Dendrimers complexed with HIV-1 peptides interact with liposomes and lipid monolayers

AimsWe have investigated the effect of surface charge of model lipid membranes on their interactions with dendriplexes formed by HIV-derived peptides and 2 types of positively charged carbosilane dendrimers (CBD).MethodsInteraction of dendriplexes with lipid membranes was measured by fluorescence anisotropy, dynamic light scattering and Langmuir–Blodgett techniques. The morphology of the complexes was examined by transmission electron microscopy.ResultsAll dendriplexes independent of the type of peptide interacted with model lipid membranes. Negatively charged vesicles composed of a mixture of DMPC/DPPG interacted more strongly, and it was accompanied by an increase in anisotropy of the fluorescent probe localized in polar domain of lipid bilayers. There was also an increase in surface pressure of the lipid monolayers. Mixing negatively charged liposomes with dendriplexes increased liposome size and made their surface charges more positive.ConclusionsHIV-peptide/dendrimer complexes interact with model lipid membranes depending on their surface charge. Carbosilane dendrimers can be useful as non-viral carriers for delivering HIV-peptides into cells.     

Interactions of a synthetic Leu–Lys-rich antimicrobial peptide with phospholipid bilayers

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH₂) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the β-sheet content to ~20%. Addition of negatively charged LUV, DMPC–dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d₅₄-DMPC) and mixed d₅₄-DMPC–DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC–DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by ³¹P NMR. However, no perturbations in the T ₁ relaxation nor the T ₂- values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).     

Influence of molecular weight and pH on adsorption of chitosan at the surface of large and giant vesicles

This paper describes the mechanisms of adsorption of chitosan, a positively charged polyelectrolyte, on the DOPC lipid membrane of large and giant unilamellar vesicles (respectively, LUVs and GUVs). We observe that the variation of the zeta potential of LUVs as a function of chitosan concentration is independent on the chitosan molecular weight (Mw). This result is interpreted in terms of electrostatic interactions, which induce a flat adsorption of the chitosan on the surface of the membrane. The role of electrostatic interactions is further studied by observing the variation of the zeta potential as a function of the chitosan concentration for two different charge densities tuned by the pH. Results show a stronger chitosan-membrane affinity at pH 6 (lipids are negatively charged, and 40% chitosan amino groups are protonated) than at pH 3.4 (100% of protonated amino groups but zwitterionic lipids are positively charged) which confirms that adsorption is of electrostatic origin. Then, we investigate the stability of decorated LUVs and GUVs in a large range of pH (6.0 < pH < 12.0) in order to complete a previous study made in acidic conditions [Quemeneur et al. Biomacromolecules 2007, 8, 2512-2519]. A comparative study of the variation of the zeta potential as a function of the pH (2.0 < pH < 12.0) reveals a difference in behavior between naked and chitosan-decorated LUVs. This result is further confirmed by a comparative observation by optical microscopy of naked and chitosan-decorated GUVs in basic conditions (6.0 < pH < 12.0): at pH > 10.0, in the absence of chitosan, the vesicles present complex shapes, contrary to the chitosan-decorated vesicles which remain spherical, confirming thus that chitosan remains adsorbed on vesicles in basic conditions up to pH = 12.0. These results, in addition with our previous data, show that the chitosan-decorated vesicles are stable over a very broad range of pH (2.0 < pH < 12.0), which holds promise for their in vivo applications. Finally, the quantification of the chitosan adsorption on a LUV membrane is performed by zeta potential and fluorescence measurements. The fraction of membrane surface covered by chitosan is estimated to be lower than 40 %, which corresponds to the formation of a flat layer of chitosan on the membrane surface on an electrostatic basis.     

The local anesthetic proparacaine modifies sodium transport in toad skin and perturbs the structures of model and cell membranes

Experimental results indicate a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of proparacaine to isolated toad skin, which may reflect an inhibition of the active transport of ions. This finding was explained on the basis of the results obtained from membrane models incubated with proparacaine. These consisted of human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), phospholipid multilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively, and in large unilamellar vesicles (LUV) of DMPC X-ray diffraction showed that proparacaine interaction with DMPC and DMPE bilayers perturbed both structures, especially DMPC. This result, confirmed by fluorescence spectroscopy of DMPC LUV at 18 degrees C, demonstrated that the local anesthetic (LA) could interact with the lipid moiety of cell membranes. However, effects observed by scanning electron microscopy (SEM) of human erythrocytes and by fluorescence spectroscopy of IUM might also imply proparacaine-protein interactions. Thus, the LA may alter epitheial sodium channels through interaction with the lipid matrix and with channel protein residues.     

Dibucaine-induced modification of sodium transport in toad skin and of model membrane structures

The interaction of the local anesthetic dibucaine with the isolated toad skin and membrane models is described. The latter consisted of human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC) and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Results indicate a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of dibucaine in toad skin, which may be interpreted as reflecting inhibition of the active transport of ions. This finding might be explained on the basis of the results obtained from fluorescence spectroscopy and X-ray diffraction studies on membrane models. In fact, dibucaine induced structural perturbations in IUM, DMPC LUV and phospholipid multilayers. Scanning electron microscopy revealed that dibucaine induced erythrocyte stomatocytosis. According to the bilayer couple hypothesis an echinocytic type of shape change would have been expected given the preferential interaction of dibucaine with DMPC. Although it is still premature to define the molecular mechanism of action of dibucaine, the experimental results confirm the important role played by the phospholipid bilayers in the association of the anesthetic with cell membranes.     

Effects of the local anesthetic benzocaine on the human erythrocyte membrane and molecular models

The interaction of the local anesthetic benzocaine with the human erythrocyte membrane and molecular models is described. The latter consisted of isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphospatidylcholine (DMPC), and phospholipid multilayers of DMPC and dimyristoylphospatidyletanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Optical and scanning electron microscopy of human erythrocytes revealed that benzocaine induced the formation of echinocytes. Experiments performed on IUM and DMPC LUV by fluorescence spectroscopy showed that benzocaine interacted with the phospholipid bilayer polar groups and hydrophobic acyl chains. X-ray diffraction analysis of DMPC confirmed these results and showed that benzocaine had no effects on DMPE. The effect on sodium transport was also studied using the isolated toad skin. Electrophysiological measurements indicated a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of benzocaine, reflecting inhibition of active ion transport.     

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3–11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events – large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.     

Large disk intermediate precedes formation of apolipoprotein A-I-dimyristoylphosphatidylcholine small disks

Small approximately 8.5 nm disks formed spontaneously when dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles (LUVs) were incubated with apolipoprotein A-I (apoA-I) (100:1 molar ratio). However, in a time course study, the transient production of approximately 11 nm large disks was detected and isolated by gel filtration. The intermediate large disks contained three apoA-I molecules and were stable over time; however, when additional apoA-I was added, they formed small disks containing two molecules of apoA-I. The reaction kinetics of apoA-I with DMPC LUVs was monitored by fluorescence resonance energy transfer, and two phases were observed, supporting the presence of the intermediate in the formation of small disks. The lipid dynamics of LUVs and disks were assayed, revealing the presence of sequestered lipid-protein domains upon apoA-I binding to DMPC LUVs. In addition, the lipids in the intermediate large disks were more constrained than those in the small disks. We propose that apoA-I binds with DMPC LUVs to form small lipid-protein domains on the LUV; then the domains are released to form large disks, which can mature in the presence of additional apoA-I to form small disks. Thus, the formation of small apoA-I lipid disks proceeds through the formation of a large disk intermediate.     

Cholesterol in Negatively Charged Lipid Bilayers Modulates the Effect of the Antimicrobial Protein Granulysin

The release of granulysin, a 9-kDa cationic protein, from lysosomal granules of cytotoxic T lymphocytes and natural killer cells plays an important role in host defense against microbial pathogens. Granulysin is endocytosed by the infected target cell via lipid rafts and kills subsequently intracellular bacteria. The mechanism by which granulysin binds to eukaryotic and prokaryotic cells but lyses only the latter is not well understood. We have studied the effect of granulysin on large unilamellar vesicles (LUVs) and supported bilayers with prokaryotic and eukaryotic lipid mixtures or model membranes with various lipid compositions and charges. Binding of granulysin to bilayers with negative charges, as typically found in bacteria and lipid rafts of eukaryotic cells, was shown by immunoblotting. Fluorescence release assays using LUV revealed an increase in permeability of prokaryotic, negatively charged and lipid raft-like bilayers devoid of cholesterol. Changes in permeability of these bilayers could be correlated to defects of various sizes penetrating supported bilayers as shown by atomic force microscopy. Based on these results, we conclude that granulysin causes defects in negatively charged cholesterol-free membranes, a membrane composition typically found in bacteria. In contrast, granulysin is able to bind to lipid rafts in eukaryotic cell membranes, where it is taken up by the endocytotic pathway, leaving the cell intact.     

Interaction of the polyene antibiotic amphotericin B with model membranes: differences between small and large unilamellar vesicles

The interaction of the polyene antibiotic amphotericin B (AmB) (Fig. 1) with large unilamellar vesicles (LUV) was monitored by circular dichroism (CD) and carboxyfluorescein (CF) release. LUV afford a far better model for biological membranes than small unilamellar vesicles (SUV) which have been used until now. With dimyristoyl phosphatidyl choline (DMPC) LUV (i.e., containing saturated acyl chains), a strong and not saturable binding for AmB/lipid ratios up to 0.5 was observed both above and below the phase transition temperature. Incorporation of cholesterol into the vesicles did not significantly change the interaction. With egg PC (EPC) LUV (i.e., containing unsaturated acyl chains), quite a different picture emerged: the binding reached saturation for AmB/lipid ratios of about 5 x 10(-3), a result not observed with EPC SUV. When sterols were introduced into membranes, the CD spectral features obtained in the presence of ergosterol were different from those obtained in the presence of cholesterol. Such a different behavior was not observed with SUV. We suggest that species whose CD spectrum was observed after 15 min in the presence of ergosterol-containing EPC LUV is the particular one which forms wide channels and induces a Ca2+ release. (H. Ramos, A. Attias, B.E. Cohen and J. Bolard, submitted for publication). The CF release from EPC LUV induced by AmB was very low, even at very high concentrations of the antibiotic (3 x 10(-4)M). In contrast, an important release of the fluorescent dye was observed with DMPC LUV at concentrations of approximately 10(-5)M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton-Induced Permeability and Fusion of Large Unilamellar Vesicles by Covalently Conjugated Poly(2-Ethylacrylic Acid)

Abstract

Proton sensitive large unilamellar vesicles (LUV) were constructed by immobilization of the pH sensitive synthetic polymer poly(2-ethylacrylic acid) onto the outer monolayer. Thiolated poly(2-ethylacrylic acid) (PEAA-SH) was covalently conjugated to the surface of LUVs composed of egg phosphatidylcholine (EPC) and cholesterol (Choi) through the thiol-reactive maleimide lipid MPB-DSPE (N-(4-(p-maleimidophenyl)butyryl)-1,2-distearoyl-sn-glyc-ero-3-phosphoethanolamine). The resulting PEAA- LUVs were shown to be stable at neutral pH (pH 7.0 to 8.0). Under acidic conditions, however, proton-ation of PEAA resulted in interaction with both the membrane it was linked to and the membrane of target vesicles, causing membrane destabilization and release of vesicle contents. Moreover, conjugated PEAA is shown to mediate fusion with target membranes in a pH dependent manner. PEAA-mediated permeabilization and vesicle-vesicle fusion occurred only when the polymer was covalently linked to the LUV surface. Proton dependent fusion of PEAA-LUVs was also observed with erythrocyte ghosts. This pH-dependent release of vesicle contents and fusion of PEAA-LUVs occurred below pH 6.8, which is well within the pH range expected to be encountered inside the endosomes in the endocytic pathway, indicating the potential of PEAA-LUVs as a drug carrier system for intracellular drug delivery.     

Measuring peptide translocation into large unilamellar vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors(1). Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors(1-3). Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms(4,5), particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes(6,7). In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides(1,8). A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)(9-13). LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies(14,15). Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II(16,17). In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties(18), to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.     

Exchange and flip-flop of dimyristoylphosphatidylcholine in liquid-crystalline, gel, and two-component, two-phase large unilamellar vesicles

The rate and extent of spontaneous exchange of dimyristoylphosphatidylcholine (DMPC) from large unilamellar vesicles (LUV) composed of either DMPC or mixtures of DMPC/distearoylphosphatidylcholine (DSPC) have been examined under equilibrium conditions. The phase state of the vesicles ranged from all-liquid-crystalline through mixed gel/liquid-crystalline to all-gel. The exchange rate of DMPC between liquid-crystalline DMPC LUV, measured between 25 and 55 degrees C, was found to have an Arrhenius activation energy of 24.9 +/- 1.4 kcal/mol. This activation energy and the exchange rates are very similar to those obtained for the exchange of DMPC between DMPC small unilamellar vesicles (SUV). The extent of exchange of DMPC in LUV was found to be approximately 90%. This is in direct contrast to the situation in DMPC SUV where only the lipid in the outer monolayer is available for exchange. Thus, transbilayer movement (flip-flop) is substantially faster in liquid-crystalline DMPC LUV than in SUV. Desorption from gel-phase LUV has a much lower rate than gel-phase SUV with an activation energy of 31.7 +/- 3.7 kcal/mol compared to 11.5 +/- 2 kcal/mol reported for SUV. A defect-mediated exchange in gel-phase SUV, which is not the major pathway for exchange in LUV, is proposed on the basis of the thermodynamic parameters of the activation process. Surprisingly, the rates of DMPC exchange between DMPC/DSPC two-component LUV, measured over a wide range of compositions and temperatures, were found to exhibit very little dependence on the composition or phase configuration of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an alpha-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Effects of divalent cations on the ultrasonic absorption coefficient of negatively charged liposomes (LUV) near their phase transition temperature

The ultrasonic absorption coefficient per wavelength (alpha lambda), as a function of temperature and frequency, was determined for large unilamellar vesicles (LUV) in the vicinity of their phospholipid phase transition temperature, using a double crystal acoustic interferometer. (The vesicles were composed of a 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). It has been found that alpha lambda reaches a maximum (alpha lambda)max at the phase transition temperature (tm) of the phospholipids in the bilayer, at an ultrasonic relaxation frequency of 2.1 MHz. Divalent cations (Ca2+ and Mg2+), added to LUV suspensions, shifted (alpha lambda)max to higher temperatures, dependent upon the concentration of divalent cation. It was also found that the shape of the alpha lambda versus t curve was significantly changed, representing changes in the Van't Hoff enthalpy of the transition, and therefore, the cooperative unit of the transition. This suggests that divalent cations interact individually with the negatively charged phospholipid headgroups of DPPG and with DPPC headgroups, thus decreasing the cooperative unit of the transition. The observed upward shift in tm suggests an interaction that increases the activation energy and, therefore, the temperature of the phase transition. However, alpha lambda as a function of frequency did not change with the addition of divalent cations and, thus, the relaxation time of the event responsible for the absorption of ultrasound is not changed by the addition of divalent cations.     

31P NMR and AFM studies on the destabilization of cell and model membranes by the major bovine seminal plasma protein, PDC-109

The effect of PDC-109 binding to dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylglycerol (DPPG) multilamellar vesicles (MLVs) and supported membranes was investigated by (31)P NMR spectroscopy and atomic force microscopy. Additionally, the effect of cholesterol on the binding of PDC-109 to phosphatidylcholine (PC) membranes was studied. Binding of PDC-109 to MLVs of DMPC and DPPG induced the formation of an isotropic signal in their (31)P NMR spectra, which increased with increasing protein/lipid ratio and temperature, consistent with protein-induced disruption of the MLVs and the formation of small unilamellar vesicles or micelles but not inverse hexagonal or cubic phases. Incorporation of cholesterol in the DMPC MLVs afforded a partial stabilization of the lamellar structure, consistent with previous reports of membrane stabilization by cholesterol. AFM results are consistent with the above findings and show that addition of PDC-109 leads to a complete breakdown of PC membranes. The fraction of isotropic signal in (31)P NMR spectra of DPPG in the presence of PDC-109 was less than that of DMPC under similar conditions, suggesting a significantly higher affinity of the protein for PC. Confocal microscopic studies showed that addition of PDC-109 to human erythrocytes results in a disruption of the plasma membrane and release of hemoglobin into the solution, which was dependent on the protein concentration and incubation time.     

Interaction of meso-tetrakis (4-N-methylpyridyl) porphyrin in its free base and as a Zn(II) derivative with large unilamellar phospholipid vesicles

Our aim was to investigate the interaction of the cationic meso-tetrakis (4-N-methylpyridyl) porphyrin, a photosensitizer used for photodynamic therapy, in its free base form (TMPyP) and complexed with Zn(II) (ZnTMPyP), with large unilamellar vesicles (LUVs), as a model for the gram-negative bacterial cell wall. Mixtures of the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) phospholipids, at different molar percentages, were used as LUVs. A significant increase of porphyrin affinity at higher POPG molar concentrations was observed from the binding constant values, K _b, estimated by optical absorption and steady-state fluorescence. Besides, as demonstrated by time-resolved fluorescence, this affinity increase is also followed by a higher fraction of vesicle-bound porphyrin in the LUVs. Moreover, based on the K _b values, we have observed a higher affinity of the ZnTMPyP to the POPG containing LUVs as compared to the TMPyP. Steady-state fluorescence quenching and zeta potential studies revealed that both porphyrins are possibly located at the LUVs Stern layer region. Therefore, the electrostatic attraction between the positively charged porphyrin peripheral groups and the negatively charged outer surface of the LUVs plays an important role in porphyrin association and localization. Our results have improved the understanding of the successful application of cationic porphyrins on the photo-inactivation of gram-negative bacteria. Since a higher accumulation of the ZnTMPyP in the bacterial cell wall would be expected, this porphyrin could be a more efficient therapeutic drug for this treatment.     

Interaction of poly(l-lysines) with negatively charged membranes: an FT-IR and DSC study

The influence of the binding of poly(l-lysine) (PLL) to negatively charged membranes containing phosphatidylglycerols (PG) was studied by DSC and FT-IR spectroscopy. We found a general increase in the main transition temperature as well as increase in hydrophobic order of the membrane upon PLL binding. Furthermore we observed stronger binding of hydration water to the lipid head groups after PLL binding. The secondary structure of the PLL after binding was studied by FT-IR spectroscopy. We found that PLL binds in an α-helical conformation to negatively charged DPPG membranes or membranes with DPPG-rich domains. Moreover we proved that PLL binding induces domain formation in the gel state of mixed DPPC/DPPG or DMPC/DPPG membranes as well as lipid remixing in the liquid–crystalline state. We studied these effects as a function of PLL chain length and found a significant dependence of the secondary structure, phase transition temperature and domain formation capacity on PLL chain length and also a correlation between the peptide secondary structure and the phase transition temperature of the membrane. We present a system in which the membrane phase transition triggers a highly cooperative secondary structure transition of the membrane-bound peptide from α-helix to random coil. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Cadmium-induced changes in the membrane of human erythrocytes and molecular models

The structural effects of cadmium on cell membranes were studied through the interaction of Cd(2+) ions with human erythrocytes and their isolated unsealed membranes (IUM). Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Cd(2+) induced shape changes in erythrocytes, which took the form of echinocytes. According to the bilayer couple hypothesis, this result meant that Cd(2+) ions located in the outer monolayer of the erythrocyte membrane. Fluorescence spectroscopy measurements in IUM indicated a disordering effect at both the polar headgroup and the acyl chain packing arrangements of the membrane phospholipid bilayer. Cd(2+) ions also interacted with molecular models of the erythrocyte membrane consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers the erythrocyte membrane, respectively. X-ray diffraction indicated that Cd(2+) ions induced structural perturbation of the polar headgroup and of the hydrophobic acyl regions of DMPC, while the effects of cadmium on DMPE bilayers were much milder. This conclusion is supported by fluorescence spectroscopy measurements on DMPC large unilamellar vesicles (LUV). All these findings point to the important role of phospholipid bilayers in the interaction of cadmium on cell membranes.