DMPG Vesicles Research Articles

Arginine Homopeptide of 11 Residues as a Model of Cell-Penetrating Peptides in the Interaction with Bacterial Membranes.

Cell-penetrating peptides rich in arginine are good candidates to be considered as antibacterial compounds, since peptides have a lower chance of generating resistance than commonly used antibiotics. Model homopeptides are a useful tool in the study of activity and its correlation with a secondary structure, constituting an initial step in the construction of functional heteropeptides. In this report, the 11-residue arginine homopeptide (R11) was used to determine its antimicrobial activity against Staphylococcus aureus and Escherichia coli and the effect on the secondary structure, caused by the substitution of the arginine residue by the amino acids Ala, Pro, Leu and Trp, using the scanning technique. As a result, most of the substitutions improved the antibacterial activity, and nine peptides were significantly more active than R11 against the two tested bacteria. The cell-penetrating characteristic of the peptides was verified by SYTOX green assay, with no disruption to the bacterial membranes. Regarding the secondary structure in four different media-PBS, TFE, E. coli membrane extracts and DMPG vesicles-the polyproline II structure, the one of the parent R11, was not altered by unique substitutions, although the secondary structure of the peptides was best defined in E. coli membrane extract. This work aimed to shed light on the behavior of the interaction model of penetrating peptides and bacterial membranes to enhance the development of functional heteropeptides.

Lysine Position Affects Binding of Aib-Rich Model Antibiotics to Lipid Vesicles

Due to the increased development of drug resistance and decrease of antibiotic drug effectiveness, there is an immediate need for new antimicrobials. A possible, promising solution is in the form of charged peptide-based molecules, which can function as antibiotics by interacting with and disturbing bacterial membranes. We are exploring these interactions using model peptides composed of hydrophobic, branched amino acid Aib (α-aminoisobutyric acid), and large unilamellar vesicles (LUVs) composed of DMPG and DMPC. These LUVs model bacterial (negatively charged) and non-bacterial (neutral) cell membranes, respectively. Aib naturally occurs in antibiotics used by some bacteria and biases peptides to adopt a helical structure. We present results using two model peptides in which two positively charged lysine molecules were placed in either adjacent positions of the helix (KK45), or a helical turn apart (KK36). Interactions between the LUVs and peptides are investigated using Isothermal Titration Calorimetry (ITC), resulting in binding enthalpies, entropies, and binding constants. Our initial results indicate that both KK45 and KK36 bind in a multi-stage interaction to the DMPG vesicles (bacterial models), while showing very little affinity when binding to DMPC (non-bacterial models). One stage appears to be enthalpy-driven and is consistent with electrostatic interactions between peptide sidechains and lipid headgroups, while the other stage appears to be entropy-driven and is consistent with hydrophobic interactions. Overall KK45 binds more favorably to DMPG vesicles, which is interesting given that the KK45 helix is kinked, while the KK36 helix is not.

Sequence Permutation of Positive Charges in a Model Peptide Antibiotic Produces Differing Enthalpic and Entropic Contributions to the Lipid-Peptide Binding Affinity

With rising disease rates and decreasing effectiveness of conventional antibiotics, there is an immediate need for new antibiotics. One promising solution is through cationic antimicrobial peptides, which act by perturbing bacterial membranes. The interaction of model peptides with membranes can provide valuable information about the role of helical structure and peptide charge distribution on peptide-membrane interactions. We are investigating model peptide antibiotics composed primarily of the hydrophobic dialkylated amino acid Aib (α-aminoisobutyric acid), which imparts a strong 310-helical bias due to steric hindrance at the α-carbon. Cationic lysine residues are placed in adjacent locations in the center of the helix (KK45) or one full turn apart (KK36). Large unilamellar vesicles (LUVs) composed of DMPC or DMPG were used as zwitterionic or anionic membrane models, respectively. NMR data indicates that in a membrane mimicing solvent (DMSO), KK45 adopts a kinked helical stucture, while KK36 adopts a canonical helical structure. We have previously seen that KK45 binding to anionic micells is more favorable than binding of KK36 to anionic micelles. However, unlike binding to micelles, NMR data suggests that binding of peptides to DMPG vesicles is more favorable for KK36 than for KK45. Here we present thermodynamic data characterizing the peptide-LUV interactions using isothermal titration calorimetry (ITC). Preliminary data suggests that binding to DMPG vesicles is exothermic, while binding to DMPC vesicles is endothermic. Complete characterization of the enthalpy of binding, entropy of binding, and thermodynamic binding constant will allow us to elucidate the relationship between peptide structure and the favorability of membrane binding.

Investigation of the mechanism of action of novel amphipathic peptides: Insights from solid-state NMR studies of oriented lipid bilayers

We have investigated in the present study the effect of both non-selective and selective cationic 14-mer peptides on the lipid orientation of DMPC bilayers by 31P solid-state nuclear magnetic resonance (NMR) spectroscopy. Depending on the position of substitution, these peptides adopt mainly either an α-helical structure able to permeabilize DMPC and DMPG vesicles (non-selective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). Several systems have been investigated, namely bilayers mechanically oriented between glass plates as well as bicelles oriented with their normal perpendicular or parallel to the external magnetic field. The results have been compared with spectral simulations with the goal of elucidating the difference in the interaction of these two types of peptides with zwitterionic lipid bilayers. The results indicate that the perturbation induced by selective peptides is much greater than that induced by non-selective peptides in all the lipid systems investigated, and this perturbation has been associated to the aggregation of the selective β-sheet peptides in these systems. On the other hand, the oriented lipid spectra obtained in the presence of non-selective peptides suggest the presence of toroidal pores. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Surface Electrostatics of Lipid Bilayers by EPR of a pH-Sensitive Spin-Labeled Lipid

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids’ polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes.

Influence of the Lipid Phase State and Electrostatic Surface Potential on the Conformations of a Peripherally Bound Membrane Protein

Avian liver bile acid-binding protein (L-BABP) binds peripherically to anionic lipid membranes. We previously showed that in the absence of added salt the binding to 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) occurs with changes in the secondary structure, the extent of which depends on the phase state of the lipid. In the present work, we used Fourier transform infrared spectroscopy to study the conformations of L-BABP bound to lipids with phosphoglycerol and phosphatidic acid polar head groups and with different transition temperatures in an aqueous medium with high ionic strength (0.1 M NaCl). When L-BABP was bound to the lipids with saturated acyl chains, DMPG, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), and 1,2-dilauroyl-sn-glycero-3-phosphate (DLPA), the conformation shifted from a native-like secondary structure to an unfolded state at the temperature of lipid chain melting. The protein was in the native-like conformation when it was bound to the unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in the liquid-crystalline phase. We also measured the electrokinetic surface potential of POPG and DMPG vesicles in the gel and in the liquid-crystalline phase and the protein binding constant to these lipid membranes. We found a correlation indicating that protein unfolding in the interface was due to the increase in the electrostatic surface potential that occurs in the lipid phase transition.

Insights on the interaction of met-enkephalin with negatively charged membranes—an infrared and solid-state NMR spectroscopic study

Enkephalins are pentapeptides found in the human nervous system, where they are involved in the relief of pain. The interaction of these neuropeptides with the nerve cell membranes would be a key-step in the receptor binding. We have used both Fourier-transform infrared and solid-state NMR spectroscopies to shed light on the interactions responsible for the association of enkephalins with negatively charged membranes. More specifically, we have investigated the interaction of methionine-enkephalin (Menk) with DMPG and DMPS vesicles. Our results suggest that Menk interacts electrostatically with both model membranes via its terminal NH 3 + group. However, the peptide induced the formation of elongated DMPG vesicles in the magnetic field. On the other hand, the association of Menk with DMPS bilayers was concentration-dependent and disrupted the membrane at high peptide concentrations. The different effect of methionine-enkephalin with the two types of anionic membranes is most likely related to the different fluidity of these systems.

Fluorescence analysis of the interaction of two peptide sequences of hepatitis GB virus C with liposomes

The physicochemical characterization of the peptide sequences E2 (39–53) and E2 (32–59) corresponding to the structural protein E2 of the GB virus C was done by studying their interaction with model membranes. The peptides showed surface activity concentration dependent when injected beneath a buffered solution. This tendency to accumulate into the air/water interface suggested a potential ability of these peptides to interact with bilayers. For that reason, Small Unilamellar Liposomes (SUVs) of 1,2-dimyiristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-dimyiristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG) were chosen as a mimetic membranes. A series of fluorescence experiments based on tryptophan peptide fluorescence or with fluorescence labeled SUVs, were done to cover different aspects of peptide interaction with bilayers. Steady state fluorescence anisotropy studies with N-(7-nitro-2-1,3-benzoxadiazol-4-yl) dioleoylphosphatidylethanolamine (NBD-PE) or 1-[4-(trimethylammonium) phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) labeled SUVs indicated that only the long peptide was able to change the lipid microenvironment of DMPG vesicles by slightly increasing the rigidity of the bilayer both above and under the lipid main transition temperature. These results were concordant with the slight blue shift of the maximum tryptophan wavelength emission after E2 (32–53) peptide incubation with DMPG vesicles. Our data provide useful information for the design of synthetic immunopeptides that can be incorporated into a liposomal system with a potential to promote a direct delivery of the membrane-incorporated immunogen to the immunocompetent cells, thus increasing the immuno response from the host.

The orientation of the antibiotic peptide maculatin 1.1 in DMPG and DMPC lipid bilayers. Support for a pore-forming mechanism

Maculatin 1.1 is an antimicrobial peptide isolated from the Australian tree frog Litoria genimaculata that adopts an amphipathic, α-helical structure in solution. Its orientation and conformation when incorporated to pre-formed DMPG (1,2-dimyristoyl- sn-glycero-3-phosphoglycerol) and DMPC (1,2-dimyristoyl- sn-glycero-3-phosphocholine) vesicles was determined using polarised Fourier transform infrared–attenuated total reflection infrared and deuterium exchange experiments. For DMPG membranes, our results show insertion of ∼70% of the maculatin 1.1 molecules, with an angle of insertion of approximately 35° to the membrane normal and with a predominant α-helical structure. These results suggest that maculatin 1.1 acts through a pore-forming mechanism to lyse bacterial membranes. A similar degree of insertion in DMPG (65%) and α-helical structure was observed for a biologically inactive, less amphipathic maculatin 1.1 analogue, P15A, although the helix tilt was found to be greater (46°) than for maculatin 1.1. Similar experiments performed using DMPC liposomes showed poor insertion, less than 5%, for both maculatin 1.1 and its analogue. In addition, the shape of the amide I band in these samples is consistent with α-helix, β-structure and disordered structures being present in similar proportion. These results clearly show that maculatin 1.1 inserts preferentially in negatively charged membranes (DMPG) which mimic the negatively charged membrane of Gram-positive bacteria. We attribute the high percentage of insertion of the biologically inactive analogue in DMPG to the fact that its concentration on the membrane surface in our experiments is likely to be much higher than that found in physiological conditions.

Effects of (+)-totarol, a diterpenoid antibacterial agent, on phospholipid model membranes

(+)-Totarol, a highly hydrophobic diterpenoid isolated from Podocarpus spp., is inhibitory towards the growth of diverse bacterial species. (+)-Totarol decreased the onset temperature of the gel to liquid-crystalline phase transition of DMPC and DMPG membranes and was immiscible with these lipids in the fluid phase at concentrations greater than 5 mol%. Different (+)-totarol/phospholipid mixtures having different stoichiometries appear to coexist with the pure phospholipid in the fluid phase. At concentrations greater than 15 mol% (+)-totarol completely suppressed the gel to liquid-crystalline phase transition in both DMPC and DMPG vesicles. Incorporation of increasing amounts of (+)-totarol into DEPE vesicles induced the appearance of the H II hexagonal phase at low temperatures in accordance with NMR data. At (+)-totarol concentrations between 5 and 35 mol% complex thermograms were observed, with new immiscible phases appearing at temperatures below the main transition of DEPE. Steady-state fluorescence anisotropy measurements showed that (+)-totarol decreased and increased the structural order of the phospholipid bilayer below and above the main gel to liquid-crystalline phase transition of DMPC respectively. The changes that (+)-totarol promotes in the physical properties of model membranes, compromising the functional integrity of the cell membrane, could explain its antibacterial effects.

Structural and morphological investigations of the formation of quasi-crystalline phases of 1,2-dimyristoyl-sn-glycero-3- phosphoglycerol (DMPG)

1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) in 100 mM NaCl at pH 7 forms a quasi-crystalline gel phase (Lc phase) when stored for several days or longer at 4°C. We studied the structure and morphology of this phase by freeze–fracture electron microscopy, synchrotron X-ray diffraction, and Fourier transform infrared spectroscopy (FTIR). The phase behavior was also followed by differential scanning calorimetry. Freeze–fracture electron microscopy revealed two different distinct morphological aggregates for the Lc phase, namely flat multilamellar sheets and cochleate cylinders in an approximately 1:1 ratio. FTIR investigations showed that the hydrocarbon chains are tightly packed within an orthorhombic subcell lattice. The frequency shift of the ester carbonyl bands to lower wavenumbers (1732 cm−1) indicates a direct involvement of the carbonyl groups in hydrogen bonding with hydroxy groups. The freeze–fracture electron microscopic and X-ray investigation reveal very tightly packed lamellar aggregates. The small lamellar repeat distance of 4.75 nm indicates that only a thin water layer is present between the lipid bilayers. The set of reflections observed in the wide angle X-ray region resembles that of the crystalline Lc phase observed in phosphatidylcholines. Upon cooling, the lamellar repeat distance observed in the liquid-crystalline phase increases drastically until the transition into the lamellar gel phase occurs. Then, the reflection in the low angle region becomes broad and diffuse. This finding agrees with the electron microscopic results that upon cooling multilamellar aggregates are destroyed and unilamellar DMPG vesicles are formed below the main phase transition. The unilamellar vesicles fuse to lamellar stacks upon low temperature storage and these then roll up into cochleate cylinders. The observed behavior is attributed to the unique properties of the glycerol headgroup in being able to replace water molecules in the hydration shell.

Intrinsic fluorescence study of the interaction of human apolipoprotein H with phospholipid vesicles.

Apolipoprotein H (ApoH) is a plasma glycoprotein with its in vivo physiological and pathogenic roles being closely related to its interaction with negatively charged membranes. In this paper, the interaction of ApoH with phospholipid vesicles was characterized by (i) detecting the wavelength shift of the fluorescence spectrum of ApoH and (ii) measuring the fluorescence quenching extent of ApoH by the membrane resident quencher 1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3-phosphocholine (DPC). The observed blue shift upon addition of DMPG vesicles indicated that the tryptophan residues of ApoH moved from a polar to a nonpolar environment. The insertion ability of ApoH into PG-containing vesicles did not depend on the PG content in a stoichiometric way as did the blue shift, indicating that the negatively charged DMPG does not serve as a specific binding site but rather provides a suitable microenvironment for ApoH interaction. The finding that the detachment effect of cations on the blue shift is remarkably different from that on the quenching extent suggests that ApoH is capable of existing in two different conformations when membrane-bound.

Structural investigations of basic amphipathic model peptides in the presence of lipid vesicles studied by circular dichroism, fluorescence, monolayer and modeling

A cationic amphiphilic peptide made of 10 leucine and 10 lysine residues, and four of its fluorescent derivatives in which leucines were substituted by Trp residues at different locations on the primary sequence have been synthesized. The interactions of these five peptides with neutral anionic or cationic vesicles were investigated using circular dichroism, steady state and time-resolved fluorescence with a combination of Trp quenching by brominated lipid probes, monolayers, modeling with minimization and simulated annealing procedures. We show that all the five peptides interact with neutral and anionic DMPC, DMPG, DOPC or egg yolk PC vesicles. The binding takes place whatever the peptide conformation in solution is. In the case of DMPC bilayers the binding free energy Δ G is estimated at −8 kcal mole −1 and the number of phospholipid molecules involved is about 20–25 per peptide molecule. Peptides are bound as single-stranded α helices orientated parallel to the bilayer surface. In the anchoring of phospholipid head groups around the peptides, the lipid molecules are not smeared out in a plane parallel to the membrane surface but are organized around the hydrophilic face of the α helices like `wheat grains around an ear' and protrude outside the bilayer towards the solvent. We suggest that such a lipid arrangement generates transient structural defects responsible for the membrane permeability enhancement. When an electrical potential is applied, the axis of the peptide helices remains parallel to the membrane surface and does not reorient to give rise to a bundle of helix monomers that forms transmembrane channels via a `barrel stave' mechanism. The penetration depth of α helices in relation to the position of phosphorus atoms in the unperturbed lipid leaflet is estimated at 3.2 Å.

Fluorescence spectroscopic study of the interaction of saporin with phospholipid vesicles.

Saporin-S6 (SO-6) is a type-1 ribosome-inactivating protein purified from the seeds of Saponaria officinalis. The fluorescence characteristics of SO-6 were studied in the presence and absence of phospholipids. The interaction of SO-6 with DMPG or DMPC vesicles results in a decrease in the fluorescence emission intensity of tryptophan without any shift in the emission maximal wavelength. The results of fluorescence titration indicate that DMPG/SO-6 saturation molar ratio is 100: 1, but the binding of DMPC with SO-6 does not reach a saturating plot. A shielding of the tryptophan fluorescence from quenching by acrylamide on interaction with the phospholipids was observed, and this shielding was more pronounced in the presence of DMPG. The interaction of SO-6 with DMPG vesicles is stronger in the liquid-crystalline phase than in the gel phase. Extrinsic fluorescence studies indicated that the interaction of the protein with DMPG vesicles does not modify the phase transition temperature of the lipid, but decreases the amplitude of the change of fluorescence anisotropy associated with the co-operative melting of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labelled vesicles. These results indicate that both electrostatic and hydrophobic components are involved in the SO-6-lipid vesicle interaction.

Conformational analysis of bioactive peptides in reverse micelles as mimics of cell membrane environments

Conformational preferences of secretin as a model peptide have been analyzed by CD and IR spectroscopy in reverse micelles of AOT/isooctane/water and compared to those in aqueous TFE, in SDS micelles and in DMPG vesicles. Among the systems examined, reverse micelles and phospholipid vesicles displayed almost identical conformational equilibria. Very high lipid-to-peptide ratios can be obtained in reverse micelles with full retention of optical transparency, even at millimolar peptide concentrations, thus indicating this system to be an interesting mimic of cell membrane environments for spectroscopic analysis of bioactive peptide conformations.

Fusion of phospholipid vesicles produced by the anti-tumour protein α-sarcin

The anti-tumour protein alpha-sarcin causes fusion of bilayers of phospholipid vesicles at neutral pH. This is demonstrated by measuring the decrease in the efficiency of the fluorescence energy transfer between N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dimyristoylphosphatidylethano lamine (NDB-PE) (donor) and N-(lissamine rhodamine B sulphonyl)-diacylphosphatidylethanolamine (Rh-PE) (acceptor) incorporated in dimyristoylphosphatidylcholine (DMPG) vesicles. The effect of alpha-sarcin is a maximum at 0.15 M ionic strength and is abolished at basic pH. alpha-Sarcin promotes fusion between 1,6-diphenylhexa-1,3,5-triene (DPH)-labelled DMPG and dipalmitoyl-PG (DPPG) vesicles, resulting in a single thermotropic transition for the population of fused phospholipid vesicles. Bilayers composed of DMPC and DMPG, at different molar ratios in the range 1:1 to 1:10 PC/PG, are also fused by alpha-sarcin. Freeze-fracture electron micrographs corroborate the occurrence of fusion induced by the protein. alpha-Sarcin also modifies the permeability of the bilayers, causing the leakage of calcein in dye-trapped PG vesicles. All of the observed effects reach saturation at a 50:1 phospholipid/protein molar ratio, which is coincident with the binding stoichiometry previously described.

Role of the polar head group stereoconfiguration in the cation-induced aggregation of dimyristoylphosphatidylglycerol vesicles.

Cation-induced aggregation of small unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-sn-1'-glycerol (1'-DMPG), the corresponding 3' stereoisomer (3'-DMPG), and their 1:1 mixture was studied as a function of the concentration of different mono- and divalent cations. The order of efficiency, Na+ greater than Li+ greater than K+ greater than Cs+, of the monovalent cations to induce the aggregation of DMPG vesicles is the same for both stereoisomers and their mixture. However, significant differences in the Na+-induced aggregation of 1'-DMPG and 3'-DMPG were evident. The threshold concentration of aggregation by Na+ was 0.35 M for 3'-DMPG, 0.55 M for 1'-DMPG, and 0.50 M for the mixed liposomes. Such difference in the aggregation of DMPG stereoisomers was not observed for the other mono- and divalent cations. The higher affinity of 3'-DMPG for Na+ is suggested to be due to a slightly different favored conformation of the head group glycerol moiety. Aggregation of the stereoisomers by 1 M NaCl was identical, indicating that the differences in the affinity of 1'-DMPG and 3'-DMPG for sodium can be overcome by very high ionic strength. Inclusion of 20 mol % cholesterol in vesicles enhanced the aggregation of 1'-DMPG and decreased the aggregation of 3'-DMPG by Na+ and thus abolished the difference between the two stereoisomers.