An NMR study of pyridine associated with DMPC liposomes and magnetically ordered DMPC-surfactant mixed micelles.

With molecular dynamics simulations of phospholipid membranes becoming a reality, there is a growing need for experiments that provide the molecular details necessary to test these computational results. Pyridine is used here to explore the interaction of planar aromatic groups with the water-lipid interface of membranes. It is shown by magic angle spinning 13C nuclear magnetic resonance (NMR) to bind between the glycerol and choline groups of dimyristoylphosphatidylcholine (DMPC) liposomes. The axial pattern for the 31P NMR spectrum of DMPC liposomes is preserved even with more than half of the interfacial sites occupied, indicating that pyridine does not disrupt the lamellar phase of this lipid. 2H NMR experiments of liposomes in deuterium oxide demonstrate that pyridine might promote greater penetration of water into restricted regions in the interface. Magnetically oriented DMPC/surfactant micelles were investigated as a means for improving resolution and sensitivity in NMR studies of species bound to bilayers. The quadrupolar splittings in the 2H NMR spectra of d5-pyridine in DMPC liposomes and magnetically oriented DMPC/Trixon X-100 micelles indicate a common bound state for the two bilayer systems. The well resolved quadrupolar splittings of d5-pyridine in oriented micelles were used to establish the tilt of the pyridine ring relative to the bilayer plane.     

High-resolution structural profile of hylaseptin-4: Aggregation,membrane topology and pH dependence of overall membrane binding process

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH₂) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d₃₈ micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.     

A comparative negative staining study of aqueous suspensions of sphingomyelin

Bovine brain sphingomyelin liposomes have been studied by TEM when negatively stained at 4, 22 and 60δC with uranyl acetate,sodium phosphotungstate and amonium molybdate. The liposomes images vary slightly in the different negative stains, yet overall agreement exists once the different permeability and ionic propreties of the stains and the orietation of the liposomes are taken into account. Sodium phosphotungstate possesses an undesirable aggregative action on sphingomylin liposomes, but no aggregation has been encountered with the other stains. The liposomes images at 4 and 22δC are very similar, since both temperatures are beneath the crystalline-liqiud crystalline phase transition temperature (Tc). At 60δC, wich is above the Tc for sphingomyelin, the liposomal particles appear to be much more flexible and accordingly present a more varied shape than at the lower temperatured. The overall conformation of the sphingomyelin liposome is thought to be a ca 50 nm flattened single bilayer vesicle. Nevertheless, data are presented which suggest that some single bilayer disc-like micelles of sphingomyelin are also present.The larger multi-lamellar particular structures or myelion bodies, which are present when solid sphingomyelin is simply disperesed in water, have been studied by negative staining at 22 and 60δC. At 22δC, sphingomylin myelin bodies contain bilayers which exhibit the 26.5 nm undulatory P⨿β′pre-transition phase. The periodic feature is revealed particularly clearly by uranyl acetate. Considerable image complexity is usually present, because of overlapping information from more than one bilayer. Myelin bodies are occasionally split open during the negative staining and the single bilayer regions then reveal the image periodicity with superior clarity. Ammonium molybdate reveals the Pβ′ pre-transition phase undulations rather faintly owing to the permeation of this across the phospholipid bilayers. Sodium phosphotungstate has not been found to reveal this structural feature of the phospholipid bilayer. Ar 22δC some liposomes from spontaneously from the larger lipid bodies, but at 60δC their vesicularization occurs very much more rapidly.     

Interactions between biliary lipid micelles and intestinal brush border membranes investigated by 1H and 31P nuclear magnetic resonance

The effect of taurocholate and lecithincholesterol-taurocholate mixed micelles on the structure of isolated intestinal brush border membranes was investigated by nuclear magnetic resonance (NMR). Rabbit brush border membranes isolated by a Mg²⁺ precipitation step were chosen for this study because of their stability and integrity as revealed by ³¹P NMR. Incubation of taurocholate with the brush border membranes does not induce significant solubilization of these membranes even when the taurocholate/phospholipid ratio reaches 3.0 ¹H NMR studies indicate that taurocholate is included in the membrane bilayer at low concentration (3 mM). However this biliary salt produces a size diminution of the vesicles when its concentration increases. Incorporation of lecithin or lecithin-cholesterol in micelles of taurocholate and subsequent incubation with brush border membranes lead simultaneously to a decrease in the ³¹P NMR isotropic/bilayer line ratio, and to an increase in . These results indicate a protective effect of these compounds against lytic damage of taurocholate. Futhermore the equilibrium distribution of lecithin between mixed micelles and the membrane bilayer is strongly in favour of complete integration of micellar components in the bilayer. These data suggest that uptake of lipids from the micellar phase by isolated brush border membranes involves an interaction of the micelles with membranes followed by a fusion process.     

Antimicrobial and Membrane Disrupting Activities of a Peptide Derived from the Human Cathelicidin Antimicrobial Peptide LL37

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an α-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. ¹⁵N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. ³¹P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.     

Phospholipid vesicle solubilization and reconstitution by detergents. Symmetrical analysis of the two processes using octaethylene glycol mono-n-dodecyl ether

The processes of liposome solubilization and reconstitution were studied by using n-dodecyl octaethylene glycol monoether (C12E8). The solubilization of large unilamellar liposomes prepared by reverse-phase evaporation was systematically investigated by turbidity, 31P nuclear magnetic resonance, and centrifugation experiments. The solubilization process is well described by the three-stage model previously proposed for other detergents, and our results further demonstrate the validity of some of the postulates related to this model. In stage I, the detergent distributes between the bilayers and the aqueous solution with a partition coefficient of 1.6 mM-1. In stage II, the detergent-saturated liposomes convert into mixed micelles, the conversion being complete by stage III where all the phospholipids are present as mixed micelles. The agreement between the three methods was excellent, and the results allowed quantitative determination of the effective detergent to phospholipid ratios at which the lamellar to micellar transformation begins and is complete, which amounted to 0.66 and 2.2 (mol/mol), respectively. Furthermore, compositional analysis determined from centrifugation experiments directly demonstrate that the properties of detergent-saturated liposomes and mixed micelles remain constant throughout most of stage II: the C12E8 to phospholipid ratios in the pelleted vesicles and in micelles are constant during stage II and similar to the ratios at which stage II was initiated and complete, respectively. On the other hand, bilayer formation upon detergent removal from mixed C12E8-phospholipid micelles by SM2 Bio-Beads is demonstrated to be the symmetrical opposite of bilayer solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of the post-insertion method for the formation of ligand-coupled liposomes

A new technique is described for the formation of ligand-targeted liposomes that can be used with whole antibodies, antibody fragments, peptides or other ligands. The ligands are coupled to polyethylene glycol micelles and then transferred in a simple incubation step from the micelles into the outer monolayer of pre-formed, drug-loaded liposomes. This versatile method allows a combinatorial approach to the design of targeted liposomes that minimises manufacturing complexities, allowing a variety of ligands to be inserted into a variety of pre-formed liposomes containing a variety of drugs. This allows the ligand-targeted therapeutics to be tailored to the needs of individual patients.     

Membrane-Mimicking Entities Induce Structuring of the Two-Peptide Bacteriocins Plantaricin E/F and Plantaricin J/K

Lactobacillus plantarum C11 produces plantaricin E/F (PlnE/F) and plantaricin J/K (PlnJ/K), two bacteriocins whose activity depends on the complementary action of two peptides (PlnE and PlnF; PlnJ and PlnK). Three of the individual Pln peptides possess some antimicrobial activity, but the highest bacteriocin activity is obtained by combining complementary peptides in about a one-to-one ratio. Circular dichroism was used to study the structure of the Pln peptides under various conditions. All four peptides were unstructured under aqueous conditions but adopted a partly alpha-helical structure in the presence of trifluoroethanol, micelles of dodecylphosphocholine, and negatively charged dioleoylphosphoglycerol (DOPG) liposomes. Far less structure was induced by zwitterionic dioleoylglycerophosphocholine liposomes, indicating that a net negative charge on the phospholipid bilayer is important for a structure-inducing interaction between (positively charged) Pln peptides and a membrane. The structuring of complementary peptides was considerably enhanced when both (PlnE and PlnF or PlnJ and PlnK) were added simultaneously to DOPG liposomes. Such additional structuring was not observed in experiments with trifluoroethanol or dodecylphosphocholine, indicating that the apparent structure-inducing interaction between complementary Pln peptides requires the presence of a phospholipid bilayer. The amino acid sequences of the Pln peptides are such that the alpha-helical structures adopted upon interaction with the membrane and each other are amphiphilic in nature, thus enabling membrane interactions.     

Phospholipid restoration of microsome membranes damaged by Fe2+--ascorbate-dependent lipid peroxidation

The incubation of microsomes damaged by Fe2+--ascorbate-dependent lipid peroxidation with phosphatidylcholine liposomes and micelles is accompanied by the rate decrease of the reduced cytochrome P450 inactivation in microsome membranes. It indicates the elimination of lipid bilayer injuries. The results of study of the saturation degree, surface charge and size of liposomes and micelles influence on the ability to reconstruct the damaged lipid bilayer are presented.     

Amiodarone-liposome interaction: a multinuclear NMR and X-ray diffraction study

Amiodarone, a potent antiarrhythmic drug, is widely used in cardiology. Its electrophysiological effects, as well as many of its side effects, seem to involve lipids. We report here a multinuclear NMR and X-ray diffraction study of amiodarone in egg phosphatidylcholine liposomes and lipid multilayers. In proton NMR experiments, amiodarone alters the signal from the lipid trimethyl ammonium group for pH values ranging from 3.2 to 8.4; cholesterol does not cause this alteration. The addition of SCN- changes both the proton and phosphorus NMR spectra of liposomes containing amiodarone. For both proton and carbon NMR, amiodarone modifies the signal from the lipid methylene groups, but to a far lesser extent than does cholesterol. Incorporation of amiodarone in EPC bilayers also modifies the low-angle X-ray diffraction patterns, decreasing the lamellar repeat period at low water contents, but swelling the fluid spaces between bilayers at high water contents. Electron density profiles and modeling studies using the X-ray data indicate that amiodarone decreases the bilayer thickness and adds electron density at the interfacial region of the bilayer. Our analysis of the NMR and X-ray data indicates that the iodine atoms of amiodarone are located near the hydrocarbon/water interface and that the tertiary amine of amiodarone is in the headgroup region of the bilayer.     

Interaction study between maltose-modified PPI dendrimers and lipidic model membranes

The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers — OS) or completely (dense shell glycodendrimers — DS) modified with maltose residues. As a model membrane, two types of 100 nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D₂O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.     

Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis

The surrounding environment has significant consequences for the structural and functional properties of membrane proteins. While native structure and function can be reconstituted in lipid bilayer membranes, the detergents used for protein solubilization are not always compatible with biological activity and, hence, not always appropriate for direct detection of ligand binding by NMR spectroscopy. Here we describe how the sample environment affects the activity of the outer membrane protein Ail (attachment invasion locus) from Yersinia pestis. Although Ail adopts the correct β-barrel fold in micelles, the high detergent concentrations required for NMR structural studies are not compatible with the ligand binding functionality of the protein. We also describe preparations of Ail embedded in phospholipid bilayer nanodiscs, optimized for NMR studies and ligand binding activity assays. Ail in nanodiscs is capable of binding its human ligand fibronectin and also yields high quality NMR spectra that reflect the proper fold. Binding activity assays, developed to be performed directly with the NMR samples, show that ligand binding involves the extracellular loops of Ail. The data show that even when detergent micelles support the protein fold, detergents can interfere with activity in subtle ways.     

Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K channels

The membrane location of two fragments in two different K⁺-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative “paddle” domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T₁ and ¹³C-¹H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. ²H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.     

Interleukin-2 binds to gangliosides in micelles and lipid bilayers

Gangliosides shed from the surface of tumour cells may be involved in tumour-induced immunosuppression. These anionic sialoglycolipids are known to be potent inhibitors of lymphocyte proliferation, and it has been suggested that they interfere with processes mediated by the growth factor interleukin-2 (IL-2). We have thus investigated the interaction of IL-2 with gangliosides in micelles and lipid bilayers. Gel filtration FPLC showed that 125I-IL-2 can bind to micellar gangliosides in aqueous solution, and this interaction was strongly promoted by low concentrations of serum. Binding to ganglioside micelles was specific in that it required a native IL-2 molecule. IL-2 binding remained unchanged in the presence of 40% ethylene glycol, suggesting that it was not due to hydrophobic interactions. Ganglioside oligosaccharides alone were not able to bind to IL-2. Direct binding studies and gel filtration chromatography indicated that both multilamellar liposomes and 100 nm unilamellar vesicles containing gangliosides were able to interact with IL-2. Bilayers of lipid alone showed no binding. The interaction of IL-2 with bilayer gangliosides was highly dependent on the bilayer lipid composition, but appeared independent of lipid phase state. These results suggest that gangliosides may be a physiologically relevant target for IL-2 binding.     

A New Water-Soluble Analogue of the Fusion Peptide of Influenza Virus Hemagglutinin: Synthesis and Properties

A water-soluble analogue F32 of the fusion peptide from influenza virus hemagglutinin was synthesized. It consisted of 32 aa residues and retained the ability to interact with lipid membranes; its N-terminal sequence 1–24 coincided with that of the fusion protein from hemagglutinin (strain A/PR/8/34), whereas residues 25–32 (GGGKKKKK) provided its solubility in water. The peptide induced the conductivity fluctuations in planar bilayer lipid membranes characteristic of active fusion peptides. Conditions were found using CD spectroscopy under which the structure of F32 inside detergent micelles, where it can be studied by high-resolution ¹H NMR spectroscopy, is close to the structure of the peptide during its interaction with phospholipid liposomes.     

How lipophilic cannabinergic ligands reach their receptor sites

It is postulated that lipophilic ligands reach their sites of action on membrane-bound functional proteins through fast lateral diffusion across the membrane bilayer. We have shown using NMR experiments that such ligands when incorporated in a membrane system assume a preferred orientation and conformation. While occupying a specific location within the bilayer, these molecules undergo fast lateral diffusion which allows them to engage in productive interactions with their respective protein sites of action. The proposed model is discussed using a group of classical and non-classical cannabinoids as well as the endogenous cannabinoid ligand anandamide.     

Asymmetric disposition of detergents within vesicle bilayer and its effect on ion permeation through the membrane

Phospholipid vesicles were prepared by detergent removal using hydrophobic porous beads, Amberlite XAD-2, or dialysis from detergent-phospholipid mixed micelles. The liposomes formed were found to be mostly unilammellar vesicles. The vesicle diameter was estimated, by both quasi-elastic light-scattering and gel-exclusion chromatography on Sephacryl S-1000, to be 80 nm for the vesicles formed by removal of octaethylene glycol monododecyl ether by the bead method. The effect of detergents within a bilayer on ion permeation was demonstrated. When the content of octaethylene glycol monododecyl ether reached a molar ratio of 0.2, the intrinsic ion selectivity of the phospholipid membrane between anion and cation was diminished. The ion permeability measured for vesicles with detergent incorporated into initially detergent-free vesicles was about 10-times greater than that for vesicles with detergent remaining following the process of detergent removal. This observation was explained by the different disposition of the detergent in the bilayer, that is, when vesicles were formed by the removal of detergent from mixed micelles, the residual detergent became distributed in both the outer and inner leaflets, and when the detergent was incorporated into initially detergent-free vesicles, the detergent became distributed only in the outer leaflet within the experimental time limits. This idea was supported by the NMR studies. It was also found that, as a detergent, octaethylene glycol monododecyl ether has a stronger effect on ion permeation than octyl glucoside.     

Structurally related glucosylated liposomes: Correlation of physicochemical and biological features

Liposomes functionalized on their surface with carbohydrates (glycoliposomes) represent an optimal approach for targeting of drugs to diseased tissues in vivo, thanks to biocompatibility, low toxicity and easy manufacturing of these lipid nanoparticles. Here we report on the study of liposomes including a novel glycosylated amphiphile and on the comparison of their features with those of glycosylated analogues described previously. Further, the capability of the different glucosylated formulations to interact with three breast cancer cell lines was investigated. Our results show that the hydrophobic portion of the lipid bilayer strongly influences both the properties and the internalization of glycosylated liposomes.     

Ionophore-catalyzed cation transport between phospholipid inverted micelles manifest in DNMR

Studies of hyperfine shifts of lipid 31P resonances due to hydrated phospholipid inverted micelles in benzene are presented. Systems with distinct resonances from micelles containing no paramagnetic ions, and from micelles containing a single praseodymium(III) or a single europium(III) ion (and three nitrate counterions) have been generated. The addition of an ionophoric antibiotic from Streptomyces lasaliensis, lasalocid-A (X537A). causes both resonances to broaden and. with further additions, coalesce and eventually resharpen as a single line. Dilution of only the ionophore reverses these spectral changes. This is interpreted as a manifestation of dynamic NMR (DNMR. exchange broadening): i.e., that the ionophore catalyzes the equilibrium exchange of metal ions from micelle to micelle to the point where it becomes fast on the NMR time scale. This exchange is inhibited by protons or other competitive metal ions. We have simulated the spectra with a total lineshape analysis program and have thus extracted the average preexchange lifetimes for various concentrations of the antibiotic. We find a reasonably good first-order dependence on lasalocid-A concentration in each of several different experiments. This is in contrast to the higher order concentration dependences often observed by others using different techniques employing bilayer membranes. We favor a diffusional carrier mechanism involving surface aggregates of lasalocid-A for our process. This leads to the implication that a higher order concentration dependence found for a bilayer system arises from a different mechanism. The ionophore valinomycin does not catalyze rapid exchange in our system.     

The adjuvant effect of selenium nanoparticles,Triton X-114 detergent micelles,and lecithin liposomes for <Emphasis Type="Italic">Escherichia coli</Emphasis> antigens

We studied the adjuvant properties of micelles from nonionogenic detergents, liposome, and selenium nanoparticles containing extracellular and intracellular vaccine antigens of a weakly virulent α-hemolytic Escherichia coli B-5 strain used for the immunization of experimental animals. Triton X-100 was used as a nonionogenic detergent for micelle preparation. The liposomes were obtained on the basis of lecithin from a chicken egg and E. coli B-5 membrane lipids. Native lipoproteins of E. coli B-5 cells and peptides for the proteolytic hydrolysis of toxin-containing culture liquid were used as antigens for micelles and liposomes. The obtained data suggested that micelles, liposomes, and selenium nanoparticles can be used for immunization with cellular and extracellular E. coli antigens.