Membrane-active peptides and the clustering of anionic lipids.

There is some overlap in the biological activities of cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs). We compared nine AMPs, seven CPPs, and a fusion peptide with regard to their ability to cluster anionic lipids in a mixture mimicking the cytoplasmic membrane of Gram-negative bacteria, as measured by differential scanning calorimetry. We also studied their bacteriostatic effect on several bacterial strains, and examined their conformational changes upon membrane binding using circular dichroism. A remarkable correlation was found between the net positive charge of the peptides and their capacity to induce anionic lipid clustering, which was independent of their secondary structure. Among the peptides studied, six AMPs and four CPPs were found to have strong anionic lipid clustering activity. These peptides also had bacteriostatic activity against several strains (particularly Gram-negative Escherichia coli) that are sensitive to lipid clustering agents. AMPs and CPPs that did not cluster anionic lipids were not toxic to E. coli. As shown previously for several types of AMPs, anionic lipid clustering likely contributes to the mechanism of antibacterial action of highly cationic CPPs. The same mechanism could explain the escape of CPPs from intracellular endosomes that are enriched with anionic lipids.

The oxysterol 3ß-hydroxy-5-oxo-5,6-secocholestan-6-al changes the phase behavior and structure of phosphatidylethanolamine-phosphatidylcholine mixtures.

An oxidized form of cholesterol, atheronal, is a form found in vivo that has been associated with human pathologies. We have studied mixtures of this oxidized sterol with the phospholipids phosphatidylethanolamine and phosphatidylcholine. We used phospholipids either with palmitoyl and oleoyl acyl chains on the C1 and C2 carbon atoms of glycerol or with both acyl chains being palmitoleoyl. We also compared the effects of atheronal on the curvature properties of these lipids with the action of cholesterol. We studied the bilayer to hexagonal phase transition temperature of mixtures of these lipids using differential scanning calorimetry as well as the dimensions of the hexagonal phase cylinders using X-ray diffraction. Disordering of the lamellar phase was also qualitatively assessed by the loss of sharp diffraction peaks. Our results demonstrate that the modulation of membrane curvature in these systems depends not only on the nature of the sterol, but also on the acyl chain composition of the phospholipids used. In addition, some of the effects of atheronal could be ascribed to reaction of the aldehyde and ketone groups of this oxidized sterol with the amino group of phosphatidylethanolamine.

Physical properties affecting cochleate formation and morphology using antimicrobial oligo-acyl-lysyl peptide mimetics and mixtures mimicking the composition of bacterial membranes in the absence of divalent cations.

Several cationic antimicrobial oligo-acyl-lysyl (OAK) peptide mimetics can form cochleate structures, that is, elongated multilayered cylindrical structures, with lipid mixtures mimicking the composition of bacterial cytoplasmic membranes. These cochleate structures do not require divalent cations for their assembly. In the present work, we use light microscopy to screen for cochleate formation in several OAK-lipid systems and freeze-fracture electron microscopy to assess their morphological features and size. We identify several factors that facilitate a structural change in these assemblies. Dehydration of the membrane interface and a high melting temperature are features of the lipids that enhance cochleate formation in OAK-based lipid systems. In addition, we observed that there is a specific length of the hydrocarbon linker in the OAK of 8-9 carbon atoms that provides optimal formation of these structures. The biophysical properties established in this study will allow for a better understanding of their role and suitability for biological studies.

OAK-based cochleates as a novel approach to overcome multidrug resistance in bacteria.

Antibiotic resistance has become a worldwide medical problem. To find new ways of overcoming this phenomenon, we investigated the role of the membrane-active oligo-acyl-lysyl (OAK) sequence C(12)K-7α(8), in combination with essentially ineffective antibiotics. Determination of minimal inhibitory concentration (MIC) against gram-negative multidrug-resistant strains of Escherichia coli revealed combinations with sub-MIC OAK levels that acted synergistically with several antibiotics, thus lowering their MICs by several orders of magnitude. To shed light into the molecular basis for this synergism, we used both mutant strains and biochemical assays. Our results suggest that bacterial sensitization to antibiotics was derived mainly from the OAK's capacity to overcome the efflux-enhanced resistance mechanism, by promoting backdoor entry of otherwise excluded antibiotics. To facilitate simultaneous delivery of the pooled drugs to an infection site, we developed a novel OAK-based cochleate system with demonstrable stability in whole blood. To assess the potential therapeutic use of such cochleates, we performed preliminary experiments that imitate systemic treatment of neutropenic mice infected with lethal inoculums of multidrug resistance E. coli. Single-dose administration of erythromycin coencapsulated in OAK-based cochleates has decreased drug toxicity and increased therapeutic efficacy in a dose-dependent manner. Collectively, our findings suggest a potentially useful approach for fighting efflux-enhanced resistance mechanisms.

Electrodeposition of hyaluronic acid and hyaluronic acid-bovine serum albumin films from aqueous solutions.

Hyaluronic acid (HYH) films were prepared from aqueous sodium hyaluronate (HYNa) solutions by anodic electrodeposition. The film thickness was varied in the range of 0-20 microm by the variation of the deposition time and HYNa concentration. The deposition rate was low at HYNa concentration below 1 g L(-1) and increased significantly in the range of 3-5 g L(-1). The addition of bovine serum albumin (BSA) to the HYNa solutions resulted in increased deposition yield, which was attributed to the formation of composite HYH-BSA films. The thickness of the HYH-BSA films deposited by anodic electrodeposition was varied in the range of 0-80 microm. The HYH and composite HYH-BSA films were studied by scanning electron microscopy, thermogravimetric analysis, differential thermal analysis, Fourier transform infrared spectroscopy and circular dichroism spectroscopy. The deposition mechanism and kinetics of deposition are discussed.

The oxidized form of cholesterol 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al induces structural and thermotropic changes in phospholipid membranes.

The effect of an oxidized form of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al on the thermotropic and structural properties of phospholipid membranes was investigated by differential scanning calorimetry and X-ray diffraction and compared with that of cholesterol. The phospholipids studied included 1-palmitoyl-2-oleoylphosphatidylserine, dipalmitoleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, dipalmitoleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine. Depending on the constituent phospholipids, the oxidized cholesterol is observed to shift phase transitions, disrupt stacking, modify interbilayer spacings and promote increased negative membrane curvature. We determined by absorption spectroscopy that the amino group of phosphatidylserine forms a Schiff base with the aldehyde group of the 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al as was previously found for the amino group of phosphatidylethanolamine. This result strengthens the biologically significant finding that not only amino groups of proteins but also amino groups of phospholipids are able to form a Schiff base with oxidized cholesterol. The marked triangular shape of the Schiff base complex with phosphatidylethanolamine may explain how 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al can promote increased negative curvature in the hexagonal phase, as compared to cholesterol, even though its increased polarity would favor a location closer to the interface with water.

Membrane lipid composition and the interaction of pardaxin: the role of cholesterol.

Modulation by pardaxin of the phase transitions of dimyristoyl phosphatidylcholine, 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-oleoyl phosphatidylglycerol in the presence or absence of cholesterol was studied by differential scanning calorimetry. The transition enthalpy of each of the phospholipids was lowered by pardaxin and there was a small decrease in the transition temperature. Addition of cholesterol and pardaxin to dimyristoyl phosphatidylcholine resulted in a very marked lowering of the transition temperature. Although the peptide broadens the transition of the pure phospholipids, it sharpens the transition of mixtures of the phospholipids with cholesterol. This and the observation that pardaxin also causes the formation of crystallites of anhydrous cholesterol, suggest that the peptide promotes redistribution of cholesterol in the membrane.

Specificity of membrane binding of the neuronal protein NAP-22.

NAP-22, a major protein of neuronal rafts is known to preferentially bind to membranes containing cholesterol. In this work we establish the requirements for membrane binding of NAP-22. We find that other sterols can replace cholesterol to promote binding. In addition, bilayers containing phosphatidylethanolamine bind NAP-22 in the absence of cholesterol. Thus, there is not a specific interaction of NAP-22 with cholesterol that determines its binding to membranes. Addition of a mol fraction of phosphatidylserine of 0.05 to membranes of phosphatidylcholine and cholesterol enhances the membrane binding of NAP-22. The dependence of binding on the mol fraction of phosphatidylserine indicates that NAP-22 binds to membranes with its amino-terminal segment closer to the membrane than the remainder of the protein. We have also determined which segments of NAP-22 are required for membrane binding. A non-myristoylated form binds only weakly to membranes. Truncating the protein from 226 amino acids to the myristoylated aminoterminal 60 amino acids does not prevent binding to membranes in a cholesterol-dependent manner, but this binding is of weaker affinity. However, myristoylation is not sufficient to promote binding to cholesterol-rich domains. An N-terminal 19-amino-acid, myristoylated peptide binds to membranes but without requiring specific lipids. Thus, the remainder of the protein contributes to the lipid specificity of the membrane binding of NAP-22.

Design and function of a conformationally restricted analog of the influenza virus fusion peptide.

A conformationally restricted analog of the N-terminal 12-residue peptide segment of the HA2 subunit of the PPV/34, PR/8/34, and Jap/57 strains of influenza virus hemagglutinin was synthesized containing three residues of Calpha-methyl-valine. This peptide has a higher content of helical structure in the presence of 50% trifluoroethanol or when interacting with liposomes of egg phosphatidylcholine compared with the conformationally more flexible control peptide with the native sequence. The control and analog peptides had opposite effects on membrane curvature as measured by shifts in the bilayer-to-hexagonal phase transition temperature of a synthetic phosphatidylethanolamine derivative. The control peptide promoted more negative curvature, particularly at acidic pH and was also more potent than the analog in promoting lipid mixing. The results indicate that the ability of the peptide to sample a broader range of conformations is required for the influenza fusion peptide to destabilize membranes and that a rigid helical structure is less fusogenic. The difference between the two peptides and between pH 7.4 and pH 5.0 show a correlation between the ability to promote negative curvature and to accelerate lipid mixing.

Role of the Glu residues of the influenza hemagglutinin fusion peptide in the pH dependence of fusion activity.

To elucidate the role of the fusion peptide in influenza hemagglutinin (HA)-mediated fusion, we compared pH-dependent conformational changes and fusion mediated by wild-type and a mutant HA in which Glu residues at positions 11 and 15 of the fusion peptide are substituted for valine. The pH dependence of conformational changes and kinetics of fusion with erythrocytes was the same for both forms of HA. The time for commitment and the temperature dependence of HA-mediated fusion were also the same. However, striking differences were observed between wild-type and mutant fusion peptides in their interactions with lipid membranes at neutral and acidic pH. Since elimination of the negatively charged residues allows the exposed fusion peptide to penetrate the bilayer at pH values closer to neutral, but does not affect conformational changes and fusion activity in intact HA, we conclude that conformational changes are tightly coupled to fusion peptide insertion in the overall HA-mediated fusion cascade.

Lipopeptaibols, a novel family of membrane active, antimicrobial peptides.

Lipopeptaibols are members of a novel group of naturally occurring, short peptides with antimicrobial activity, characterized by a lipophilic acyl chain at the N-terminus, a high content of the turn/helix forming alpha-aminoisobutyric acid and a 1,2-amino alcohol at the C-terminus. The amino acid sequences range from 6 to 10 residues and the fatty acyl moieties from 8 to 15 carbon atoms. The peptide portion of lipopeptaibols can be shorter than those of the nonlipidated peptaibols that range from 10 to 19 amino acid residues. The longest peptides fold into a mixed 3(10)/alpha helix, whereas the shortest peptides tend to adopt a beta-turn/sheet structure. Using solution methodologies, a series of analogues of trichogin GA IV was synthesized which allowed determination of the minimal lipid chain and peptide main-chain lengths for the onset of membrane activity and exploitation of a number of spectroscopic techniques aimed at determining its preferred conformation under a variety of conditions and investigating in detail its mode of interaction with, and its effect on, the phospholipid membranes.

Protein-induced formation of cholesterol-rich domains.

A major protein of neuronal rafts, NAP-22, binds specifically to cholesterol. We demonstrate by circular dichroism that NAP-22 contains a significant amount of beta-structure that is not sensitive to binding of the protein to membranes, suggesting that a major portion of the protein does not insert deeply into the membrane. The free energy of binding of NAP-22 to liposomes of dioleoylphosphatidylcholine with 40% cholesterol is -7.3 +/- 0.5 kcal/mol. NAP-22 mixed with dipalmitoylphosphatidylcholine and 40% cholesterol partitions into the detergent insoluble fraction in the presence of 1% Triton X-100. NAP-22 also causes this insoluble fraction to become enriched in cholesterol relative to phospholipid, again demonstrating the ability of this protein to segregate cholesterol and phospholipids into domains. Differential scanning calorimetry results demonstrate that NAP-22 promotes domain formation in liposomes composed of cholesterol and phosphatidylcholine. This is shown by NAP-22-promoted changes in the shape and enthalpy of the phase transition of phosphatidylcholine as well as by the appearance of cholesterol crystallite transitions in membranes composed of phosphatidylcholine with either saturated or unsaturated acyl chains. In situ atomic force microscopy revealed a marked change in the surface morphology of a supported bilayer of dioleoylphosphatidylcholine with 0.4 mole fraction of cholesterol upon addition of NAP-22. Prior to the addition of the protein, the bilayer appears to be a molecularly smooth structure with uniform thickness. Addition of NAP-22 resulted in the rapid formation of localized raised bilayer domains. Remarkably, there was no gross disruption or erosion of the bilayer but rather simply an apparent rearrangement of the lipid bilayer structure due to the interaction of NAP-22 with the lipid. Our results demonstrate that NAP-22 can induce the formation of cholesterol-rich domains in membranes. This is likely to be relevant in neuronal membrane domains that are rich in NAP-22.

A new high-temperature transition of crystalline cholesterol in mixtures with phosphatidylserine.

Phosphatidylserine and cholesterol are two major components of the cytoplasmic leaflet of the plasma membrane. The arrangement of cholesterol is markedly affected by the presence of phosphatidylserine in model membranes. At relatively low mol fractions of cholesterol in phosphatidylserine, compared with other phospholipids, cholesterol crystallites are formed that exhibit both thermotropic phase transitions as well as diffraction of x-rays. In the present study we have observed and characterized a novel thermotropic transition occurring in mixtures of phosphatidylserine and cholesterol. This new transition is observed at 96 degrees C by differential scanning calorimetry (DSC), using a heating scan rate of 2 degrees C/min. Observation of the transition requires that the hydrated lipid mixture be incubated for several days, depending on the temperature of incubation. The rate of formation of the material exhibiting a transition at 96 degrees C is more rapid at higher incubation temperatures. At 37 degrees C the half-time of conversion is approximately 7 days. Concomitant with the appearance of the 96 degrees C peak the previously known transitions of cholesterol, occurring at approximately 38 degrees C and 75 degrees C on heating scans of freshly prepared suspensions, disappear. These two transitions correspond to the polymorphic transition of anhydrous cholesterol and to the dehydration of cholesterol monohydrate, respectively. The loss of the 75 degrees C peak takes a longer time than that of the 38 degrees C peak, indicating that anhydrous cholesterol first gets hydrated to the monohydrate form exhibiting a transition at 75 degrees C and subsequently is converted by additional time of incubation to an altered form of the monohydrate, showing a phase transition at 96 degrees C. After several weeks of incubation at 37 degrees C, only the form with a phase transition at 96 degrees C remains. If such a sample undergoes several successive heating and cooling cycles, the 96 degrees C peak disappears and the 38 degrees C transition reappears on heating. For samples of 1-palmitoyl-2-oleoyl phosphatidylserine or of 1-stearoyl-2-oleoyl phosphatidylserine having mol fractions of cholesterol between 0.4 and 0.7, the 38 degrees C transition that reappears after the melting of the 96 degrees C component generally has the same enthalpy as do freshly prepared samples. This demonstrates that, at least for these samples, the amount of anhydrous cholesterol crystallites formed is indeed a property of the lipid mixture. We have also examined variations in the method of preparation of the sample and find similar behavior in all cases, although there are quantitative differences. The 96 degrees C transition is partially reversible on cooling and reheating. This transition is also scan rate dependent, indicating that it is, at least in part, kinetically determined. The enthalpy of the 96 degrees C transition, after incubation of the sample for 3 weeks at 37 degrees C is dependent on the ratio of cholesterol to 1-palmitoyl-2-oleoyl phosphatidylserine or to 1-stearoyl-2-oleoyl phosphatidylserine, with the enthalpy per mole cholesterol increasing between cholesterol mol fractions of 0.2 and 0.5. Dimyristoyl phosphatidylserine at a 1:1 molar ratio with cholesterol, after incubation at 37 degrees C, exhibits a transition at 95 degrees C that reverses on cooling at 44 degrees C, instead of 60 degrees C, as observed with either 1-palmitoyl-2-oleoyl phosphatidylserine or 1-stearoyl-2-oleoyl phosphatidylserine. These findings along with the essential absence of the 96 degrees C transition in pure cholesterol or in cholesterol/phosphatidylcholine mixtures, indicates that the phospholipid affects the characteristics of the transition, and therefore the cholesterol crystallites must be in direct contact with the phospholipid and are not simply in the form of pure crystals of cholesterol. These observations are particularly important in view of recent observations of the presence of cholesterol crystals in biological systems.

Effects of increasing hydrophobicity on the physical-chemical and biological properties of a class A amphipathic helical peptide.

We have recently shown that a class A amphipathic peptide 5F with increased amphipathicity protected mice from diet-induced atherosclerosis (Garber et al. J. Lipid Res. 2001. 42: 545-552). We have now examined the effects of increasing the hydrophobicity of a series of homologous class A amphipathic peptides, including 5F, on physical and functional properties related to atherosclerosis inhibition by systematically replacing existing nonpolar amino acids with phenylalanine. The peptides, based on the sequence Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH(2) (Ac-18A-NH(2) or 2F) were: 3F(3)(Ac-F(3)18A-NH(2)), 3F(14)(Ac-F(14)18A-NH(2)), 4F(Ac-F(3,14)18A-NH(2)), 5F(Ac-F(11,14,17) 18A-NH(2)), 6F(Ac-F(10,11,14,17)18A-NH(2)), and 7F(Ac-F(3,10,11,14,17) 18A-NH(2)). Measurements of aqueous solubility, HPLC retention time, exclusion pressure for penetration into an egg phosphatidylcholine (EPC) monolayer, and rates of EPC solubilization revealed an abrupt increase in the hydrophobicity between peptides 4F and 5F; this was accompanied by increased ability to associate with phospholipids. The peptides 6F and 7F were less effective, indicating a limit to increased hydrophobicity for promoting lipid interaction in these peptides. Despite this marked increase in lipid affinity, these peptides were less effective than apoA-I in activating the plasma enzyme, lecithin:cholesterol acyltransferase, with 5F activating LCAT the best (80% of apoA-I). Peptides 4F, 5F, and 6F were equally potent in inhibiting LDL-induced monocyte chemotactic activity. These studies suggest that an appropriate balance between peptide-peptide and peptide-lipid interactions is required for optimal biological activity of amphipathic peptides. These studies provide a rationale for the design of small apoA-I-mimetics with increased potency for atherosclerosis inhibition.

Membrane interactions of mutated forms of the influenza fusion peptide.

We have studied a group of fusion peptides of influenza hemagglutinin in which the N-terminal amino acid, Gly (found in the wild-type peptide), has been systematically substituted with Ala, Ser, Val, or Glu. The activity of the intact hemagglutinin protein with these same substitutions has already been reported. As a measure of the extent of modulation of intrinsic membrane curvature by these peptides, we determined their effects on the polymorphic phase transition of dipalmitoleoylphosphatidylethanolamine. The wild-type peptide is the only one that, at pH 5, can substantially decrease the temperature of this transition. This is also the only form in which the intact protein promotes contents mixing in cells. The Ala and Ser mutant hemagglutinins exhibit a hemifusion phenotype, and their fusion peptides have little effect on lipid polymorphism at low pH. The two mutant proteins that are completely fusion inactive are the Val and Glu mutant hemagglutinins. The fusion peptides from these forms significantly increase the polymorphic phase transition temperature at low pH. We find that the effect of the fusion peptides on membrane curvature, as monitored by a shift in the temperature of this polymorphic phase transition, correlates better with the fusogenic activities of the corresponding protein than do measurements of the isotropic (31)P NMR signals or the ability to induce the fusion of liposomes. The inactivity of the hemagglutinin protein with the hydrophobic Val mutation can be explained by the change in the angle of membrane insertion of the helical fusion peptide as measured by polarized FTIR. Thus, the nature of the interactions of the fusion peptides with membranes can, in large part, explain the differences in the fusogenic activity of the intact protein.

Self-assembly of influenza hemagglutinin: studies of ectodomain aggregation by in situ atomic force microscopy.

We have used in situ tapping mode atomic force microscopy (AFM) to study the structural morphology of two fragments of the influenza hemagglutinin protein bound to supported bilayers. The two proteins that we studied are the bromelain-cleaved hemagglutinin (BHA), corresponding to the full ectodomain of the hemagglutinin protein, and FHA2, the 127 amino acid N-terminal fragment of the HA2 subunit of the hemagglutinin protein. While BHA is water soluble at neutral pH and is known to bind to membranes via specific interactions with a viral receptor, FHA2 can only be solubilized in water with an appropriate detergent. Furthermore, FHA2 is known to readily bind to membranes at neutral pH in the absence of a receptor. Our in situ AFM studies demonstrated that, when bound to supported bilayers at neutral pH, both these proteins are self-assembled as single trimeric molecules. In situ acidification resulted in further lateral association of the FHA2 without a large perturbation of the bilayer. In contrast, BHA remained largely unaffected by acidification, except in areas of exposed mica where it is aggregated. Remarkably, these results are consistent with previous observations that FHA2 promotes membrane fusion while BHA only induces liposome leakage at low pH. The results presented here are the first example of in situ imaging of the ectodomain of a viral envelope protein allowing characterization of the real-time self-assembly of a membrane fusion protein.

Factors contributing to the fusogenic potency of foamy virus.

Three model peptides have been studied in an effort to understand the molecular basis for the fusogenic potency of foamy virus. These peptides corresponded to a 23 amino acid helical segment close to the amino terminus, a shortened 17 amino acid, more hydrophobic homolog of this peptide, and an 18-amino-acid peptide close to or within the transmembrane domain. The peptides have a conformation containing both alpha-helical and beta-structure in aqueous solution but are predominantly alpha-helical in solutions of trifluoroethanol, as assessed by circular dichroism. In common with other viruses, the most fusogenic peptide was the one closest to the amino terminus. However, unlike several other fusion peptides that have been studied previously, this peptide did not promote increase negative membrane curvature as assessed by effects of the peptide on lipid polymorphism. Nevertheless, the foamy virus fusion peptide promotes membrane fusion, apparently by a mechanism that is independent of changes in membrane curvature. We demonstrate that there is a synergistic action in the promotion of membrane fusion between the peptide from the amino terminal region and the one from the region adjacent to the transmembrane segment.

Ligand-modulation of the stability of the glucose transporter GLUT 1.

The glucose transporter GLUT 1 was isolated from human erythrocytes and reconstituted into endogenous membrane lipids. Results from thermal denaturation studies, using differential scanning calorimetry, indicate that the thermal denaturation temperature of GLUT 1 is significantly lower in the presence of ATP. The lowering of this transition temperature is very dependent on pH. At more acidic pH, ATP has a greater effect of lowering the thermal denaturation temperature of the protein. For example, with 4.8 mM ATP, the denaturation endotherm is lowered by over 10 degrees at pH 4.3, whereas at pH 7.4, ATP does not alter this transition temperature. However, a change in pH alone, in the absence of ATP, has very little effect on the denaturation temperature. Both glucose and salt partially reverse the lowering of the temperature of thermal denaturation caused by ATP. Studies of acrylamide quenching of the Trp residues of GLUT 1 indicate that at neutral pH, ATP increases the Stern-Volmer quenching constant, while glucose lowers it. The results indicate that ATP binds to GLUT 1 and destabilizes the native structure, leading to a lowering of the thermal denaturation temperature and an increase in acrylamide quenching. The effects of ATP are reversed in part by glucose and are also partly electrostatic in nature.

A novel linear amphipathic beta-sheet cationic antimicrobial peptide with enhanced selectivity for bacterial lipids.

All known naturally occurring linear cationic peptides adopt an amphipathic alpha-helical conformation upon binding to lipids as an initial step in the induction of cell leakage. We designed an 18-residue peptide, (KIGAKI)3-NH2, that has no amphipathic character as an alpha-helix but can form a highly amphipathic beta-sheet. When bound to lipids, (KIGAKI)3-NH2 did indeed form a beta-sheet structure as evidenced by Fourier transform infrared and circular dichroism spectroscopy. The antimicrobial activity of this peptide was compared with that of (KIAGKIA)3-NH2, and it was better than that of GMASKAGAIAGKIAKVALKAL-NH2 (PGLa) and (KLAGLAK)3-NH2, all of which form amphipathic alpha-helices when bound to membranes. (KIGAKI)3-NH2 was much less effective at inducing leakage in lipid vesicles composed of mixtures of the acidic lipid, phosphatidylglycerol, and the neutral lipid, phosphatidylcholine, as compared with the other peptides. However, when phosphatidylethanolamine replaced phosphatidylcholine, the lytic potency of PGLa and the alpha-helical model peptides was reduced, whereas that of (KIGAKI)3-NH2 was improved. Fluorescence experiments using analogs containing a single tryptophan residue showed significant differences between (KIGAKI)3-NH2 and the alpha-helical peptides in their interactions with lipid vesicles. Because the data suggest enhanced selectivity between bacterial and mammalian lipids, linear amphipathic beta-sheet peptides such as (KIGAKI)3-NH2 warrant further investigation as potential antimicrobial agents.

Membrane perturbing properties of sucrose polyesters.

Sucrose polyester (SPE), in the form of sucrose octaesters and sucrose hexaesters of palmitic (16:0), stearic (18:0), oleic (18:1cis), and linoleic (18:2cis) acids, have many uses. Applications include: a non-caloric fat substitute, detoxification agent, and oral contrast agent for human abdominal (MRI) magnetic resonance imaging. However, it has been shown that the ingestion of SPE was shown to generate a depletion of physiologically important lipidic vitamins and other lipophilic molecules. In order to better understand, at the molecular level, the type of interaction between SPE and lipid membrane, we have, first synthesized different type of labelled and non-labelled SPEs. Secondly, we have studied the effect of SPEs on multilamellar dispersions of dielaidoylphosphatidylethanolamine (DEPE) and dipalmitoylphosphocholine (DPPC) as a function of temperature, SPE composition and concentration. The effects of SPEs were studied by differential scanning calorimetry (DSC), X-ray diffraction, 2H and 31P NMR spectroscopy. At low concentration (< 1 mol%) all of the SPEs lowered the bilayer to the inverted hexagonal phase transition temperature of DEPE and induced the formation of a cubic phase in a composition dependent manner. At the same low concentration, SPEs in DPPC induce the formation of a non-bilayer phase as seen by 31P NMR. Order parameter measurements of DPPC-d62/SPE mixtures show that the SPE effect on the DPPC monolayer thickness is dependent on the SPE, concentration, chains length and saturation level. At higher concentration (> or = 10 mol%) SPE are very potent DEPE bilayer to HII phase transition promoters, although at that concentration the SPE have lost the ability to form cubic phases. SPEs have profound effects on the phase behaviour of model membrane systems, and may be important to consider when developing current and potential industrial and medical applications.