Pathways of lipid vesicle deposition on solid surfaces: a combined QCM-D and AFM study

Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications, yet the mechanism of SLB formation is only partially understood. In this study, the adsorption and subsequent conformational changes of sonicated unilamellar vesicles on silica supports were investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy, using mixtures of zwitterionic, negatively charged, and positively charged lipids, both in the presence and in the absence of Ca(2+) ions. Four different pathways of vesicle deposition could be distinguished. Depending on their charge, vesicles i). did not adsorb; ii). formed a stable vesicular layer; or iii). decomposed into an SLB after adsorption at high critical coverage or iv). at low coverage. Calcium was shown to enhance the tendency of SLB formation for negatively charged and zwitterionic vesicles. The role of vesicle-support, interbilayer, and intrabilayer interactions in the formation of SLBs is discussed.     

Diacyl glycerol arginine-based surfactants: biological and physicochemical properties of catanionic formulations

In this paper, we report on a catanionic vesicles-based strategy to reduce the cytotoxicity of the diacyl glycerol arginine-based synthetic surfactants 1,2-dimyristoyl-rac-glycero-3-O-(N ^α-acetyl-l-arginine) hydrochloride (1414RAc) and 1,2-dilauroyl-rac-glycero-3-O-(N ^α-acetyl-l-arginine) hydrochloride (1212RAc). The behavior of these surfactants was studied either as pure components or after their formulation as pseudo-tetra-chain catanionic mixtures with phosphatidylglycerol (PG) and as cationic mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) used as control. The antimicrobial activity of the negatively charged formulations against Acinetobacter baumannii was maintained with respect to the surfactant alone, while a significant improvement of the antimicrobial activity against Staphylococcus aureus was observed, together with a strong decrease of hemolytic activity. The influence of the net charge of the catanionic vesicles on membrane selectivity was studied using model membranes. The dynamics of surface tension changes induced by the addition of 1414RAc/PG aqueous dispersions into phospholipid monolayers composed of zwitterionic DPPC as model system for mammalian membranes and of negatively charged PG mimicking cytoplasmic membrane of Gram-positive bacteria was followed by tensiometry. Our results constitute a proof of principle that tuning formulation can reduce the cytotoxicity of many surfactants, opening their possible biological applications.     

Changes in conformation, stability and condensation of DNA by univalent and divalent cations in methanol-water mixtures

Circular dichroism spectroscopy, absorption spectroscopy, measurements of Tm values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na+ or Cs+) concentration and also in the presence of divalent cations Ca2+, Mg2+, and Mn2+. In the absence of divalent cations only slight conformational changes occurred and no condensation and/or aggregation could be detected. The Tm values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs+ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occurred and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.     

Conformational transition of DNA induced by cationic lipid vesicle in acidic solution: spectroscopy investigation

The conformational transition of DNA induced by the interaction between DNA and a cationic lipid vesicle, didodecyldimethylammonium bromide (DDAB), had been investigated by circular dichroism (CD) and UV spectroscopy methods. We used singular value decomposition least squares method (SVDLS) to analyze the experimental CD spectra. Although pH value influenced the conformation of DNA in solution, the results showed that upon binding to double helical DNA, positively charged liposomes induced a conformational transition of DNA molecules from the native B-form to more compact conformations. At the same time, no obvious conformational changes occurred at single-strand DNA (ssDNA). While the cationic lipid vesicles and double-strand DNA (dsDNA) were mixed at a high molar ratio of DDAB vesicles to dsDNA, the conformation of dsDNA transformed from the B-form to the C-form resulting in an increase in duplex stability (DeltaT(m)=8+/-0.4 degrees C). An increasing in T(m) was also observed while the cationic lipid vesicles interacted with ssDNA.     

Lipid-induced conformational transition of the amyloid core fragment Abeta(28-35) and its A30G and A30I mutants

The interaction of the beta-amyloid peptide (Abeta) with neuronal membranes could play a key role in the pathogenesis of Alzheimer's disease. Recent studies have focused on the interactions of Abeta oligomers to explain the neuronal toxicity accompanying Alzheimer's disease. In our study, we have investigated the role of lipid interactions with soluble Abeta(28-35) (wild-type) and its mutants A30G and A30I in their aggregation and conformational preferences. CD and Trp fluorescence spectroscopic studies indicated that, immediately on dissolution, these peptides adopted a random coil structure. Upon addition of negatively charged 1,2-dipalmitoyl-syn-glycero-3-phospho-rac-(glycerol) sodium salt (PG) lipid, the wild-type and A30I mutant underwent reorganization into a predominant beta-sheet structure. However, no conformational changes were observed in the A30G mutant on interaction with PG. In contrast, the presence of zwitterionic 1,2-dipalmitoyl-syn-glycero-3-phosphatidylcholine (PC) lipid had no effect on the conformation of these three peptides. These observations were also confirmed with atomic force microscopy and the thioflavin-T assay. In the presence of PG vesicles, both the wild-type and A30I mutant formed fibrillar structures within 2 days of incubation in NaCl/P(i), but not in their absence. Again, no oligomerization was observed with PC vesicles. The Trp studies also revealed that both ends of the three peptides are not buried deep in the vesicle membrane. Furthermore, fluorescence spectroscopy using the environment-sensitive probe 1,6-diphenyl-1,3,5-hexatriene showed an increase in the membrane fluidity upon exposure of the vesicles to the peptides. The latter effect may result from the lipid head group interactions with the peptides. Fluorescence resonance energy transfer experiments revealed that these peptides undergo a random coil-to-sheet conversion in solution on aging and that this process is accelerated by negatively charged lipid vesicles. These results indicate that aggregation depends on hydrophobicity and propensity to form beta-sheets of the amyloid peptide, and thus offer new insights into the mechanism of amyloid neurodegenerative disease.     

Conformational Transition of Giant DNA in a Confined Space Surrounded by a Phospholipid Membrane

It has been established that a long DNA molecule exhibits a large discrete conformational change from a coiled state to a highly folded state in aqueous solution, depending on the presence of various condensing agents such as polyamines. In this study, T4 DNA labeled with fluorescent dyes was encapsulated in a cell-sized microdroplet covered with a phospholipid membrane to investigate the conformational behavior of a DNA molecule in such a confined space. Fluorescence microscopy showed that the presence of Mg²⁺ induced the adsorption of DNA onto the membrane inner-surface of a droplet composed of phosphatidylethanolamine, while no adsorption was observed onto a phosphatidylcholine membrane. Under the presence of spermine (tetravalent amine), DNA had a folded conformation in the bulk solution. However, when these molecules were encapsulated in the microdroplet, DNA adsorbed onto the membrane surface accompanied by unfolding of its structure into an extended coil conformation under high concentrations of Mg²⁺. In addition, DNA molecules trapped in large droplets tended not to be adsorbed on the membrane, i.e., no conformational transition occurred. A thermodynamic analysis suggests that the translational entropy loss of a DNA molecule that is accompanied by adsorption is a key factor in these phenomena under micrometer-scale confinement.     

Effect of water soluble non-membrane proteins on the phospholipid structure

The interaction of water-soluble nonmembraneous proteins (trypsin and the basic pancreatic trypsin inhibitor (BPTI)) with soybean phospholipids was studied using multilamellar vesicles. Multilamellar vesicles were obtained from soybean lipid extracts and mixtures of individual phospholipids based on phosphatidylcholine. These mixtures contain different phospholipids: "bilayer", "nonbilayer", and negatively charged. It was shown that the content of both proteins in the complex depends on pH and the presence of negatively charged components. On the basis of this finding, the conclusion about the electrostatic nature of lipid-protein interaction was made. The structural organization of soybean phospholipids in multilamellar vesicles was studied in the presence and absence of the proteins using broad-line 31P-NMR spectroscopy. It was found that, in mixtures of phospholipids of complex composition, different types of phases coexist, and phospholipids of different classes can compensate the effects of each other. Trypsin and BPTI affect the structure of phospholipids in a similar way, inducing considerable structural changes in multilamellar vesicles of preparations containing negatively charged components in whose structure there coexisted primordially the bilayer and isotropic phases.     

Detection of peptide-lipid interactions in mixed monolayers, using isotherms, atomic force microscopy, and fourier transform infrared analyses

To improve the understanding of the membrane uptake of an amphipathic and positively charged vector peptide, we studied the interactions of this peptide with different phospholipids, the nature of whose polar headgroups and physical states were varied. Three lipids were considered: dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and dioleoylphosphatidylglycerol (DOPG). The approach was carried out by three complementary methods: compression isotherms of monolayers and atomic force microscopy observations associated with Fourier transform infrared investigations. From analysis of the compression isotherms, it was concluded that the peptide interacts with all lipids and with an expansion of the mean molecular area, implying that both components form nonideal mixtures. The expansion was larger in the case of DOPG than for DPPC and DPPG because of an alpha to beta conformational transition with an increase in the peptide molar fraction. Atomic force microscopy observations showed that the presence of small amounts of peptide led to the appearance of bowl-like particles and that an increase in the peptide amounts generated the formation of filaments. In the case of DOPG, filaments were found at higher peptide molar fractions than already observed for DOPC because of the presence of negatively charged lipid headgroups.     

The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.     

Vesicles with charged domains

This work summarizes results obtained on membranes composed of the ternary mixture dioleoylphosphatidylglycerol (DOPG), egg sphingomyelin (eSM) and cholesterol (Chol). The membrane phase state as a function of composition is characterized from data collected with fluorescence microscopy on giant unilamellar vesicles. The results suggest that the presence of the charged DOPG significantly decreases the composition region of coexistence of liquid ordered and liquid disordered phases as compared to that in the ternary mixture of dioleoylphosphatidycholine, sphingomyelin and cholesterol. The addition of calcium chloride to DOPG:eSM:Chol vesicles, and to a lesser extent the addition of sodium chloride, leads to the stabilization of the two-phase coexistence region, which is expressed in an increase in the miscibility temperature. On the other hand, addition of the chelating agent EDTA has the opposite effect, suggesting that impurities of divalent cations in preparations of giant vesicles contribute to the stabilization of charged domains. We also explore the behavior of these membranes in the presence of extruded unilamellar vesicles made of the positively charged lipid dioleoyltrimethylammoniumpropane (DOTAP). The latter can induce domain formation in DOPG:eSM:Chol vesicles with initial composition in the one-phase region.     

Conformational status of apomyoglobin in the presence of phospholipid vesicles at neutral pH

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near- and far-UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.     

The Conformational State of Apomyoglobin in the Presence of Phospholipid Vesicles at Neutral pH

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near and far UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.     

Changes in Conformation,Stability and Condensation of DNA by Univalent and Divalent Cations in Methanol-Water Mixtures

Abstract

Circular dichroism spectroscopy, absorption spectroscopy, measurements of T_m values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na⁺ or Cs⁺) concentration and also in the presence of divalent cations Ca²⁺, Mg²⁺, and Mn²⁺. In the absence of divalent cations only slight conformational changes occured and no condensation and/or aggregation could be detected. The T_m values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs⁺ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occured and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.     

Lipid-DNA complex formation: reorganization and rupture of lipid vesicles in the presence of DNA as observed by cryoelectron microscopy.

Cryoelectron microscopy has been used to study the reorganization of unilamellar cationic lipid vesicles upon the addition of DNA. Unilamellar DNA-coated vesicles, as well as multilamellar DNA lipid complexes, could be observed. Also, DNA induced fusion of unilamellar vesicles was found. DNA appears to adsorb to the oppositely charged lipid bilayer in a monolayer of parallel helices and can act as a molecular "glue" enforcing close apposition of neighboring vesicle membranes. In samples with relatively high DNA content, there is evidence for DNA-induced aggregation and flattening of unilamellar vesicles. In these samples, multilamellar complexes are rare and contain only a small number of lamellae. At lower DNA contents, large multilamellar CL-DNA complexes, often with >10 bilayers, are formed. The multilamellar complexes in both types of sample frequently exhibit partially open bilayer segments on their outside surfaces. DNA seems to accumulate or coil near the edges of such unusually terminated membranes. Multilamellar lipid-DNA complexes appear to form by a mechanism that involves the rupture of an approaching vesicle and subsequent adsorption of its membrane to a "template" vesicle or a lipid-DNA complex.     

Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and 31P-nuclear magnetic resonance

 It has been reported that repetitive freeze-thaw cycles of aqueous suspensions of dioleoylphosphatidylcholine form vesicles with a diameter smaller than 200 nm. We have applied the same treatment to a series of phospholipid suspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoylphosphatidic acid (DOPC/DOPA) mixtures. Freeze-fracture electron microscopy revealed that these unsaturated lipids form unilamellar vesicles after 10 cycles of freeze-thawing. Both electron microscopy and broad-band ³¹P NMR spectra indicated a disparity of the vesicle sizes with a highest frequency for small unilamellar vesicles (diameters ≤30 nm) and a population of larger vesicles with a frequency decreasing exponentially as the diameter increases. From ³¹P NMR investigations we inferred that the average diameter of DOPC/DOPA vesicles calculated on the basis of an exponential size distribution was of the order of 100 nm after 10 freeze-thaw cycles and only 60 nm after 50 cycles. Fragmentation by repeated freeze-thawing does not have the same efficiency for all lipid mixtures. As found already by others, fragmentation into small vesicles requires the presence of salt and does not take place in pure water. Repetitive freeze-thawing is also efficient to fragment large unilamellar vesicles obtained by filtration. If applied to sonicated DOPC vesicles, freeze-thawing treatment causes fusion of sonicated unilamellar vesicles into larger vesicles only in pure water. These experiments show the usefulness of NMR as a complementary technique to electron microscopy for size determination of lipid vesicles. The applicability of the freeze-thaw technique to different lipid mixtures confirms that this procedure is a simple way to obtain unilamellar vesicles. Received: 2 September 1999 / Revised version: 27 February 2000 / Accepted: 27 February 2000     

Spontaneous vesiculation of aqueous lipid dispersions

The swelling properties of lipid mixtures consisting of phosphatidylcholine and a charged single-chain detergent have been studied. The work presented here is confined to lipid mixtures forming smectic lamellar phases in H2O. These mixtures exhibit continuous swelling with increasing water content, provided the surface charge density exceeds a threshold value of about 1-2 microC/cm2. In excess H2O, such mixtures undergo spontaneous vesiculation: unilamellar vesicles form spontaneously when excess H2O or salt solutions of moderate ionic strength (I less than 0.2) are added to the dried film of such lipid mixtures. The resulting dispersion of unilamellar vesicles is usually polydisperse. Its average size depends on the detergent/phospholipid mole ratio, decreasing with increasing detergent content. It is shown that in the phase diagram of three-component systems consisting of phosphatidylcholine, a charged single-chain detergent, and excess H2O there is a compositional range, though narrow, within which the small unilamellar vesicle (diameter less than 100 nm) is the thermodynamically most stable structure. This behavior is characteristic of charged, single-chain detergents of 14 and more C atoms. Many pharmacologically active compounds are amphiphilic and surface-active, and as such, they will orient at phospholipid-water interfaces, imparting a net surface charge to neutral lipid surfaces. It is shown that such drugs exhibit detergent-like action. Mixed films of phosphatidylcholine and a pharmacologically active compound behave similarly to phosphatidylcholine-detergent mixtures: they undergo spontaneous vesiculation when excess H2O or salt solutions of moderate ionic strength are added. In this case, the drug itself induces vesiculation; possible pharmacological implications of this finding are discussed.     

Conformational states of the water-soluble fragment of cytochrome b5. I. pH-induced denaturation

Cytochrome b5 is a membrane protein that comprises two fragments: one is water-soluble and heme-containing, and the other is hydrophobic and membrane-embedded. The function of electron transfer is performed by the former whose crystal structure is known; however, its conformational states when in the membrane field and interacting with other proteins are still to be studied. Previously, we proposed water-alcohol mixtures for modeling the effect of membrane surface on proteins, and used this approach to study the conformational behavior of positively charged cytochrome c as well as relatively neutral retinol-binding protein also functioning in the field of negatively charged membrane. The current study describes the conformational behavior of the negatively charged water-soluble fragment of cytochrome b5 as dependent on pH. Decreasing pH was shown to transform the fragment state from native to intermediate, similar to the molten globule reported earlier for other proteins in aqueous solutions: at pH 3.0, the fragment preserved a pronounced secondary structure and compactness but lost its rigid tertiary structure. A possible role of this intermediate state in cytochrome b5 functioning is discussed.     

Biophysical characterization of quaternary pyridinium functionalized polynorbornenes for DNA complexation and their cellular interactions

Cationic polymers with hydrophobic side chains have gained great interest as DNA carriers since they form a compact complex with negatively charged DNA phosphate groups and interact with the cell membrane. Amphiphilic polyoxanorbornenes with different quaternary alkyl pyridinium side chains with ethyl‐p(OPy2) and hexyl units‐p(OPy6) bearing 10 kDa MWT were synthesized by living Ring‐Opening Metathesis Polymerization method. The physicochemical characteristics: critical micellar concentration, size distribution, surface charge, and condensation of polymer/DNA complex were investigated. Morphology of complexes was monitored by Atomic force microscopy. Cytotoxicity and interaction of these complexes with model lipid vesicles mimicking the cell membrane were examined. These polymers were enabled to form small sized complexes of DNA, which interact with model membrane vesicles. It was found that the nature of hydrophobicity of the homopolymers significantly impacts rates of DNA complexation and the surface charge of the resulting complexes. These results highlight the prospect of the further examinations of these polymers as gene carriers.     

Influence of phospholipids on the formation and stability of vimentin-type intermediate filaments

The interaction of vesicles produced from individual phospholipids and mixtures thereof with preformed vimentin filaments as well as the influence of these vesicles on filament assembly were investigated employing negative stain electron microscopy and sucrose density gradient equilibrium centrifugation. Liposomes with a phospholipid composition characteristic of Ehrlich ascites tumor cells were able to bind efficiently to vimentin filaments without significantly affecting their morphology at higher concentrations. However, in sucrose density gradient centrifugation partial disintegration of the filaments was observed. In addition, larger quantities of phospholipid mixture totally blocked intermediate filament (IF) formation. Using vesicles of individual phospholipids, these effects could be shown to be due to the presence of negatively charged lipid species in the phospholipid mixture. While these were highly active in preventing filament assembly and in dissociating preformed filaments, electrically uncharged phospholipids were virtually inactive. The highest efficiency was shown by phosphatidylinositol-4,5-diphosphate. These results demonstrate that a negative surface charge of liposomes is an essential prerequisite for their successful and tight association with vimentin filaments. However, the high susceptibility of these filaments to photoaffinity labeling with the membrane-penetrating reagent 1-azidopyrene in the presence of phospholipid vesicles, points to additional interactions between hydrophobic regions of both reactants. Finally, the data also suggest a direct relationship between IFs and the lipid bilayer as the active principle underlying the association of IFs with natural membranes as observed by electron and immunofluorescence microscopy.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.