Anionic Bilayers Research Articles

Chitosan derivatives targeting lipid bilayers: Synthesis, biological activity and interaction with model membranes

The antimicrobial activity of chitosan and derivatives to human and plant pathogens represents a high-valued prospective market. Presently, two low molecular weight derivatives, endowed with hydrophobic and cationic character at different ratios were synthesized and characterized. They exhibit antimicrobial activity and increased performance in relation to the intermediate and starting compounds. However, just the derivative with higher cationic character showed cytotoxicity towards human cervical carcinoma cells. Considering cell membranes as targets, the mode of action was investigated through the interaction with model lipid vesicles mimicking bacterial, tumoral and erythrocyte membranes. Intense lytic activity and binding are demonstrated for both derivatives in anionic bilayers. The less charged compound exhibits slightly improved selectivity towards bacterial model membranes, suggesting that balancing its hydrophobic/hydrophilic character may improve efficiency. Observing the aggregation of vesicles, we hypothesize that the “charge cluster mechanism”, ascribed to some antimicrobial peptides, could be applied to these chitosan derivatives.

Insight into the interactions, residue snorkeling, and membrane disordering potency of a single antimicrobial peptide into different lipid bilayers.

Pardaxin, with a bend-helix-bend-helix structure, is a membrane-active antimicrobial peptide that its membrane activity depends on the lipid bilayer composition. Herein, all-atom molecular dynamics (MD) simulations were performed to provide further molecular insight into the interactions, structural dynamics, orientation behavior, and cationic residues snorkeling of pardaxin in the DMPC, DPPC, POPC, POPG, POPG/POPE (3:1), and POPG/POPE (1:3) lipid bilayers. The results showed that the C-terminal helix of the peptide was maintained in all six types of the model-bilayers and pardaxin was tilted into the DMPC, DPPC, and POPG/POPE mixed bilayers more than the POPC and POPG bilayers. As well as, the structure of zwitterionic membranes was more affected by the peptide than the anionic bilayers. Taken together, the study demonstrated that the cationic residues of pardaxin snorkeled toward the interface of lipid bilayers and all phenylalanine residues of the peptide played important roles in the peptide-membrane interactions. We hope that this work will provide a better understanding of the interactions of antimicrobial peptides with the membranes.

Effects of Hydrophobic Amino Acid Substitutions on Antimicrobial Peptide Behavior.

Antimicrobial peptides (AMPs) are naturally occurring components of the immune system that act against bacteria in a variety of organisms throughout the evolutionary hierarchy. There have been many studies focused on the activity of AMPs using biophysical and microbiological techniques; however, a clear and predictive mechanism toward determining if a peptide will exhibit antimicrobial activity is still elusive, in addition to the fact that the mechanism of action of AMPs has been shown to vary between peptides, targets, and experimental conditions. Nonetheless, the majority of AMPs contain hydrophobic amino acids to facilitate partitioning into bacterial membranes and a net cationic charge to promote selective binding to the anionic surfaces of bacteria over the zwitterionic host cell surfaces. This study explores the role of hydrophobic amino acids using the peptide C18G as a model system. These changes were evaluated for the effects on antimicrobial activity, peptide-lipid interactions using Trp fluorescence spectroscopy, peptide secondary structure formation, and bacterial membrane permeabilization. The results show that while secondary structure formation was not significantly impacted by the substitutions, antibacterial activity and binding to model lipid membranes were well correlated. The variants containing Leu or Phe as the sole hydrophobic groups bound bilayers with highest affinity and were most effective at inhibiting bacterial growth. Peptides with Ile exhibited intermediate behavior while those with Val or α-aminoisobutyric acid (Aib) showed poor binding and activity. The Leu, Phe, and Ile peptides demonstrated a clear preference for anionic bilayers, exhibiting significant emission spectrum shifts upon binding. Similarly, the Leu, Phe, and Ile peptides demonstrated greater ability to disrupt lipid vesicles and bacterial membranes. In total, the data indicate that hydrophobic moieties in the AMP sequence play a significant role in the binding and ability of the peptide to exhibit antibacterial activity.

Enhanced Pb2 + removal by anionic surfactant bilayer anchored on chitosan bead surface

Although chitosan is an effective sorbent for the removal of contaminants from wastewater, however, the performance of pure chitosan is limited in many cases (such as for cations) because of its positive charge. Thus modification of chitosan remains challenging to remove cations (such as metal ions) in a better way. The present study has been carried out for the successful removal of lead ions (Pb2+) from aqueous solution by modified chitosan beads. Surface modification of chitosan (CS) beads was performed by using anionic surfactant (AS), sodium dodecyl sulfate (SDS). Micelle aggregation property of SDS has been utilized for the formation of bilayer over the CS beads to produce surfactant modified chitosan (SMCS) beads. Prepared adsorbents were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). SMCS beads when compared to the pure CS beads showed three times higher adsorption of Pb2+. This enhanced adsorption is believed to be due to the adsolubilization of Pb2+on SDS bilayer. The bilayer provides more adsorption sites for quick and effective removal of Pb2+ from the aqueous phase. The adsorption followed pseudo-second order kinetic and Langmuir isotherm model. The maximum adsorption capacity obtained was 100.0mg/g at the dosage of 0.675g/L for 50mg/L initial concentration of Pb2+. Studies on the thermodynamic parameters showed that, the adsorption was exothermic. The interference studies conducted on several anions and cations indicated that, the anions had less interference compared to those of cations. Desorption study confirmed 30–40% recovery of Pb2+ using 0.15N HNO3 and 0.1N HCl.

Electrostatic double-layer interaction between stacked charged bilayers.

The inapplicability of the DLVO theory to multilayered anionic bilayers is found in terms of the co-ion-valence dependence of the lamellar repeat distance. Most of the added salt is expelled from the interlamellar space to the bulk due to the Gibbs-Donnan effect on multiple bilayers with the bulk. The electrostatic double-layer interaction is well expressed by the formula recently proposed by Trefalt. The osmotic pressure due to the expelled ions, rather than the van der Waals interaction, is the main origin of the attractive force between the bilayers.

Membrane perturbing activities and structural properties of the frog-skin derived peptide Esculentin-1a(1-21)NH2 and its Diastereomer Esc(1-21)-1c: Correlation with their antipseudomonal and cytotoxic activity

Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d-amino acids i.e. DLeu14 and DSer17 revealed to be less cytotoxic, more stable to proteolytic degradation and more efficient in eradicating Pseudomonas biofilm. When tested in vitro against the free-living form of this pathogen, it displayed potent bactericidal activity, but this was weaker than that of the all-l peptide. To investigate the reason accounting for this difference, mechanistic studies were performed on Pseudomonas spheroplasts and anionic or zwitterionic membranes, mimicking the composition of microbial and mammalian membranes, respectively. Furthermore, structural studies by means of optical and nuclear magnetic resonance spectroscopies were carried out. Our results suggest that the different extent in the bactericidal activity between the two isomers is principally due to differences in their interaction with the bacterial cell wall components. Indeed, the lower ability in binding and perturbing anionic phospholipid bilayers for Esc(1-21)-1c contributes only in a small part to this difference, while the final effect of membrane thinning once the peptide is inserted into the membrane is identical to that provoked by Esc(1-21). In addition, the presence of two d-amino acids is sufficient to reduce the α-helical content of the peptide, in parallel with its lower cytotoxicity.

Binding, Folding, and Insertion of a β-Hairpin Peptide at a Lipid Bilayer Surface: Molecular Dynamics Simulations

Antimicrobial peptides (AMPs) are common in many biological systems and act as host defenses against microbial pathogens. Existing theories on how AMPs permeabilize lipid membranes still lack mechanistic detail. In this study, we used atomistic molecular dynamics (MD) simulations to investigate the interactions of SVS-1 (KVKVKVKVPDPLTKVKVKVK), an anti-cancer β-hairpin peptide, with a fully hydrated neutral POPC bilayer and a mixture of 1:1 anionic POPC/POPG bilayer. In agreement with experiment, peptides in simulation bind more strongly to anionic bilayers than to neutral bilayers. Unfolded peptides at the anionic surface were observed to undergo several folding and mis-folding pathways. Folded peptides at the surface did not spontaneously insert their hydrophobic faces into the bilayer on the 1 µs timescale, but kept their Lys-rich face downward toward the lipid head groups. Upon the application of sufficient surface tension, the peptides were observed to “flip and dip” into the bilayer within ∼100 ns. Pre-folded SVS-1 peptides were observed to insert in neutral membranes with ample surface tension, but remained at the surface or in solution if unfolded. We associate this insertion phenomenon to increased hydrophobic exposure of lipid tails due to tension. Our simulations suggest electrostatics interactions and hydrophobic exposure are key factors in witnessing an insertion event. The apparent absence of a pathway for a single peptide to produce a defect or pore in the bilayer suggests that the mechanism involves cooperative action of multiple peptides.

Diffusion of Fluorescently Labeled Bacteriocin from Edible Nanomaterials and Embedded Nano-Bioactive Coatings.

Application of nano-biotechnology to improve the controlled release of drugs or functional agents is widely anticipated to transform the biomedical, pharmaceutical, and food safety trends. The purpose of the current study was to assess and compare the release rates of fluorescently labeled antimicrobial peptide nisin (lantibiotic/biopreservative) from liposomal nanocarriers. The elevated temperature, high electrostatic attraction between anionic bilayers and cationic nisin, larger size, and higher encapsulation efficiency resulted in rapid and elevated release through pore formation. However, acidic pH and optimal ethanol concentration in food simulating liquid (FSL) improved the stability and retention capacity of loaded drug. Thus, controlling various factors had provided partition coefficient K values from 0.23 to 8.78 indicating variation in nisin affinity toward encapsulating macromolecule or FSL. Interaction between nisin and nanoscale bilayer systems by atomic force (AFM) and transmission electron microscopy demonstrated membrane activity of nisin from adsorption and aggregation to pore formation. Novel nanoactive films with preloaded nanoliposomes embedded in biodegradable polymer revealed improved morphological, topographic, and roughness parameters studied by confocal microscopy and AFM. Pre-encapsulated nanoactive biopolymer demonstrated excellent retention capacity as drug carriers by decreasing the partition coefficient value from 1.8 to 0.66 (∼30%) due to improved stability of nanoliposomes embedded in biopolymer network.

Toward More Free-Floating Model Cell Membranes: Method Development and Application to Their Interaction with Nanoparticles.

Identifying the mechanisms of nanoparticle (NP) interactions with cell membranes is key to understanding potential NP cytotoxicity and applications as nanocarriers for targeted drug delivery. To elucidate these mechanisms of interaction, supported phospholipid bilayers (SPBs) are commonly used as models of cell membranes. However, SPBs are soft thin films, and, as such, their properties can be significantly affected by the underlying substrate. Free-floating cell membranes would be best modeled by weakly adhered SPBs; thus, we propose a method for tailoring the interfacial interaction of an electrically charged SPB-substrate system based on modulations in the solution chemistry. Using the dissipation signal of the quartz crystal microbalance with dissipation monitoring (QCM-D), we show that the method can be used to tailor SPB-substrate interactions without the loss of its structural integrity. To demonstrate the application of the method, SPBs are exposed to cationic and anionic polystyrene latex NPs. These studies reveal that the bilayer response to the modulations in the interfacial interaction with its underlying substrate can be used as a sensitive tool to probe the integrity of SPBs upon exposure to NPs. As expected, anionic NPs tend to impart no significant damage to the anionic bilayers, whereas cationic NPs can be detrimental to bilayer integrity. This is the first report of a QCM-D based method to probe bilayer integrity following exposure to NPs. Importantly, the degree of SPB interaction with its underlying substrate is shown to be a critical factor in the kinetics of bilayer disruption by cationic NPs, whereby weakly adhered bilayers are prone to significantly faster breakup. Since free-floating cell membranes are better represented by a weakly adhered SPB, the results of this work critically influence paradigms in experimental studies involving SPBs as models for cell membranes.

Interactions of Polyethylenimines with Zwitterionic and Anionic Lipid Membranes.

Interactions between polyethylenimines (PEIs) and phospholipid membranes are of fundamental importance for various biophysical applications of these polymers such as gene delivery. Despite investigations into the nature of these interactions, their molecular basis remains poorly understood. In this article, we combined experimental methods and atomistic molecular dynamics (MD) simulations to obtain comprehensive insight into the effect of linear and branched PEIs on zwitterionic and anionic bilayers used as simple models of mammalian cellular membranes. Our results show that PEIs adsorb only partially on the surface of zwitterionic membranes by forming hydrogen bonds to the lipid headgroups, whereas a large part of the polymer chains dangles freely in the aqueous phase. In contrast, PEIs readily adhere to and insert into the anionic membrane. The attraction of the polymer chains to the membrane is due to electrostatic interactions as well as hydrogen bonding between the amine groups of PEI and the phosphate groups of lipids. These interactions were found to induce a substantial reorganization of the bilayer in the polymer vicinity due to the reorientation of lipid molecules. The lipid headgroups were pulled toward the center of the membrane, which can facilitate transmembrane translocations of anionic lipids. Furthermore, the PEI-lipid interactions affect the stability of liposomal dispersions, but we did not see any evidence of disruption of the vesicular structures into small fragments at polymer concentrations typically used in gene therapy. Our results provide a detailed molecular-level description of the lipid organization in the membrane in the presence of polycations that can be useful in understanding their mechanisms of in vitro and in vivo cytotoxicity.

The Alzheimer's disease Aβ peptide binds to the anionic DMPS lipid bilayer

We have applied isobaric–isothermal replica exchange molecular dynamics (REMD) and the all-atom explicit water model to study binding of Aβ10–40 peptide to the anionic DMPS bilayer. To provide comparison with a zwitterionic bilayer, we used our previous REMD simulations probing binding of the same peptide to the DMPC bilayer. Using two sets of simulations, we comparatively analyzed the equilibrium Aβ conformational ensemble, peptide–bilayer interactions, and changes in the bilayer structure induced by Aβ binding. Our results are six-fold. (1) Binding to the DMPS bilayer triggers the formation of stable helix in the Aβ C-terminal, although the helix-inducing effect caused by DMPS lipids is weaker than that of DMPC. (2) Compared to the DMPC-bound Aβ monomer, the anionic bilayer weakens intrapeptide interactions, particularly, formed by charged amino acids. (3) Binding of Aβ peptide to the DMPS bilayer is primarily governed by electrostatic interactions between charged amino acids and charged lipid groups. In contrast, these interactions play minor role in Aβ binding to the DMPC bilayer. (4) Aβ peptide generally resides on the DMPS bilayer surface causing relatively minor bilayer thinning. The opposite scenario applies to Aβ binding to the DMPC bilayer. (5) In contrast to DMPC simulations, Aβ largely expels anionic lipids from its binding “footprint” forming a ring of charged amino acids mixed with charged lipid groups around the peptide. (6) Aβ binding disorders proximal DMPS lipids more strongly than their DMPC counterparts. Our simulations show that Aβ monomers fail to perturb anionic or zwitterionic bilayers across both leaflets.

The Position of Aβ22-40 and Aβ1-42 in Anionic Lipid Membranes Containing Cholesterol.

Amyloid-β peptides interact with cell membranes in the human brain and are associated with neurodegenerative diseases, such as Alzheimer’s disease. An emerging explanation of the molecular mechanism, which results in neurodegeneration, places the cause of neurotoxicity of the amyloid-β peptides on their potentially negative interaction with neuronal membranes. It is known that amyloid-β peptides interact with the membrane, modifying the membrane’s structural and dynamic properties. We present a series of X-ray diffraction experiments on anionic model lipid membranes containing various amounts of cholesterol. These experiments provide experimental evidence for an interaction of both the full length amyloid- peptide, and the peptide fragment amyloid- with anionic bilayer containing cholesterol. The location of the amyloid-β peptides was determined from these experiments, with the full length peptide embedding into the membrane, and the peptide fragment occupying 2 positions—on the membrane surface and embedded into the membrane core.

2D metal–organic networks based on asymmetric hexa-carboxylates and tetra-carboxylates ligands: A microporous Cl-pillared anionic bilayer and a neutral (4, 8)-net

New asymmetric tetra-carboxylates ligand, 2,2′-((6-chloro-1,3,5-triazine-2,4-diyl)bis(azanediyl))diterephthalic acid (H4L1) and hexa-carboxylates ligand, 2,2′,2″-((1,3,5-triazine-2,4,6-triyl)(azanediyl))-triterephthalic acid (H6L2), were designed and used for the assemble reactions with Cd2+ ions. [Cd2(L1)(H2O)2]·2.5DMSO·2H2O (1) and (Me2NH2)9(H3O)2[Cd2Cl3(L2)2]·5H2O (2) have been synthesized and structurally characterized. Single crystal X-ray analysis show that compound 1 bears a 2D neutral (4, 8)-net based on 3-connected {Cd2} nodes and 3-connected spacers. 2 is the first bilayer MOFs pillared by monatomic Cl linker and its structure is built of 4-connected single metal nodes and 3-connected spacers. The topology of the resulted (3, 4)-connected anionic framework could be described as (63)(66) (Schläfli symbol). N2 sorption isotherm of 2 shows typical type I behavior with BET surface area of 599m2g−1 and a micropore volume of 0.2345cm3g−1. Luminescent analysis indicates both compounds exhibit linker-localized emission. Furthermore, infrared (IR), thermogravimetric analyses (TGA) properties of the compounds were also investigated.

Distinct helix propensities and membrane interactions of human and rat IAPP(1-19) monomers in anionic lipid bilayers.

Islet amyloid polypeptide, IAPP or amylin, is a 37-residue peptide hormone coexpressed with insulin by pancreatic β-cells. The aggregation of human IAPP (hIAPP) into amyloid deposits is associated with type II diabetes. Substantial evidence suggests that the interaction of anionic membranes with hIAPP may facilitate peptide aggregation and the N-terminal 1-19 fragment (IAPP(1-19)) plays an important role in peptide-membrane interaction. As a first step to understand how structural differences between human and rat IAPP peptides in membranes may influence the later oligomerization process, we have investigated the structures and orientations of hIAPP(1-19) and the less toxic rIAPP(1-19) (i.e., the H18R mutant of hIAPP(1-19)) monomers in anionic POPG bilayers by performing replica exchange molecular dynamics (REMD) simulations. On the basis of ∼20 μs REMD simulations started from a random coil conformation of the peptide placed in water, we find that unfolded h(r)IAPP(1-19) can insert into the bilayers and the membrane-bound peptide stays mainly at the lipid head-tail interface. hIAPP(1-19) displays higher helix propensity than rIAPP(1-19), especially in the L12-L16 region. The helix is oriented parallel to the bilayer surface and buried in the membrane 0.3-0.8 nm below the phosphorus atoms, consistent with previous electron paramagnetic resonance data. The helical conformation is an amphiphilic helix with its hydrophilic and hydrophobic faces pointing, respectively, to the lipid head and tail regions. The H18R substitution enhances the electrostatic interactions of IAPP(1-19) with the membrane, while it weakens the intrapeptide interactions crucial for helix formation, thus leading to lower helix propensity of rIAPP(1-19). Implications of our simulation results on the membrane-mediated IAPP(1-19) oligomerization are discussed.

Nucleotide-Dependent Membrane Interaction and Dimerization of K-Ras4B

Ras proteins are membrane-associated, small guanine nucleotide-binding GTPases that control cell survival and proliferation. They consist of highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ significantly across Ras isoforms, H-Ras, N-Ras, K-Ras4A and K-Ras4B, where KRas4B is among the frequently mutated oncogenes in human tumors. Recent NMR experiments discovered that the HVR of GDP-bound K-Ras4B extensively interacts with the catalytic domain. However, the HVR weakly interacts with the catalytic domain when the protein is in the GTP-bound state. Molecular dynamics (MD) simulations confirmed tight interaction of HVR with catalytic domain in the GDP-bound state, but not GTP-bound K-Ras4B, suggesting that in the GDP-bound state, an HVR domain could adopt a β-strand conformation, extending the β-sheet in the active site of the catalytic domain. Here we modeled K-Ras4B membrane interaction and dimerization. Membrane binding of K-Ras4B through the anchoring of the positively charged HVR is critical to its function as an oncogene and initiates signaling events. Recent studies showed that post-translationally modified HVR peptide spontaneously inserts the farnesyl group into the zwitterionic (DOPC) and anionic (DOPC:DOPS=4:1) bilayers in the liquid phase, but not into the DPPC bilayer in the gel phase. Further, spontaneous membrane insertion of the farnesyl group in full-length K-Ras4B strongly depends on the nucleotide type. The HVR of K-Ras4B-GTP preferentially interacts with lipids through the farnesyl insertion, while the HVR of K-Ras4B-GDP rather binds the catalytic domain and inserts less frequently the farnesyl into the lipid bilayer. Remarkably, K-Ras4B-GTP, but not GDP-bound, is able to form stable homodimers with different dimer interfaces, suggesting that the nucleotide-dependent dimerization with various dimer interfaces can resolve nanoclustering and cluster reorganization accomplishment with Raf's activation. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E.

Preparation of pH-sensitive anionic liposomes designed for drug delivery system (DDS) application.

We prepared pH-sensitive anionic liposomes composed solely of anionic bilayer membrane components that were designed to promote efficient release of entrapped agents in response to acidic pH. The pH-sensitive anionic liposomes showed high dispersion stability at neutral pH, but the fluidity of the bilayer membrane was enhanced in an acidic environment. These liposomes were rather simple and were composed of dimyristoylphosphatidylcholine (DMPC), an anionic bilayer membrane component, and polyoxyethylene sorbitan monostearate (Tween 80). In particular, the present pH-sensitive anionic liposomes showed higher temporal stability than those of conventional DMPC/DPPC liposomes. We found that pHsensitive properties strongly depended on the molecular structure, pKa value, and amount of an incorporated anionic bilayer membrane component, such as sodium oleate (SO), dimyristoylphosphatidylserine (DMPS), or sodium β-sitosterol sulfate (SS). These results provide an opportunity to manipulate liposomal stability in a pH-dependent manner, which could lead to the formulation of a high performance drug delivery system (DDS).

Combining experimental evidence and molecular dynamic simulations to understand the mechanism of action of the antimicrobial octapeptide jelleine-I.

Jelleines are four naturally occurring peptides that comprise approximately eight or nine C-terminal residues in the sequence of the major royal jelly protein 1 precursor (Apis mellifera). The difference between these peptides is limited to one residue in the sequence, but this residue has a significant impact in their efficacy as antimicrobials. In peptide-bilayer experiments, we demonstrated that the lytic, pore-forming activity of Jelleine-I is similar to that of other cationic antimicrobial peptides, which exhibit stronger activity on anionic bilayers. Results from molecular dynamics simulations suggested that the presence of a proline residue at the first position is the underlying reason for the higher efficacy of Jelleine-I compared with the other jelleines. Additionally, simulations suggested that Jelleine-I tends to form aggregates in water and in the presence of mimetic membrane environments. Combined experimental evidence and simulations showed that the protonation of the histidine residue potentiates the interaction with anionic palmitoyl-oleoyl-phosphatidylcholine/palmitoyl-oleoyl-phosphatidylglycerol (POPC/POPG) (70:30) bilayers and reduces the free energy barrier for water passage. The interaction is driven by electrostatic interactions with the headgroup region of the bilayer with some disturbance of the acyl chain region. Our findings point to a mechanism of action by which aggregated Jelleine-I accumulates on the headgroup region of the membrane. Remaining in this form, Jelleine-I could exert pressure to accommodate its polar and nonpolar residues on the amphiphilic environment of the membrane. This pressure could open pores or defects, could disturb the bilayer continuity, and leakage would be observed. The agreement between experimental data and simulations in mimetic membranes suggests that this approach may be a valuable tool to the understanding of the molecular mechanisms of action.

On the freezing behavior and diffusion of water in proximity to single-supported zwitterionic and anionic bilayer lipid membranes

We compare the freezing/melting behavior of water hydrating single-supported bilayers of a zwitterionic lipid DMPC with that of an anionic lipid DMPG. For both membranes, the temperature dependence of the elastically scattered neutron intensity indicates distinct water types undergoing translational diffusion: bulk-like water probably located above the membrane and two types of confined water closer to the lipid head groups. The membranes differ in the greater width of the water freezing transition near the anionic DMPG bilayer compared to zwitterionic DMPC as well as in the abruptness of the freezing/melting transitions of the bulk-like water.

Structure–activity relationships of the antimicrobial peptide gramicidin S and its analogs: Aqueous solubility, self-association, conformation, antimicrobial activity and interaction with model lipid membranes

GS10 [cyclo-(VKLdYPVKLdYP)] is a synthetic analog of the naturally occurring antimicrobial peptide gramicidin (GS) in which the two positively charged ornithine (Orn) residues are replaced by two positively charged lysine (Lys) residues and the two less polar aromatic phenylalanine (Phe) residues are replaced by the more polar tyrosine (Tyr) residues. In this study, we examine the effects of these seemingly conservative modifications to the parent GS molecule on the physical properties of the peptide, and on its interactions with lipid bilayer model and biological membranes, by a variety of biophysical techniques. We show that although GS10 retains the largely β-sheet conformation characteristic of GS, it is less structured in both water and membrane-mimetic solvents. GS10 is also more water soluble and less hydrophobic than GS, as predicted, and also exhibits a reduced tendency for self-association in aqueous solution. Surprisingly, GS10 associates more strongly with zwitterionic and anionic phospholipid bilayer model membranes than does GS, despite its greater water solubility, and the presence of anionic phospholipids and cholesterol (Chol) modestly reduces the association of both GS10 and GS to these model membranes. The strong partitioning of both peptides into lipid bilayers is driven by a large favorable entropy change opposed by a much smaller unfavorable enthalpy change. However, GS10 is also less potent than GS at inducing inverted cubic phases in phospholipid bilayer model membranes and at inhibiting the growth of the cell wall-less bacterium Acholeplasma laidlawii B. These results are discussed in terms of the comparative antibiotic and hemolytic activities of these peptides.

Dynamics of Bilayer Interactions at the Air-Water Interface

A novel supported bilayer system at the air-water interface has been developed for use in synchrotron X-ray scattering experiments in order to drastically increase signal to noise ratio. This system gives a significant resolution improvement comparing to more traditional solid-supported bilayer system and is better suited for specular X-Ray reflectivity and grazing incidence X-ray off-specular scattering. Using this system we have successfully characterized anionic bilayers mimetic of Gram-positive bacterial membranes interacting with engineered biomimetic oligomers (peptoids), zwitterionic bilayers interacting with human islet amyloid polypeptide (IAPP), and insertion differences between zwitterionic membranes with and without cholesterol and the glycoprotein-41 subunit fragment of the viral envelope glycoprotein-160. Results from anionic and net neutral bilayers correspond to previous studies on monolayer insertion studies, where peptoid ML 2-6 acts by associating throughout the lipophilic regions and zwitterionic bilayers without cholesterol were completely penetrated by the GP-41 fusion peptide. Resolution of the dynamic formation of a zwitterionic lipid bilayer system in minute scale time resolution via Grazing Incidence X-Ray Off-Specular Scattering (GIXOS) was also observed, and found to be in agreement with the proposed mechanisms associated with vesicle fusion on a solid support.