Lipid Headgroup Region Research Articles

Membrane-bound peptides mimicking transmembrane Vph1p helix 7 of yeast V-ATPase: A spectroscopic and polarity mismatch study

The V-ATPases are a family of ATP-dependent proton pumps, involved in a variety of cellular processes, including bone breakdown. V-ATPase enzymes that are too active in the latter process can result in osteoporosis, and inhibitors of the enzyme could be used to treat this disease. As a first step in studying the structure and function of the membrane-embedded interface at which proton translocation takes place, and its role in V-ATPase inhibition, synthetic peptides P1 and P2 consisting of 25 amino acid residues are presented here that mimic Vph1p helix 7 of yeast V-ATPase. A single mutation R10A between peptide P1 and P2 makes it possible to focus on the role of the essential arginine residue R735 in proton translocation. In the present work, we use a novel combination of spectroscopic techniques, such as CD spectroscopy, tryptophan emission spectra, acrylamide quenching and parallax analysis, and polarity mismatch modeling to characterize the peptides P1 and P2 in lipid bilayer systems. Based on both the spectroscopic experiments and the polarity mismatch modeling, P1 and P2 adopt a similar transmembrane conformation, with a mainly α-helical structure in the central part, placing the tryptophan residue at position 12 at a location 4 ± 2 Å from the centre of the lipid bilayer. Furthermore, the arginine at position 10 in P1 does not have an effect on the bilayer topology of the peptide, showing that the long, flexible side chain of this residue is able to snorkel towards the lipid headgroup region. This large flexibility of R735 might be important for its function in proton translocation in the V-ATPase enzyme.

Membrane permeabilization, orientation, and antimicrobial mechanism of subtilosin A

Subtilosin A is an antimicrobial peptide produced by the soil bacterium Bacillus subtilis that possesses bactericidal activity against a diverse range of bacteria, including Listeria monocytogenes. Recent structural studies have found that subtilosin A is posttranslationally modified in a unique way, placing it in a new class of bacteriocins. In this study, in order to understand the mechanism of membrane-disruption by subtilosin A, the interaction of the peptide with model phospholipid bilayers is characterized using fluorescence, solid-state NMR and differential scanning calorimetry (DSC) experiments. Our results in this study show that subtilosin A interacts with the lipid head group region of bilayer membranes in a concentration dependent manner. Fluorescence experiments reveal the interaction of subtilosin A with small unilamellar vesicles (SUVs) composed of POPC, POPG and E. coli total lipids, and that at least one edge of the molecule is buried in membrane bilayers. At high concentrations, it induces leakage from SUVs of POPC and POPE/POPG (7:3) mixture. 15N solid-state NMR data suggests that the cyclic peptide is partially inserted into bilayers, which is in agreement with the fluorescence data. 31P and 2H NMR experiments and DSC data support the hypothesis that subtilosin A adopts a partially buried orientation in lipid bilayers, by showing that it induces a conformational change in the lipid headgroup and disordering in the hydrophobic region of bilayers. These results suggest that the lipid perturbation observed in this study may be one of the consequences of subtilosin A binding to lipid bilayers, which results in membrane permeabilization at high peptide concentrations.

Membrane interaction of neuropeptide Y detected by EPR and NMR spectroscopy

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals. It belongs to the best-conserved peptides in nature, i.e., the amino acid sequences of even evolutionary widely separated species are very similar to each other. Using porcine NPY, which differs from human NPY only at position 17 (a leucine residue exchanged for a methionine), labeled with a TOAC spin probe at the 2nd, 32nd, or 34th positions of the peptide backbone, the membrane binding and penetration of NPY was determined using EPR and NMR spectroscopy. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the analysis of the EPR line shapes, the spectral contributions of free, dimerized, and membrane bound NPY could be separated. This analysis was further supported by quenching experiments, which selected the contributions of the bound NPY fraction. The results of this study give rise to a model where the α-helical part of NPY (amino acids 13–36) penetrates the membrane interface. The unstructured N-terminal part (amino acids 1–12) extends into the aqueous phase with occasional contacts with the lipid headgroup region. Besides the mixing ratio of zwitterionic and negatively charged phospholipid species, the electrostatic peptide membrane interactions are influenced by the pH value, which determines the net charge of the peptide resulting in a modified membrane binding affinity. The results of these variations indicate that NPY binding to phospholipid membranes depends strongly on the electrostatic interactions. An estimation of the transfer energy of the peptide from aqueous solution to the membrane interface Δ G supports the preferential interaction of NPY with negatively charged membranes.

Identification of the Hydrophobic Thickness of a Membrane Protein Using Fluorescence Spectroscopy: Studies with the Mechanosensitive Channel MscL,1

The hydrophobic thickness of a membrane protein is an important parameter, defining how the protein sits within the hydrocarbon core of the lipid bilayer that surrounds it in a membrane. Here we show that Trp scanning mutagenesis combined with fluorescence spectroscopy can be used to define the hydrophobic thickness of a membrane protein. The mechanosensitive channel of large conductance (MscL) contains two transmembrane alpha-helices, of which the second (TM2) is lipid-exposed. The region of TM2 that spans the hydrocarbon core of the bilayer when MscL is reconstituted into bilayers of dioleoylphosphatidylcholine runs from Leu-69 to Leu-92, giving a hydrophobic thickness of ca. 25 A. The results obtained using Trp scanning mutagenesis were confirmed using Cys residues labeled with the N-methyl-amino-7-nitroben-2-oxa-1,3-diazole [NBD] group; both fluorescence emission maxima and fluorescence lifetimes for the NBD group are sensitive to solvent dielectric constant over the range (2-40) thought to span the lipid headgroup region of a lipid bilayer. Changing phospholipid fatty acyl chain lengths from C14 and C24 results in no significant change for the fluorescence of the interfacial residues, suggesting very efficient hydrophobic matching between the protein and the surrounding lipid bilayer.

Composition of Supported Model Membranes Determined by Neutron Reflection

We have investigated the formation of supported bilayers by coadsorption of dipalmitoyl phosphatidylcholine (DPPC) with the nonionic surfactant beta-D-dodecyl maltoside. The adsorption of mixed phospholipid-surfactant micelles on hydrophilic silica surfaces at 25 degrees C was followed as a function of bulk concentration by neutron reflection. Using chain-deuterated d(25)-beta-D-dodecyl maltoside and d(62)-DPPC, we demonstrate that it is possible to determine the composition of the bilayers at each stage of a sequential dilution process, which enriches the adsorbed layer in phospholipid and leads to complete elimination of the surfactant. The final supported bilayers have thicknesses of 51 +/- 3 A and are stable to heating to 37 degrees C once all surfactant has been removed, and the structures agree well with other published data on DPPC supported bilayers. The coadsorption of cholesterol in a DPPC-surfactant mixture was also achieved, and the location and volume fraction of cholesterol in the DPPC bilayer was determined. Cholesterol is located in a 18 +/- 1 A thick layer below the lipid headgroup region and leads to an increased bilayer thickness of 58 +/- 2 A at 26 mol % of cholesterol.

Binding and insertion of α-helical anti-microbial peptides in POPC bilayers studied by molecular dynamics simulations

We have performed molecular dynamics simulations of the interactions of two α-helical anti-microbial peptides, magainin2 and its synthetic analog of MSI-78, with palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayers. We used various initial positions and orientations of the peptide with respect to the lipid bilayer, including a surface-bound state parallel to the interface, a trans-membrane state, and a partially inserted state. Our 20 ns long simulations show that both magainin2 and MSI-78 are most stable in the lipid environment, with the peptide destabilized to different extents in both aqueous and lipid/water interfacial environments. We found that there are strong specific interactions between the lysine residues of the peptides and the lipid head-group regions. MSI-78, owing to its large number of lysines, shows better binding characteristics and overall stability when compared to magainin2. We also find that both peptides destabilize the bilayer environment, as observed by the increase in lipid tail disorder and the induction of local curvature on the lipid head-groups by the peptides. From all the simulations, we conclude that the hydrogen bonding interactions between the lysines of the peptides and the oxygens of the polar lipid head-groups are the strongest and determine the overall peptide binding characteristics to the lipids.

Investigation of the interaction of myelin basic protein with phospholipid bilayers using solid-state NMR spectroscopy

Interaction of bovine myelin basic protein and its constituent charge isomers (C1–C3) with phospholipid bilayers was studied using solid-state NMR experiments on model membranes. 31P NMR experiments on multilamellar vesicles and mechanically aligned bilayers were used to measure the degree of protein-induced disorder in the lipid headgroup region while 2H NMR data provided the disorder caused by the protein in the hydrophobic core of the bilayers. Our results suggest that MBP and its charge isomers neither fragment nor significantly disrupt DMPC, POPC, POPC:POPG, and POPE bilayers. These results demonstrate that the MBP-induced fragmentation of POPC bilayers is due to the freeze-thaw cycles used in the preparation of multilamellar vesicles and not due to intrinsic protein–lipid interactions.

The Influence of Short-Chain Alcohols on Interfacial Tension, Mechanical Properties, Area/Molecule, and Permeability of Fluid Lipid Bilayers

We used micropipette aspiration to directly measure the area compressibility modulus, bending modulus, lysis tension, lysis strain, and area expansion of fluid phase 1-stearoyl, 2-oleoyl phosphatidylcholine (SOPC) lipid bilayers exposed to aqueous solutions of short-chain alcohols at alcohol concentrations ranging from 0.1 to 9.8 M. The order of effectiveness in decreasing mechanical properties and increasing area per molecule was butanol> propanol> ethanol> methanol, although the lysis strain was invariant to alcohol chain-length. Quantitatively, the trend in area compressibility modulus follows Traube’s rule of interfacial tension reduction, i.e., for each additional alcohol CH 2 group, the concentration required to reach the same area compressibility modulus was reduced roughly by a factor of 3. We convert our area compressibility data into interfacial tension values to: confirm that Traube’s rule is followed for bilayers; show that alcohols decrease the interfacial tension of bilayer-water interfaces less effectively than oil-water interfaces; determine the partition coefficients and standard Gibbs adsorption energy per CH 2 group for adsorption of alcohol into the lipid headgroup region; and predict the increase in area per headgroup as well as the critical radius and line tension of a membrane pore for each concentration and chain-length of alcohol. The area expansion predictions were confirmed by direct measurements of the area expansion of vesicles exposed to flowing alcohol solutions. These measurements were fitted to a membrane kinetic model to find membrane permeability coefficients of short-chain alcohols. Taken together, the evidence presented here supports a view that alcohol partitioning into the bilayer headgroup region, with enhanced partitioning as the chain-length of the alcohol increases, results in chain-length-dependent interfacial tension reduction with concomitant chain-length-dependent reduction in mechanical moduli and membrane thickness.

2D IR analyses of rate processes in lipid–antibiotic monomolecular films

Polarization modulation infrared reflection spectra of a 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) monolayer on a subphase containing 5 mM tetracycline hydrochloride (TC) were collected under varying surface pressures at the air–water interface. Statistical correlation spectroscopy using 2D IR, βν and kν correlation analyses were performed on these spectra to gain a better understanding of the surface pressure-induced effects on the interaction between the phospholipid and antibiotic. Conventional 2D IR correlation maps revealed strong correlation behavior between the vibrational modes of the lipid and antibiotic. βν correlation plots provided information about the relative rates of occurrence of the coupled responses noted in the conventional 2D IR plots. These calculations indicated that molecular reorientation occurs at lower surface pressures for the modes in Ring A than for the modes in Ring C. A new model-dependent two-dimensional correlation method, exponential kν correlation analysis, confirmed the results from the previous correlation methods and confirmed that the lipid–antibiotic interactions occurred in a bimodal fashion, depending upon surface pressure. The conclusions of the correlation analysis of the surface-pressure induced changes in the DPPA–TC monolayer system lead to the following model for lipid–antibiotic interaction. Initial interaction between the tetracycline molecule and the DPPA molecule occurs at low surface pressures primarily between Ring A of the tetracycline molecule and the lipid headgroup region. However, with increasing surface pressure, the mode of interaction changes, and the strongest interaction at high surface pressures occurs between Ring C of tetracycline and the DPPA headgroup.

Design, syntheses and in vitro gene delivery efficacies of novel mono-, di- and trilysinated cationic lipids: a structure-activity investigation.

Structure-activity investigation including design, syntheses, and evaluation of relative in vitro gene delivery efficacies of a novel series of cationic amphiphiles (1-10) containing mono-, di-, and trilysine headgroups are described in CHO, COS-1, and HepG2 cells. Several interesting and rather unexpected transfection profiles were observed. In general, lipid 1 with the myristyl tail used in combination with DOPE as colipid exhibited superior transfection properties compared to (a) the monolysinated analogues with longer hydrocarbon tails (lipids 2-4), (b) the dilysine (lipids 5-7) and the trilysine headgroup analogues (lipids 8-10), and (c) commercially available LipofectAmine with multiple positive charges in its polar region. As a preliminary estimate of the relative DNA-compacting efficacies of these new lysinated cationic lipids, the hydrodynamic diameters of representative lipoplexes were measured using dynamic laser light scattering technique. Our lipoplex size data are consistent with the notion that covalent grafting of an increasing number of positively charged functional groups in the headgroup region of cationic lipids need not necessarily result in more compacted lipoplexes. Both gel retardation and DNase I sensitivity assays indicated similar lipid/DNA binding interactions for all the novel mono-, di-, and trilysinated cationic lipids. MTT-assay-based cell viability results clearly demonstrate that the overall lower transfection properties of trilysine analogues (8-10) compared to their mono- (1-4) and dilysinated (5-7) counterparts are unlikely to originate from differential toxicity related effects. Taken together, the present findings support the notion that caution needs to be exercised in ensuring enhanced gene delivery efficacies of cationic lipids through covalent grafting of multiple lysine functionalities in the headgroup region.

The Effect of Fructan on Membrane Lipid Organization and Dynamics in the Dry State

Fructans are a group of fructose-based oligo- and polysaccharides, which appear to be involved in membrane preservation during dehydration by interacting with the membrane lipids. To get further understanding of the protective mechanism, the consequences of the fructan-membrane lipid interaction for the molecular organization and dynamics in the dry state were studied. POPC and DMPC were investigated in the dry state by 2H, 31P NMR, and Fourier transform infrared spectroscopy using two types of fructan and dextran. The order-disorder transition temperature of dry POPC was reduced by 70°C in the presence of fructan. Fructan increased the mobility of the acyl chains, but immobilized the lipid headgroup region. Most likely, fructans insert between the headgroups of lipids, thereby spacing the acyl chains. This results in a much lower phase transition temperature. The headgroup is immobilized by the interaction with fructan. The location of the interaction with the lipid headgroup is different for the inulin-type fructan compared to the levan-type fructan, since inulin shows interaction with the lipid phosphate group, whereas levan does not. Dextran did not influence the phase transition temperature of dry POPC showing that reduction of this temperature is not a general property of polysaccharides.

Interfacial Anchor Properties of Tryptophan Residues in Transmembrane Peptides Can Dominate over Hydrophobic Matching Effects in Peptide−Lipid Interactions

Membrane model systems consisting of phosphatidylcholines and hydrophobic alpha-helical peptides with tryptophan flanking residues, a characteristic motif for transmembrane protein segments, were used to investigate the contribution of tryptophans to peptide-lipid interactions. Peptides of different lengths and with the flanking tryptophans at different positions in the sequence were incorporated in relatively thick or thin lipid bilayers. The organization of the systems was assessed by NMR methods and by hydrogen/deuterium exchange in combination with mass spectrometry. Previously, it was found that relatively short peptides induce nonlamellar phases and that relatively long analogues order the lipid acyl chains in response to peptide-bilayer mismatch. Here it is shown that these effects do not correlate with the total hydrophobic peptide length, but instead with the length of the stretch between the flanking tryptophan residues. The tryptophan indole ring was consistently found to be positioned near the lipid carbonyl moieties, regardless of the peptide-lipid combination, as indicated by magic angle spinning NMR measurements. These observations suggest that the lipid adaptations are not primarily directed to avoid a peptide-lipid hydrophobic mismatch, but instead to prevent displacement of the tryptophan side chains from the polar-apolar interface. In contrast, long lysine-flanked analogues fully associate with a bilayer without significant lipid adaptations, and hydrogen/deuterium exchange experiments indicate that this is achieved by simply exposing more (hydrophobic) residues to the lipid headgroup region. The results highlight the specific properties that are imposed on transmembrane protein segments by flanking tryptophan residues.

Spectroscopic analysis of the interaction of a peptide sequence of Hepatitis G virus with bilayers

Merocyanine 540 (MC540) has been used as external probe to determine the interaction of the peptide sequence 125–139 corresponding to the E2 protein of Hepatitis G virus, with lipid bilayers. The probe was incorporated into large unilamellar vesicles (LUVs) or small unilamellar vesicles (SUVs) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). When incorporated into bilayers, MC540 shows two absorption maxima corresponding to the monomer (570 nm) and dimer (530 nm) form of the probe. Changes in the probe microenvironment are reflected by a modification in the position and/or intensity of these maxima. Addition of increasing amounts of peptide resulted in a slight decrease of the ratio A570/A530 thus indicating a change in MC540 partition into the membrane, going from a hydrophobic to a more hydrophilic environment. This effect was concomitant with an increase in dimer formation as stated from the values of the apparent dimerization constant ( K app) obtained. Fluorescence spectra as well as steady state anisotropy measurements were in agreement with the above results indicating that the peptide was able to relocate the probe and displacing MC540 from its initial location into the bilayer. Results with SUVs or LUVs were similar for what curvature does not seem to play any role on peptide activity. These results reflect the ability of peptide to interact with biomimetic membranes in the lipid head group region.

C2 domain of protein kinase C alpha: elucidation of the membrane docking surface by site-directed fluorescence and spin labeling.

The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca(2+)-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase C alpha (PKC alpha) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca(2+) binding loops and an anion binding site on beta-strands 3-4, such that the beta-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca(2+) binding loops and the beta-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKC alpha C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca(2+) activation or membrane docking trigger measurable fluorescence changes. Ca(2+) binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca(2+) binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Phi, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca(2+) binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca(2+) first and third Ca(2+) binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on beta-strands 3-4 lies near the headgroups. The data support a model in which the beta-strands are tilted toward the parallel orientation relative to the membrane surface.

Investigations of the Orientation of a Membrane Peptide by Sum Frequency Spectroscopy

Sum frequency spectra of gramicidin A at the air/water interface in partially deuterated dimyristoylphosphatildycholine monolayers were collected as a function of surface pressure and mole fraction of the peptide. Reorganization of the lipid and peptide molecules were followed between 5 and 35 mN/m. Selective deuteration of the lipid enabled independent observation of the ordering of the lipid tail and headgroup regions. The results from pure lipid monolayer studies revealed that both portions of the amphiphile rearranged roughly in tandem as a function of pressure. When the membrane peptide was added to the monolayer, changes in its orientation were noted at mole fractions below 0.5, but not above this value. Specifically, two sets of data for the peptide could be obtained. One was for the aliphatic residues and the other for the Trp residues as each gave a distinct sum frequency signal in the CH stretch range. The aliphatic residues, which are dispersed throughout gramicidin, revealed that the whole peptide could facilely track changes in lipid orientation at 0.1 mole fraction but did so less readily at 0.4 mole fraction. On the other hand, the Trp residues, which all reside near the C-terminus and interact strongly with the lipid headgroups, showed a greater propensity to track lipid reorientation at the higher mole fraction. These results indicated that changes in the C-terminus probably precede realignment in other parts of the membrane peptide at higher peptide concentration. Furthermore, peptide-peptide interactions probably inhibit gramicidin A reorientation.

β-Amyloid 25 to 35 Is Intercalated in Anionic and Zwitterionic Lipid Membranes to Different Extents

Neuronal plasma membranes are thought to be the primary target of the neurotoxic β-amyloid peptides (A β) in the pathogenesis of the Alzheimer's disease. Histologically, A β peptides are observed as extracellular macroscopic senile plaques, and most biophysical techniques have indicated the presence of A β close to the lipid headgroup region but not in the core of the membrane bilayers. The focus of this study is an investigation of the interaction between A β and lipid bilayers from a structural point of view. Neutron diffraction with the use of selectively deuterated amino acids has allowed us to determine unambiguously the position of the neurotoxic fragment A β (25–35) in the membrane. Two populations of the peptide are detected, one in the aqueous vicinity of the membrane surface and the second inside the hydrophobic core of the lipid membrane. The location of the C terminus was studied in two different lipid compositions and was found to be dependent on the surface charge of the membrane. The localization of β-amyloid peptides in cell membranes will offer new insights on their mechanism in the neurodegenerative process associated with Alzheimer's disease and might provide clues for therapeutic developments.

Domain V of m-calpain shows the potential to form an oblique-orientated alpha-helix, which may modulate the enzyme's activity via interactions with anionic lipid.

The activity of m-calpain, a heterodimeric, Ca2+-dependent cysteine protease appears to be modulated by membrane interactions involving oblique-orientated alpha-helix formation by a segment, GTAMRILGGVI, in the protein's smaller subunit. Here, graphical and hydrophobic moment-based analyses predicted that this segment may form an alpha-helix with strong structural resemblance to the influenza virus peptide, HA2, a known oblique-orientated alpha-helix former. Fourier transform infrared spectroscopy showed that a peptide homologue of the GTAMRILGGVI segment, VP1, adopted low levels of alpha-helical structure ( approximately 20%) in the presence of zwitterionic lipid and induced a minor decrease (3 degrees C) in the gel to liquid-crystalline phase transition temperature, TC, of the hydrocarbon chains of zwitterionic membranes, suggesting interaction with the lipid headgroup region. In contrast, VP1 adopted high levels of alpha-helical structure (65%) in the presence of anionic lipid, induced a large increase (10 degrees C) in the TC of anionic membranes, and showed high levels of anionic lipid monolayer penetration (DeltaSP = 5.5 mN.m-1), suggesting deep levels of membrane penetration. VP1 showed strong haemolytic ability (LD50 = 1.45 mm), but in the presence of ionic agents, this ability, and that of VP1 to penetrate anionic lipid monolayers, was greatly reduced. In combination, our results suggest that m-calpain domain V may penetrate membranes via the adoption of an oblique-orientated alpha-helix and electrostatic interactions. We speculate that these interactions may involve snorkelling by an arginine residue located in the polar face of this alpha-helix.

Design, Synthesis, and Transfection Biology of Novel Cationic Glycolipids for Use in Liposomal Gene Delivery

The molecular structure of the cationic lipids used in gene transfection strongly influences their transfection efficiency. High transfection efficiencies of non-glycerol-based simple monocationic transfection lipids with hydroxyethyl headgroups recently reported by us (Banerjee et al. J. Med. Chem. 1999, 42, 4292-4299) are consistent with the earlier observations that the presence of hydroxyl functionalities in the headgroup region of a cationic lipid contributes favorably in liposomal gene delivery. Using simple sugar molecules as the source of multiple hydroxyl functionalities in the headgroup region of the transfection lipids, we have synthesized four novel simple monocationic transfection lipids, namely, 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol (1), 1-deoxy-1-[methyl(ditetradecyl)ammonio]-D-arabinitol (2), 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol (3) and 1-deoxy-1-[methyl(dioctadecyl)ammonio]-D-arabinitol (4), containing hydrophobic aliphatic tails and the hydrophilic arabinosyl or xylose sugar groups linked directly to the positively charged nitrogen atom. Syntheses, chemical characterizations, and the transfection biology of these novel transfection lipids 1-4 are described in this paper. Lipid 1, the xylosyl derivative, showed maximum transfection on COS-1 cells. All the lipids showed transfection with cholesterol as colipid and not with dioleoylphosphatidylethanolamine (DOPE). Radioactive quantitation of free and complexed DNA combined with ethidium bromide exclusion measurements suggest that though nearly 70% of the DNA exists as complexed DNA, the DNA may not have condensed as was observed with other cationic lipids. Presence of additional (more than two) hydroxyl functionalities in the headgroup of the cationic lipids appears to have improved the transfection efficiency and made these lipids less cytotoxic compared to two-hydroxyl derivatives.

An Amino-Substituted Phenylethynyl-anthracene Probe Shows a Sensitivity to Changes in the Lipid Monolayer Curvature of Nonlamellar Lipid/Water Phases

The fluorescent excitation and emission spectra of an amphiphilic 2-amino-substituted bis(phenylethynyl)anthracene derivative (APA) are studied in self-organized supramolecular assemblies of the hydrated lipids dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), and monooleoylglycerol (MO), which form, under well-defined conditions, one-dimensional lamellar, two-dimensional inverted hexagonal, and three-dimensional inverted cubic structures, respectively. A comparison is made with APA spectra in organic solvents. Because the investigated fluorescent molecule is water-insoluble and is favorably located in the hydrocarbon-chain region proximate to the lipid headgroup/water interfaces of the host lipid/water structures, its spectral properties are anticipated to be influenced by the lipid monolayer curvature changes of the periodic nonlamellar organizations (as induced for instance by temperature changes). The results indicate that the temperature-induced variations of the excitation and emission maxima of APA in lipid phases are distinct from those in organic solvent and that the hydrophobic fluorophore is sensitive to the polarity of the microenvironment in the proximate vicinity of the headgroup region of the lipid assemblies. Correlations are established between the temperature dependences of the fluorescence maxima of APA and the temperature-induced changes in the curvatures of the lipid headgroup regions of the nonlamellar MO and DOPE lyotropic phases.

Action of antimicrobial peptides: two-state model.

The argument and experimental evidence are presented for a two-state model that explains the action of both helical and beta-sheet antimicrobial peptides after they bind to the plasma membranes of cells. Each peptide has two distinct physical states of binding to lipid bilayers. At low peptide-to-lipid ratios (P/L), the peptide tends to adsorb in the lipid headgroup region in a functionally inactive state. At a P/L above a threshold value P/L, the peptide forms a multiple-pore state that is lethal to a cell. The susceptibility of a cell to an antimicrobial peptide depends on the value of P/L that is determined by the lipid composition of the cell membrane. This model provides plausible explanations for the experimental findings that the susceptibility of different bacteria to a peptide is not directly correlated to its binding affinity, different peptides preferentially kill different pathogens, and peptides exhibit varying levels of lytic activity against different eukaryotic cells.