Phosphatidylglycerol Vesicles Research Articles

Single-vesicle imaging reveals lipid-selective and stepwise membrane disruption by monomeric α-synuclein

The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson's disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction).

Apolipophorin III interaction with phosphatidylglycerol and lipopolysaccharide: A potential mechanism for antimicrobial activity

Apolipophorin III (apoLp-III) is a model insect apolipoprotein to study structure-function relationships of exchangeable apolipoproteins. The protein associates with lipoproteins to aid in the transport of neutral lipids, and also interacts with the bacterial membrane. To better understand a potential role as an antimicrobial protein, the binding interaction of apoLp-III from Locust migratoria and Galleria mellonella with phosphatidylglycerol and lipopolysaccharides was analyzed. ApoLp-III from either species induced a robust release of calcein from phosphatidylglycerol vesicles, but was ineffective for phosphatidylcholine vesicles with comparable side-chain architecture. Acetylation of L. migratoria apoLp-III lysine residues greatly reduced the calcein release from phosphatidylglycerol vesicles, indicating a critical role of lysine side-chains in phosphatidylglycerol vesicles interaction. Isothermal calorimetry provided Kd values of 0.26 μM (L. migratoria) and 0.50 μM (G. mellonella) for binding to dimyristoylphosphatidylglycerol vesicles, which is an order of magnitude stronger compared to zwitterionic vesicles. A strong preference of apoLp-III for dimyristoylphosphatidylglycerol vesicles was also observed with differential scanning calorimetry with a concentration dependent shift in the lipid phase transition temperature. Native PAGE analysis showed that LPS binding was significantly weaker for L. migratoria apoLp-III compared to G. mellonella apoLp-III. This difference was confirmed by fluorescence titration analysis of L. migratoria apoLp-III, which also indicated that acetylation of the apolipoprotein did not affect LPS binding. Taken together, the results indicate that apoLp-III phosphatidylglycerol interaction may follow a detergent model with an important electrostatic binding component. Since lipopolysaccharide binding was not affected by neutralization of apoLp-III lysine-side chains, the binding interaction may be distinctly different from that of phosphatidylglycerol.

Lipid-bound apoLp-III is less effective in binding to lipopolysaccharides and phosphatidylglycerol vesicles compared to the lipid-free protein.

Apolipophorin III (apoLp-III) is an insect apolipoprotein that is predominantly present in a lipid-free state in the hemolymph. ApoLp-III from Galleria mellonella is able to interact with membrane components of Gram-negative bacteria, as part of an innate immune response to infection. The protein also exists in a lipoprotein-associated state when large amounts of lipids are mobilized. Therefore, lipid-bound apoLp-III was generated to analyze the binding interaction with lipopolysaccharides and phosphatidylglycerol, both abundantly present in membranes of Gram-negative bacteria. G. mellonella apoLp-III was lipidated with palmitoyl-2-oleoyl-glycero-3-phosphocholine to form lipid-protein complexes. The particle shape was discoidal with a 16.4nm diameter, a molecular mass of 460kDa, and contained 4 apoLp-III molecules. These discoidal lipoproteins were used to compare the lipopolysaccharide and phosphatidylglycerol binding activity with lipid-free apoLp-III. Lipopolysaccharide binding interaction was analyzed by non-denaturing PAGE, showing reduced ability of the lipid-bound protein to form lipopolysaccharide-protein complexes and to disaggregate lipopolysaccharide micelles. The apoLp-III-induced release of calcein from phosphatidylglycerol vesicles was decreased approximately fivefold when the protein was in the lipid-bound form, indicating reduced binding interaction with the phosphatidylglycerol membrane surface. These results show that when apoLp-III adopts a lipid-bound conformation, it is markedly less effective in interacting with lipopolysaccharides and phosphatidylglycerol vesicles. Thus, in order to be an effective antimicrobial protein, apoLp-III needs to be in a lipid-free state.

The membrane phospholipid cardiolipin plays a pivotal role in bile acid adaptation by Lactobacillus gasseri JCM1131T

Bile acids exhibit strong antimicrobial activity as natural detergents, and are involved in lipid digestion and absorption. We investigated the mechanism of bile acid adaptation in Lactobacillus gasseri JCM1131T. Exposure to sublethal concentrations of cholic acid (CA), a major bile acid in humans, resulted in development of resistance to otherwise-lethal concentrations of CA by this intestinal lactic acid bacterium. As this adaptation was accompanied by decreased cell-membrane damage, we analyzed the membrane lipid composition of L. gasseri. Although there was no difference in the proportions of glycolipids (~70%) and phospholipids (~20%), adaptation resulted in an increased abundance of long-sugar-chain glycolipids and a 100% increase in cardiolipin (CL) content (to ~50% of phospholipids) at the expense of phosphatidylglycerol (PG). In model vesicles, the resistance of PG vesicles to solubilization by CA increased with increasing CL/PG ratio. Deletion of the two putative CL synthase genes, the products of which are responsible for CL synthesis from PG, decreased the CL content of the mutants, but did not affect their ability to adapt to CA. Exposure to CA restored the CL content of the two single-deletion mutants, likely due to the activities of the remaining CL synthase. In contrast, the CL content of the double-deletion mutant was not restored, and the lipid composition was modified such that PG predominated (~45% of total lipids) at the expense of glycolipids. Therefore, CL plays important roles in bile acid resistance and maintenance of the membrane lipid composition in L. gasseri.

Interactions between Surface-Immobilized Antimicrobial Peptides and Model Bacterial Cell Membranes.

Sum frequency generation (SFG) vibrational spectroscopy was used to study surface immobilization effects on the interactions between antimicrobial peptide cecropin P1 (CP1) and model cell membranes. While free CP1 in solution interacted with a model cell membrane composed of a phosphatidylglycerol (PG) bilayer, electrostatic interaction led to the attachment of CP1 molecules onto the PG surface and the hydrophobic domain in the lipid bilayer enabled the peptides to insert into the bilayer and form α-helices from random coil structures. While CP1 molecules immobilized on a self-assembled monolayer interacted with PG lipid vesicles, the intensity of the SFG peak for the peptide α-helix decreased as the PG vesicle concentration increased. It was believed that when surface-immobilized CP1 molecules interacted with lipid vesicles, they lay down on the surface or became random coils. When the immobilized CP1 interacted with a PG lipid monolayer on water, the strong interaction led to the lying-down orientation of all of the surface-immobilized peptides as well. Differently, no significant interactions between surface-immobilized CP1 with the mammalian cell membrane model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer were observed. Our results suggest that, instead of membrane insertion, the electrostatic interactions between the surface cationic charges of CP1 and anionic bacterial membranes may play an important role in the antimicrobial activity of the surface-immobilized CP1 peptide.

Copper-phospholipid interaction at cell membrane model hydrophobic surfaces

Detailed investigation of Cu (II) binding with natural lipid phosphatidylglycerol (PG) in aqueous solution was carried out by voltammetric measurements at the mercury drop electrode, complemented by monolayer studies in a Langmuir trough and electrophoretic measurements, all used as models for hydrophobic cell membranes. Penetration of copper ions into the PG layer was facilitated by the formation of hydrophilic Cu-Phenanthroline (Phen) complex in the subphase, followed by the mixed ligand Cu-Phen-PG complex formation at the hydrophobic interface. Electrophoretic measurements indicated a comparatively low abundance of the formed mixed ligand complex within the PG vesicles, resulting it the zeta potential change of +0.83mV, while monolayer studies confirmed their co-existence at the interface. The Cu-Phen-PG complex was identified in the pH range from 6 to 9. The stoichiometry of the complex ([PhenCuOHPG]), as well as its stability and kinetics of formation, were determined at the mercury drop electrode. Cu-Phen-PG reduces quasireversibly at about −0.7V vs. Ag/AgCl including reactant adsorption, followed by irreversible mixed complex dissociation, indicating a two-electron transfer - chemical reaction (EC mechanism). Consequently, the surface concentration (γ) of the adsorbed [PhenCuOHPG] complex at the hydrophobic electrode surface was calculated to be (3.35±0.67)×10−11molcm−2. Information on the mechanism of Cu (II) - lipid complex formation is a significant contribution to the understanding of complex processes at natural cell membranes.

Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity

Apolipophorin III (apoLp-III) is an insect apolipoprotein (18kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28kDa two-domain protein: an α-helical N-terminal domain (residues 1–189) and a less structured C-terminal domain (residues 190–243). To better understand the apolipoprotein domain organization, a novel chimeric protein was engineered by attaching residues 179 to 243 of apoA-I to the C-terminal end of apoLp-III. The apoLp-III/apoA-I chimera was successfully expressed and purified in E. coli. Western blot analysis and mass spectrometry confirmed the presence of the C-terminal domain of apoA-I within the chimera. While parent apoLp-III did not self-associate, the chimera formed oligomers similar to apoA-I. The chimera displayed a lower α-helical content, but the stability remained similar compared to apoLp-III, consistent with the addition of a less structured domain. The chimera was able to solubilize phospholipid vesicles at a significantly higher rate compared to apoLp-III, approaching that of apoA-I. The chimera was more effective in protecting phospholipase C-treated low density lipoprotein from aggregation compared to apoLp-III. In addition, binding interaction of the chimera with phosphatidylglycerol vesicles and lipopolysaccharides was considerably improved compared to apoLp-III. Thus, addition of the C-terminal domain of apoA-I to apoLp-III created a two-domain protein, with self-association, lipid and lipopolysaccharide binding properties similar to apoA-I. The apoA-I like behavior of the chimera indicate that these properties are independent from residues residing in the N-terminal domain of apoA-I, and that they can be transferred from apoA-I to apoLp-III.

Binding Interaction of Lipid‐bound ApoA–I with Lipopolysaccharides and Phosphatidylglycerol

Apolipoprotein A‐I (apoA‐I) is a major component of high‐density lipoprotein (HDL), an anti‐antherogenic complex responsible for reverse cholesterol transport from peripheral tissues to liver, and mediates several essential metabolic functions related to heart disease. Human apoA‐I is 28 kDa protein found in lipid‐free and lipid‐bound forms. Previous studies have shown that lipid‐free apoA‐I binds and neutralizes lipopolysaccharides (LPS), which are major constituents of the outer bacterial membrane of gram‐negative bacteria, therefore reducing endotoxic effects. In addition, the protein destabilizes bilayers of negatively charged phosphatidylglycerol (PG), a phospholipid found in the inner bacterial membrane, which may result in cell lysis. However, the majority of apoA‐I is found in the lipid‐bound form as HDL; therefore binding interactions with LPS and PG were studied. To this end reconstituted HDL (rHDL) was produced with 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) and apoA‐I, which was isolated by KBr density ultracentrifugation. rHDL was analyzed by native PAGE and showed three distinct lipid‐protein complexes of 246, 203, and 149 kDa. To compare the binding of lipid‐free and lipid bound apoA‐I to LPS, non‐denaturing PAGE was employed. This showed that LPS binding was decreased for rHDL in comparison to lipid‐free apoA‐I. Both forms of apoA‐I were able to disaggregate LPS micelles, as shown by LPS carbohydrate staining of native PAGE. To investigate binding to PG, unilamellar PG vesicles with encapsulated calcein were prepared by extrusion. Calcein is a fluorescent dye and used as an indicator of lipid vesicle leakage, and is self‐quenched at a concentration above 70 mM. Binding to the vesicles was measured by the increase in calcein fluorescence, which was released upon protein‐vesicle binding. Lipid‐free apoA‐I induced the release of 60 to 70% calcein from PG vesicles; however, calcein release was reduced to 20 to 30% for rHDL. These results show that when apoA‐I is in the lipid‐bound state, binding interaction with LPS and PG bilayer vesicles was decreased significantly. Thus in order to be most effective as an antimicrobial protein, apoA‐I needs to be in the lipid‐free conformation.Support or Funding InformationThis research was supported by a grant from the National Institutes of Health (NIGMS #GM089564).

Binding of lysine residues of apolipophorin III to phosphatidylglycerol membranes

Apolipoproteins are the protein components of lipoproteins, which are responsible for transport of lipids to target tissues through the aqueous environment of the body. Apolipophorin III (apoLp‐III) is an abundant apolipoprotein of the hemolymph of insects. The 18 kDa protein is made of a bundle of five amphipathic α‐helices, which are the structural elements responsible for lipid binding. ApoLp‐III facilitates the lipoprotein mediated transport of diacylglycerols, which are released from fat body tissue for transport to the flight muscles during flight. Another novel function performed by apoLp‐III is modulating the cellular and humoral immune response. The protein up‐regulates expression of antimicrobial proteins, neutralizes lipopolysaccharides, and binds and deforms bacterial membranes. Lipopolysaccharides and phosphatidylglycerol (PG) are abundant components of the bacterial membrane, and our studies aim to better understand the antimicrobial properties of apoLp‐III of Locusta migratoria. This eventually may lead to improved strategies to treat bacterial infection and sepsis. ApoLp‐III contains 8 lysine residues, and the objective of this study was to characterize the ionic interactions between lysine residues of apoLp‐III and the negatively charged membrane components of gram‐negative bacteria, lipopolysaccharides and PG. To mimic bacterial membranes, PG vesicles with encapsulated calcein were prepared by extrusion. Protein‐induced membrane perturbation causes release of calcein which fluoresces intensively. When lysine side‐chains were neutralized by acetylation, binding to PG membranes was greatly reduced as indicated by the large reduction in calcein release from 80 ± 3.3% (unmodified apoLp‐III) to 21 ± 0.2% (acetylated apoLp‐III). In contrast, no effect on lipopolysaccharide binding was observed. To identify the lysine residues responsible for PG binding, site‐directed mutagenesis was used to change lysine into glutamine at position 52, 54, 63, 68 and 121. The proteins were expressed in bacteria and purified to homogeneity. The lysine variants were all able to release calcein, with values for most mutants around 70 to 80%. Since this is similar to the wild‐type protein, this indicates that these residues are not critical for PG binding. The remaining lysine residues at positions 143,145 and 161 were also changed into glutamine; the mutant proteins are currently expressed and purified. In addition, studies are underway to specifically acetylate the N‐terminal amino group by lowering the pH since the pKa for α amino groups is lower than the lysine ɛ‐amino group and hence making it a better nucleophile.

The lipid interaction capacity of Sin a 2 and Ara h 1, major mustard and peanut allergens of the cupin superfamily, endorses allergenicity.

Sin a 2 (11S globulin) and Ara h 1 (7S globulin) are major allergens from yellow mustard seeds and peanut, respectively. The ability of these two allergens to interact with lipid components remains unknown. To study the capacity of Sin a 2 and Ara h 1 to interact with lipid components and the potential effects of such interaction in their allergenic capacity. Spectroscopic and SDS-PAGE binding assays of Sin a 2 and Ara h 1 with different phospholipid vesicles and gastrointestinal and endolysosomal digestions in the presence or absence of lipids were performed. The capacity of human monocyte-derived dendritic cells (hmoDCs) to capture food allergens in the presence or absence of lipids, the induced cytokine signature, and the effect of allergens and lipids to regulate TLR2-L-induced NF-kB/AP-1 activation in THP1 cells were analyzed. Sin a 2 and Ara h 1 bind phosphatidylglycerol (PG) acid but not phosphatidylcholine (PC) vesicles in a pH-dependent manner. The interaction of these two allergens with lipid components confers resistance to gastrointestinal digestion, reduces their uptake by hmoDCs, and enhances their stability to microsomal degradation. Mustard and peanut lipids favor a proinflammatory environment by increasing the IL-4/IL-10 ratio and IL-1β production by hmoDCs. The presence of mustard lipids and PG vesicles inhibits TLR2-L-induced NF-kB/AP-1 activation in THP1 cells. Sin a 2 and Ara h 1 interact with lipid components, which might well contribute to explain the potent allergenic capacity of these two clinically relevant allergens belonging to the cupin superfamily.

Fluorescence depolarization analysis of thermal phase transition in DPPC and DMPG aqueous dispersions

We performed an overall analysis of steady state, kinetic and dynamical parameters of phospholipids labeled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), to investigate the structural changes accompanying the phase transition of dimyristoyl phosphatidylglycerol (DMPG) vesicles, under low and high ionic strength conditions. For comparison, we also performed experiments on dimyristoyl phosphatidylcholine (DPPC) vesicles, which exhibit a well-defined thermal phase transition. Fluorescence parameters alone (lifetime, pre-exponential factor, rotational correlation times, and initial anisotropy) do not describe the thermal behavior of the vesicles. Combination of intensity decay and anisotropy decay data allows the calculation of mean anisotropy values, and among the several parameters obtained from time-resolved measurements, the main contribution to the mean anisotropy comes from the residual anisotropy, obtained as the limit value at long times. The results of calculations were comparable to the steady state measurements, and allowed the observation of the dependence between the thermal phase transition in DMPG and the ionic strength of the medium. The presence of NaCl affects the lipid packing leading to structural constraints onto the probes that are systematically higher than those observed in low ionic strength. In low ionic strength the long rotational correlation time of the NBD-PE (NBD-phosphatidylethanolamine) probe presents peculiar behavior, showing transient changes along the broad gel–fluid transition, that occurs parallel to the modifications in the scattering intensity.

Spin Labels Detect the Coexistence of Two Lipid Domains Along the Anomalous Gel-Fluid Transition of Anionic Dmpg Bilayers

Vesicles of the acidic lipid dimyristoyl phosphatidylglycerol (DMPG), at physiological conditions, show a highly cooperative gel-fluid transition, around 23°C. However, at low ionic strength, DMPG bilayers display a peculiar thermo-structural behavior, presenting a broad gel-fluid transition, between 18°C and 35°C. Several interesting properties of this region led to different models for DMPG dispersion at low ionic strength. Here, we use computer simulations (1) of the electron spin resonance signal of spin labels incorporated into DMPG vesicles to evaluate the structure of the vesicles along the gel-fluid transition. At temperatures below and above the phase transition, in the gel and fluid phases of the bilayer, only one lipid population can be detected. But along the phase transition region, two lipid populations coexist: one very mobile, even more fluid than lipids in the bilayer fluid phase, and another one more rigid and organized, typical of lipids in the gel phase. The more mobile population appears at ca. 18°C and disappears by the end of the phase transition. In accord with previous works (2-4), we propose that this highly mobile lipid population is associated with the formation of pores at the bilayer, and these mobile lipids are located at the edges of the pores. Hence, spin labels can monitor the increase and decrease of bilayer perforations along the membrane phase transition.Financially supported by USP, FAPESP, CNPq.(1)Schneider, Freed, Biological Magnetic Resonance, vol. 8, 1, 1989; (2) Riske, Fernandez, Nascimento, Bales, Lamy-Freund, Chem. Phys. Lipids 124, 69, 2003; (3) Riske, Amaral, Dobereiner, Lamy, Biophys. J. 86, 3722, 2004; (4) Enoki, Henriques, Lamy, Chem. Phys. Lipids 165, 826, 2012.

Biophysical Investigation of the Membrane-Disrupting Mechanism of the Antimicrobial and Amyloid-Like Peptide Dermaseptin S9

Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with 2H- and 31P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors.

α-Synuclein Oligomers with Broken Helical Conformation Form Lipoprotein Nanoparticles

α-Synuclein (αS) is a membrane-binding protein with sequence similarity to apolipoproteins and other lipid-carrying proteins, which are capable of forming lipid-containing nanoparticles, sometimes referred to as "discs." Previously, it has been unclear whether αS also possesses this property. Using cryo-electron microscopy and light scattering, we found that αS can remodel phosphatidylglycerol vesicles into nanoparticles whose shape (ellipsoidal) and dimensions (in the 7-10-nm range) resemble those formed by apolipoproteins. The molar ratio of αS to lipid in nanoparticles is ∼1:20, and αS is oligomeric (including trimers and tetramers). Similar nanoparticles form when αS is added to vesicles of mitochondrial lipids. This observation suggests a mechanism for the previously reported disruption of mitochondrial membranes by αS. Circular dichroism and four-pulse double electron electron resonance experiments revealed that in nanoparticles αS assumes a broken helical conformation distinct from the extended helical conformation adopted when αS is bound to intact vesicles or membrane tubules. We also observed αS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that αS can interact with fatty acids and lipids in a similar manner. αS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein.

Light Scattering on the Structural Characterization of DMPG Vesicles along the Bilayer Anomalous Phase Transition

Highly charged vesicles of the saturated anionic lipid dimyristoyl phosphatidylglycerol (DMPG) in low ionic strength medium exhibit a very peculiar thermo-structural behavior. Along a wide gel-fluid transition region, DMPG dispersions display several anomalous characteristics, like low turbidity, high electrical conductivity and viscosity. Here, static and dynamic light scattering (SLS and DLS) were used to characterize DMPG vesicles at different temperatures. Similar experiments were performed with the largely studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC). SLS and DLS data yielded similar dimensions for DMPC vesicles at all studied temperatures. However, for DMPG, along the gel-fluid transition region, SLS indicated a threefold increase in the vesicle radius of gyration, whereas the hydrodynamic radius, as obtained from DLS, increased 30% only. Despite the anomalous increase in the radius of gyration, DMPG lipid vesicles maintain isotropy, since no light depolarization was detected. Hence, SLS data are interpreted regarding the presence of isotropic vesicles along the DMPG anomalous transition, but highly perforated vesicles, with large holes. DLS/SLS discrepancy along the DMPG transition region is discussed in terms of the interpretation of the Einstein-Stokes relation for porous vesicles. Therefore, SLS data are shown to be much more appropriate for measuring porous vesicle dimensions than the vesicle diffusion coefficient. Although the underlying microscopic process which leads to the opening of pores in charged DMPG bilayer is very intriguing and deserves further investigation, one could envisage biotechnological applications, with vesicles being produced to enlarge and perforate in a chosen temperature and/or pH value, for a desired drug delivery process.

Light scattering on the structural characterization of DMPG vesicles along the bilayer anomalous phase transition

Highly charged vesicles of the saturated anionic lipid dimyristoyl phosphatidylglycerol (DMPG) in low ionic strength medium exhibit a very peculiar thermo-structural behavior. Along a wide gel–fluid transition region, DMPG dispersions display several anomalous characteristics, like low turbidity, high electrical conductivity and viscosity. Here, static and dynamic light scattering (SLS and DLS) were used to characterize DMPG vesicles at different temperatures. Similar experiments were performed with the largely studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC). SLS and DLS data yielded similar dimensions for DMPC vesicles at all studied temperatures. However, for DMPG, along the gel–fluid transition region, SLS indicated a threefold increase in the vesicle radius of gyration, whereas the hydrodynamic radius, as obtained from DLS, increased 30% only. Despite the anomalous increase in the radius of gyration, DMPG lipid vesicles maintain isotropy, since no light depolarization was detected. Hence, SLS data are interpreted regarding the presence of isotropic vesicles within the DMPG anomalous transition, but highly perforated vesicles, with large holes. DLS/SLS discrepancy along the DMPG transition region is discussed in terms of the interpretation of the Einstein–Stokes relation for porous vesicles. Therefore, SLS data are shown to be much more appropriate for measuring porous vesicle dimensions than the vesicle diffusion coefficient. The underlying nanoscopic process which leads to the opening of pores in charged DMPG bilayer is very intriguing and deserves further investigation. One could envisage biotechnological applications, with vesicles being produced to enlarge and perforate in a chosen temperature and/or pH value.

Human apoA‐I lysine residues promote association with lipopolysaccharides and phosphatidylglycerol bilayers

Human apolipoprotein A‐I (apoA‐I) is a 28 kDa protein and a major component of high‐density lipoproteins, mediating several essential metabolic functions related to heart disease. Recently, a novel protective role against bacterial pathogens has emerged. ApoA‐I suppressed bacterial growth of log phase Escherichia coli and Klebsiella pneumoniae, resulting in a reduced colony count. We suggest that apoA‐I interacts with gram‐negative bacteria in two ways: 1) by associating with lipopolysaccharides (LPS) which are the major constituent of the outer bacterial membrane, thereby reducing endotoxic effects, and 2) destabilizing the negatively charged phospholipid bilayer of the inner bacterial membrane, resulting in cell lysis. To analyze the role of lysine in LPS and phospholipid binding, lysine side chains were acetylated using acetic anhydride in saturated sodium acetate. Electrophoresis analysis and 1‐anilino‐naphthalene‐8‐sulfonate fluorescence of apoA‐I in the absence or presence of LPS indicated decreased binding upon acetylation. ApoA‐I was able to lyse phosphatidylglycerol vesicles; however, acetylated apoA‐I showed a marked decrease in activity. These results indicate the potential for apoA‐I to function as an antimicrobial protein and the importance of lysine residues.

Spray layer-by-layer films based on phospholipid vesicles aiming sensing application via e-tongue system

The Layer-by-Layer (LbL) technique via spraying (spray-LbL) has been applied as new and alternative methodology to fabricate ultrathin films due to its versatility in relation to the conventional dipping-LbL method, mainly in terms of faster layer deposition and larger coated area. In this work, the possibility of immobilizing vesicles of dipalmitoyl phosphatidyl glycerol (DPPG) phospholipid onto alternating layers of the polyelectrolyte poly(allylamine hydrochloride) (PAH) using the spray-LbL method was investigated, being the results compared to the conventional dipping-LbL method. The growth of (PAH/DPPG)n spray-LbL films was systematically monitored by quartz crystal microbalance (QCM) and ultraviolet–visible (UV–vis) absorption spectroscopy, revealing a linear increase of the absorbance vs deposited layers. In relation to a possible electrostatic interaction between the groups PO4− (DPPG) and NH3+ (PAH), it was observed through Fourier transform infrared (FTIR) absorption spectroscopy that the spectrum recorded for the spray-LbL film is basically a simple superposition of the FTIR spectra from PAH and DPPG casting films. The latter indicates a weak interaction between both materials, differently of the trend observed for (PAH/DPPG)n grown via dipping-LbL method. Atomic force microscopy (AFM) images of spray-LbL films showed evidences that the DPPG vesicles present in the aqueous dispersion are not destroyed when submitted to pressure conditions during the spray deposition. However, comparing to dipping-LbL, the DPPG vesicles do not cover completely the PAH layer for the spray-LbL film, which was further confirmed by surface-enhanced Raman scattering (SERS) measurements. Moreover, the AFM analysis showed that the spray-LbL deposition led to thicker PAH/DPPG bilayers in average than via dipping-LbL for the same concentrations of PAH solution and DPPG dispersion, which is consistent with QCM and UV–vis absorption results. PAH/DPPG films deposited by dipping- and spray-LbL techniques and also by Langmuir–Blodgett (LB) onto Pt interdigitated electrodes composing an array of sensing units (e-tongue) were applied in the detection of a xanthene derivate (eosin) in diluted solutions (10−9, 10−7 and 10−6M). Despite the LB and LbL films are formed by the same materials (PAH and DPPG), it was found that their different molecular architectures play an important role on the electrical response of Pt interdigitated electrodes in impedance spectroscopy measurements. The high sensitivity reached by these sensing units was intimately related to changes in the film morphology caused by the adsorption of the eosin molecules onto the film surfaces during electrical measurements, as evidenced by micro-Raman technique.

Comparative study on the interaction of cell-penetrating polycationic polymers with lipid membranes

Cell-penetrating peptides are arginine- and lysine-rich cationic peptides that can readily enter cells not only by themselves but also carrying other macromolecular cargos. In fact, we have reported that polycationic polymer such as poly- l-lysine (PLL) and poly- l-arginine (PLA) translocate through negatively charged phospholipid liposome membranes. In this work, we made a comparative study of the interaction of PLL or PLA with lipid membranes consisting of negatively charged phospholipids to understand the role of basic amino acid residue (i.e. arginine and lysine) in the membrane-penetrating activity of polypeptides. PLA and PLL translocated into giant unilamellar vesicle composed of soybean phospholipids. ζ-potential and turbidity measurements demonstrated the electrostatic binding of PLL and PLA to large unilamellar vesicle (LUV). Fluorescence studies using membrane probes revealed that the binding of PLA and PLL to LUV affects the hydration and packing of the membrane interface region, in which the membrane insertion of PLA appeared to be greater than PLL. Differential scanning calorimetry showed that the enthalpy of the gel to liquid-crystalline phase transition for dipalmitoyl phosphatidylglycerol vesicle was greatly reduced by binding of PLL and PLA, in which the reduction is much larger in PLA than in PLL. Circular dichroism measurements in 2,2,2-trifluoroethanol/water mixture or in the presence of LUV indicated that the propensity of PLA to form α-helical structure is greater than PLL. Consistently, attenuated total reflection-Fourier transform infrared spectroscopy revealed that there is greater α-helical structure in PLA bound to LUV compared to PLL, which has much less ordered structure. Furthermore, isothermal titration calorimetry measurements demonstrated that the contribution of enthalpy to the energetics of binding to LUV is two-fold larger in PLA than in PLL. These results suggest that the stronger interaction of arginine residue with negatively charged phospholipid membranes compared to lysine residue appears to facilitate the conformational change in cationic polypeptide and its insertion into lipid membrane interior.

The interaction of the Bax C-terminal domain with negatively charged lipids modifies the secondary structure and changes its way of insertion into membranes

Fourier transform infrared spectroscopy (FTIR) was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domain of the pro-apoptotic protein Bax (Bax-C) when incorporated into different lipid vesicles with or without negatively charged phospholipids. The infrared spectroscopy results showed that while the beta-sheet components are predominant in the membrane-free Bax-C secondary structure as well as in the presence of phosphatidylcholine vesicles, the peptide changes its secondary structure in the presence of negatively charged membranes, including phospholipids such as phosphatidylglycerol or phosphatidylinositol, depending on both the lipid composition and their molar ratio. The negative charges in the model membrane surface caused a marked change from beta-sheet to alpha-helix structure. Moreover, using attenuated total reflection infrared spectroscopy (ATR-FTIR), we investigated the orientation of Bax-C alpha-helical structures with respect to the normal to the internal reflection element. The orientation of Bax-C in membranes was also affected by negatively charged lipids, the presence of phosphatidylglycerol reduced the angle it forms with the normal to the germanium plate from 45 degrees in phosphatidylcholine to 27 degrees in phosphatidylglycerol vesicles. These results highlight the importance of lipid-protein interaction for the correct folding of membrane proteins and they suggest that the C-terminal domain of Bax will only span membranes with a net negative charge in their surface.