis at reduced pH (6.5 and 5.4). The pH-sensitive cell membrane disruption can facilitate endosomal-lysosomal escape of siRNA delivery systems at the endosomal-lysosomal pH. The surfactants formed compact nanoparticles (160-260 nm) with siRNA at N/P ratios of 8 and 10 via charge complexation with the amino head group, lipophilic condensation, and autoxidative polymerization of dithiols. The siRNA complexes with the surfactants demonstrated low cytotoxicity. The cellular siRNA delivery efficiency and RNAi activity of the surfactants correlated well with their pH-sensitive amphiphilic cell membrane disruption. The surfactants mediated 40-88% silencing of luciferase expression with 100 nM siRNA and 35-75% with 20 nM siRNA in U87-luc cells. Some of the surfactants resulted in similar or higher gene silencing efficiency than TransFast. EHCO with no hemolytic activity at pH 7.4 and 6.5 and high hemolytic activity at pH 5.4 resulted in the best siRNA delivery efficiency. The polymerizable surfactants with pH-sensitive amphiphilicity are promising for efficient siRNA delivery.     

Amphiphilicity index of polar amino acids as an aid in the characterization of amino acid preference at membrane-water interfaces

MOTIVATION: An amphiphilicity index of amino acid residues was developed for improving the method of transmembrane helix prediction. RESULTS: The transfer energy of a hydrocarbon stem group beyond the gamma-carbon was calculated from the accessible surface area, and used to index the amphiphilicity of the residue. Non-zero amphiphilicity index values were obtained for lysine, arginine, histidine, glutamic acid, glutamine, tyrosine and tryptophan. Those residues were found to be abundant in the end regions of transmembrane helices, indicating their preference for the membrane-water interface. The moving average of the amphiphilicity index actually showed significant peaks in the end regions of most transmembrane helices. A dispersion diagram of average amphiphilicity index versus average hydrophobicity index was devised to facilitate discrimination of transmembrane helices. AVAILABILITY: The amphiphilicity index has been incorporated into a system, SOSUI, for the discrimination of membrane proteins and the prdiction of tranmembrane helical regions (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html).     

Amphiphilic properties of polysaccharides: dextrin chains as example.

Polysaccharide chains are usually considered to be highly hydrophilic, since they contain no obvious apolar moieties. However, it is possible for even these chains to display hydrophobic character, arising out of stereochemical constraints in the chain. We had earlier shown that linear dextrin chains display amphiphilic properties, since all the hydroxyl groups are disposed on one side or face of the chain and the hydrogens disposed on the other. We provide further evidence here for this conclusion that dextrins are amphiphilic chains. In contrast, dextrans and cellulosic chains do not display amphiphilicity. Oligosaccharides that can adopt incipient helical structures might display amphiphilicity. This property might be relevant to intermolecular recognition on cell surfaces, lectin-sugar binding, antigen-antibody interactions and the like, and might be manifested more in a heteromolecular recognition process than as homomolecular self-aggregation.     

The prediction of hydrophobicity gradients within membrane interactive protein alpha-helices using a novel graphical technique

Here, it is shown that amphiphilicity profiling based on the mean hydrophobic moment provides a simple visual guide for the identification of oblique orientated alpha-helices. The methodology has an efficiency of circa 70% and predicts that approximately 40% of transmembrane alpha-helices may possess these structures.     

Streptavidin-induced lysis of homologous biotinylated erythrocytes. Evidence against the key role of the avidin charge in complement activation via the alternative pathway

It is shown that non-covalent attachment of streptavidin, as well as of avidin, to biotinylated human erythrocytes induces homologous hemolysis by complement. Rabbit antiserum against human C3 is found to inhibit the lysis specifically as compared with non-immune rabbit serum. Efficiency of lysis inhibition is greater for avidin- and streptavidin-induced lysis of biotinylated human erythrocytes than for antibody-sensitized sheep erythrocytes. In contrast to positively charged avidin (pI 11), streptavidin is a neutral protein. Hence, hemolysis of streptavidin-carrying erythrocytes is inconsistent with the suggestion on the crucial role of avidin charge in lysis. Membrane alterations (cross-linking and clusterization of biotinylated components) induced by avidin (streptavidin) seem to be a more plausible explanation for the lysis.     

Tipping the Scale from Disorder to Alpha-helix: Folding of Amphiphilic Peptides in the Presence of Macroscopic and Molecular Interfaces

Secondary amphiphilicity is inherent to the secondary structural elements of proteins. By forming energetically favorable contacts with each other these amphiphilic building blocks give rise to the formation of a tertiary structure. Small proteins and peptides, on the other hand, are usually too short to form multiple structural elements and cannot stabilize them internally. Therefore, these molecules are often found to be structurally ambiguous up to the point of a large degree of intrinsic disorder in solution. Consequently, their conformational preference is particularly susceptible to environmental conditions such as pH, salts, or presence of interfaces. In this study we use molecular dynamics simulations to analyze the conformational behavior of two synthetic peptides, LKKLLKLLKKLLKL (LK) and EAALAEALAEALAE (EALA), with built-in secondary amphiphilicity upon forming an alpha-helix. We use these model peptides to systematically study their aggregation and the influence of macroscopic and molecular interfaces on their conformational preferences. We show that the peptides are neither random coils in bulk water nor fully formed alpha helices, but adopt multiple conformations and secondary structure elements with short lifetimes. These provide a basis for conformation-selection and population-shift upon environmental changes. Differences in these peptides’ response to macroscopic and molecular interfaces (presented by an aggregation partner) can be linked to their inherent alpha-helical tendencies in bulk water. We find that the peptides’ aggregation behavior is also strongly affected by presence or absence of an interface, and rather subtly depends on their surface charge and hydrophobicity.     

Prevention of nonspecific lysis in liposomal and erythrocyte immunoassay systems by small lipid vesicles and erythrocyte ghosts

Large unilamellar liposomes prepared by the reverse-phase evaporation method (REVs) were made immunoreactive by incorporating dinitrophenylaminocaproyl-phosphatidylethanolamine (DNP-Cap-PE) or 8-(3-carboxypropyl)-theophylline-dipalmitoylphosphatidylethanolamine (Th-DPPE) into the phospholipid bilayer. Specific lysis in the presence of anti-DNP-BSA and goat anti-theophylline serum respectively, was induced by adding guinea pig serum as source for complement to these liposomes. However, specific lysis was found to be compromised by high levels of nonspecific lysis as monitored by the release of the fluorescent aqueous-space marker 6-carboxyfluorescein. Nonspecific lysis could be prevented without affecting specific lysis by pretreatment of complement or incubation of the reaction mixture with small unilamellar liposomes (SUVs). SUVs of various lipid compositions produced the desired effect; however, when the fraction of negative charge in the SUVs was increased to 30 mol%, specific lysis was inhibited as well. In a similar assay system consisting of hemolysin-sensitized sheep red blood cells it was also found that nonspecific lysis could be inhibited by addition of erythrocyte ghosts to the incubation medium, although specific lysis was somewhat depressed. However, SUVs or REVs of a composition similar to sheep erythrocytes were ineffective indicating a more selective nature of complement-mediated immunoreaction with erythrocyte membranes than with synthetic bilayer membranes.     

Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae

In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) alpha-monoglucosyldiacylglycerol (MGlcDAG) synthase (alMGS) (EC ) and the (ii) alpha-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC ), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively. Cloning of the alDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified. A strong stimulation of alDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA. Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases. Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes. Further support for this was obtained when hybrids of the N- and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of the E. coli inner membrane and cytoplasm in vivo. In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N- and (catalytic) C-domains of these structurally similar membrane-associated enzymes.     

Hydrophobic surfaces in oligosaccharides: linear dextrins are amphiphilic chains.

Polysaccharide chains are usually considered to be highly hydrophilic, since they have no obvious nonpolar moieties in them. Yet, it is possible to realise conformations in these chains wherein all the hydroxy groups are disposed in one side or face of the chain and the hydrogens disposed in the other. We experimentally demonstrate that such an amphiphilic surface is present in linear oligomeric dextrins, i.e., alpha-1,4-linked D-glucosides, but not in alpha-1,6-D-glucosides (dextrans) or in beta-1,4-D-glucosides (cellulose). This amphiphilicity is generated as a consequence of the stereochemical constraints, which vary with the structure of the sugar and with the type of linkage. Oligosaccharide chains that can adopt incipient helical structures might display amphiphilicity. This property might be relevant to intermolecular recognition on cell surfaces, lectin-sugar binding, antigen-antibody interactions and the like, and might be manifested more in heteromolecular recognition process than as homomolecular self-aggregation.     

The C-terminal sequence of the lambda holin constitutes a cytoplasmic regulatory domain

The C-terminal domains of holins are highly hydrophilic and contain clusters of consecutive basic and acidic residues, with the overall net charge predicted to be positive. The C-terminal domain of lambda S was found to be cytoplasmic, as defined by protease accessibility in spheroplasts and inverted membrane vesicles. C-terminal nonsense mutations were constructed in S and found to be lysis proficient, as long as at least one basic residue is retained at the C terminus. In general, the normal intrinsic scheduling of S function is deranged, resulting in early lysis. However, the capacity of each truncated lytic allele for inhibition by the S107 inhibitor product of S is retained. The K97am allele, when incorporated into the phage context, confers a plaque-forming defect because its early lysis significantly reduces the burst size. Finally, a C-terminal frameshift mutation was isolated as a suppressor of the even more severe early lysis defect of the mutant SA52G, which causes lysis at or before the time when the first phage particle is assembled in the cell. This mutation scrambles the C-terminal sequence of S, resulting in a predicted net charge increase of +4, and retards lysis by about 30 min, thus permitting a viable quantity of progeny to accumulate. Thus, the C-terminal domain is not involved in the formation of the lethal membrane lesion nor in the "dual-start" regulation conserved in lambdoid holins. Instead, the C-terminal sequence defines a cytoplasmic regulatory domain which affects the timing of lysis. Comparison of the C-terminal sequences of within holin families suggests that these domains have little or no structure but act as reservoirs of charged residues that interact with the membrane to effect proper lysis timing.     

Effects of peptide secondary structure on the interaction with oppositely charged microgels

The importance of peptide secondary structure on the interaction between antimicrobial peptides and oppositely charged poly(acrylic acid-co-acrylamide) microgels of various charge density was investigated for EFKRIVQRIKDFLRNLV (EFK17). Through D-enantiomer (EFK17-d/a; E(dF)KR(dI)VQR(dI)KD(dF)LRNLV) or tryptophan (EFK17-W/a; EWKRWVQRWKDFLRNLV) substitutions, both conformation-dependent and -independent amphiphilicity of this peptide could be precisely controlled. Peptide secondary structure was investigated by circular dichroism, whereas microgel deswelling and reswelling in response to peptide binding and release were studied by micromanipulator-assisted light and fluorescence microscopy, and peptide uptake in the microgels was determined from solution depletion measurements. Results show that peptide binding to the microgel is highly influenced by peptide secondary structure. EFK17-a, characterized by an idealized helix with all polar/charged amino acids located at one side of the helix, and all nonpolar/hydrophobic residues on the other, displays pronounced α-helix induction on peptide binding to the microgels. EFK17-d/a, on the other hand, displays no such amphiphilic helix induction. Mirroring this, EFK17-a displays substantially higher binding to the microgels than EFK17-d/a as well as much larger peptide-induced microgel deswelling. For EFK17-W/a, both conformation-dependent and -independent amphiphilicity effects were demonstrated. Overall, the results show that peptide conformational aspects need to be considered in peptide/microgel interactions, for example, in the design of microgel carrier systems for peptide drugs.     

A theoretical analysis of secondary structural characteristics of anticancer peptides

Here, cluster analysis showed that a database of 158 anticancer peptides formed 21 clusters based on net positive charge, hydrophobicity and amphiphilicity. In general, these clusters showed similar median toxicities (P = 0.176) against eukaryotic cell lines and no single combination of these properties was found optimal for efficacy. The database contained 14 peptides, which showed selectivity for tumour cell lines only (ACP_CT), 123 peptides with general toxicity to eukaryotic cells (ACP_GT) and 21 inactive peptides (ACP_I). Hydrophobic arc size analysis showed that there was no significant difference across the datasets although peptides with wide hydrophobic arcs (>270°) appeared to be associated with decreased toxicity. Extended hydrophobic moment plot analysis predicted that over 50% of ACP_CT and ACP_GT peptides would be surface active, which led to the suggestion that amphiphilicity is a key driver of the membrane interactions for these peptides but probably plays a role in their efficacy rather than their selectivity. This analysis also predicted that only 14% of ACP_CT peptides compared to 45% of ACP_GT peptides were candidates for tilted peptide formation, which led to the suggestion that the absence of this structure may support cancer cell selectivity. However, these analyses predicted that ACP_I peptides, which possess no anticancer activity, would also form surface active and tilted α-helices, clearly showing that other factors are involved in determining the efficacy and selectivity of ACPs.     

Charged amino-terminal amino acids affect the lethal capacity of Lambda lysis proteins S107 and S105

The lysis inhibitor protein S107 and the lysis effector protein S105 start at Met codons 1 and 3 of the Lambda S gene, respectively. The antagonistic action of both proteins precisely schedules lysis by formation of a non-specific lesion in the inner membrane through which the Lambda-encoded murein transglycosylase can pass. Here, we show that the main difference between lysis—effector and lysis—inhibitor is the degree by which an energized membrane inhibits either protein from hole formation. To dissect the structural parameters responsible for intrinsic inhibition of both proteins, charged amino acids were replaced proximal to the first putative membrane-spanning region in both S proteins. Our results show that the distribution of amino-terminal charged amino acids as well as the total amino-terminal net charge of S107 and S105 influence their lethal potential. The data are interpreted in terms of a model in which the electrostatic status of the amino-terminus of both S107 and S105 is an important feature affecting their conf or mat ional change required for formation of the S-dependent hole.     

Amphiphilicity is essential for mitochondrial presequence function.

We have shown earlier that a mitochondrial presequence peptide can form an amphiphilic helix. However, the importance of amphiphilicity for mitochondrial presequence function became doubtful when an artificial presequence, designed to be non-amphiphilic, proved to be active as a mitochondrial import signal. We now show experimentally that this 'non-amphiphilic' presequence peptide is, in fact, highly amphiphilic as measured by its ability to insert into phospholipid monolayers and to disrupt phospholipid vesicles. This result, and similar tests on three additional artificial presequences (two functionally active and one inactive), revealed that all active presequences were amphiphilic whereas the inactive presequence was non-amphiphilic. One of the active presequence peptides was non-helical in solution and in the presence of detergent micelles. We conclude that amphiphilicity is necessary for mitochondrial presequence function whereas a helical structure may not be essential.     

Reactive oxygen injury to cultured pulmonary artery endothelial cells: mediation by poly(ADP-ribose) polymerase activation causing NAD depletion and altered energy balance.

The vascular endothelium is a significant site for tissue injury following exposure to reactive oxygen species derived from a number of sources. In order to develop a better understanding of the mechanism(s) of oxidative damage, monolayer cultures of endothelial cells obtained from bovine pulmonary arteries were exposed to reactive oxygen species generated from the oxidation of dihydroxyfumarate (DHF) to diketosuccinate. Exposure to oxidizing DHF caused a loss of cell membrane integrity that was delayed in onset; that is, it did not begin until 2 h after the addition of DHF although reactive oxygen species are produced immediately by DHF in solution. Endothelial cell lysis by DHF was prevented by the simultaneous addition of superoxide dismutase (SOD), catalase (CAT), or deferoximine (DFX). This oxidant-induced lysis was unaffected by N,N,-diphenyl-p-phenylenediamine (DPPD), a potent inhibitor of lipid peroxidation. However, simultaneous addition of 3-aminobenzamide (3AB) and nicotinamide (NA), inhibitors of poly(ADP-ribose) polymerase, prevented cell lysis. Oxidant-induced loss of membrane integrity was preceded by the early appearance of DNA strand breaks, by increased levels of poly(ADP-ribose), the product of polymerase activity, and by depletion of NAD+ and ATP, followed by a decline in the energy charge ratio of the cells. None of these intracellular changes occurred when either SOD, CAT, or DFX were added at the same time as DHF, suggesting that O2-., H2O2, and HO. mediated these changes. The O2-. appears to be important in the autoxidation reaction of DHF. The latter two reactive oxygen species may be part of cellular-catalyzed Fenton chemistry. The increase in poly(ADP-ribose), depletion of NAD+, and the decline in ATP were also prevented by the addition of 3AB. The oxidant-induced DNA strand breakage was, however, unaffected by either 3AB or NA. Addition of 3AB immediately prior to the onset of cell lysis (2 h after the addition of DHF), prevented cell lysis, i.e., "rescued" the cells when neither SOD, CAT, nor DFX addition were effective. Concurrent with the "rescue" from lysis by 3AB, there was an increase in NAD+ content and a return of the energy charge ratio to control levels. The data presented in this study suggests that in endothelial cells, DNA is a very sensitive target for reactive oxygen species and HO. is the likely proximal damaging species.(ABSTRACT TRUNCATED AT 400 WORDS)     

Design and synthesis of cyclic disulfide-bonded antibacterial peptides on the basis of the alpha helical domain of Tenecin 1, an insect defensin

We synthesized cyclic disulfide-bonded (i, i+4) peptides with various net positive charges (+2-+5) from linear peptides derived from the alpha helical domain of Tenecin 1, an insect defensin, and investigated the effect of the intradisulfide bridge (i, i+4) on hydrophobicity, secondary structure, leakage activity and binding activity for large unilamellar vesicles, antimicrobial activity, and hemolytic activity. Intradisulfide bridge formation of the peptides resulted in the increase of amphiphilicity and hydrophobicity. Cyclic forms of the peptides did not deeply penetrate into PG/PC (1:1, mole ratio) large unilamellar vesicles and had a decreased lipid membrane perturbation activity for PG/PC LUVs. When the peptides interacted with PG/CL (2:1, mole ratio) LUVs, cyclic peptides with a high net positive charge (+4-+5) showed similar binding affinities and leakage activities for vesicles to those of linear forms, whereas cyclic peptides with a low net positive charge (+2-+3) exhibited lower leakage activity than their linear forms. CD spectra indicate that the intradisulfide bridge (i, i+4) provided little conformational constraint to linear peptides in buffer solution but resulted in the decrease of alpha helicity of the peptides in lipid membrane mimic conditions. The cyclic peptide with the highest net positive charge had a similar antibacterial activity to that of the linear peptide, whereas the cyclic peptides with a low net positive charge (+3-+4) exhibited lower antibacterial activity than their linear forms. The cyclic peptides of an appropriate net charge showed more potent activities against some bacteria than those of linear forms under high salt conditions.