PC Bilayers Research Articles

Structural Basis of the Interaction of the G Proteins, Gαi1, Gβ1γ2 and Gαi1β1γ2, with Membrane Microdomains and Their Relationship to Cell Localization and Activity.

GPCRs receive signals from diverse messengers and activate G proteins that regulate downstream signaling effectors. Efficient signaling is achieved through the organization of these proteins in membranes. Thus, protein-lipid interactions play a critical role in bringing G proteins together in specific membrane microdomains with signaling partners. Significantly, the molecular basis underlying the membrane distribution of each G protein isoform, fundamental to fully understanding subsequent cell signaling, remains largely unclear. We used model membranes with lipid composition resembling different membrane microdomains, and monomeric, dimeric and trimeric Gi proteins with or without single and multiple mutations to investigate the structural bases of G protein-membrane interactions. We demonstrated that cationic amino acids in the N-terminal region of the Gαi1 and C-terminal region of the Gγ2 subunit, as well as their myristoyl, palmitoyl and geranylgeranyl moieties, define the differential G protein form interactions with membranes containing different lipid classes (PC, PS, PE, SM, Cho) and the various microdomains they may form (Lo, Ld, PC bilayer, charged, etc.). These new findings in part explain the molecular basis underlying amphitropic protein translocation to membranes and localization to different membrane microdomains and the role of these interactions in cell signal propagation, pathophysiology and therapies targeted to lipid membranes.

Temperature- and Pressure-Induced Bilayer Phase Transitions of an Amide-Linked Phosphatidylcholine: A Contrasting Effect of Chain-Linkage Type

Abstract An unnatural phospholipid with two amide linkages, dipalmitoylamidodeoxyphosphatidylcholine (DPADPC), was synthesized and its bilayer phase transitions were observed by differential scanning calorimetry under atmospheric pressure and light-transmittance measurements under high pressure. The DPADPC bilayer membrane showed different phase transitions depending on the thermal history of the lipid sample under atmospheric pressure. The thermal-history dependent transitions were also observed under high pressure. The temperature–pressure phase diagram and thermodynamic quantities of the phase transitions of the DPADPC bilayer membrane were compared with those of bilayer membranes of hydrophobic chain-linkage isomers, ester-linked dipalmitoylphosphatidylcholine (DPPC) and ether-linked dihexadecylphosphatidylcholine (DHPC). The thermodynamic quantities of the main transition for these PC bilayer membranes were similar to one another, whereas the suppression of the interdigitation and great stabilization of the hydrated crystal phase were observed for the DPADPC bilayer membrane. Furthermore, the results of nuclear magnetic resonance measurements indicated that the motility of PC molecules in both gel and liquid crystal phases of the DPADPC bilayer membranes were much lower than those of the DPPC and DHPC bilayer membranes. From the above results, we concluded that the molecular interaction in the PC bilayer membranes increases in the order of ether-, ester- and amide-linkage isomers.

Mechanical properties of anionic asymmetric bilayers from atomistic simulations.

Mechanotransduction, the biological response to mechanical stress, is often initiated by activation of mechanosensitive (MS) proteins upon mechanically induced deformations of the cell membrane. A current challenge in fully understanding this process is in predicting how lipid bilayers deform upon the application of mechanical stress. In this context, it is now well established that anionic lipids influence the function of many proteins. Here, we test the hypothesis that anionic lipids could indirectly modulate MS proteins by alteration of the lipid bilayer mechanical properties. Using all-atom molecular dynamics simulations, we computed the bilayer bending rigidity (KC), the area compressibility (KA), and the surface shear viscosity (ηm) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) lipid bilayers with and without phosphatidylserine (PS) or phosphatidylinositol bisphosphate (PIP2) at physiological concentrations in the lower leaflet. Tensionless leaflets were first checked for each asymmetric bilayer model, and a formula for embedding an asymmetric channel in an asymmetric bilayer is proposed. Results from two different sized bilayers show consistently that the addition of 20% surface charge in the lower leaflet of the PC bilayer with PIP2 has minimal impact on its mechanical properties, while PS reduced the bilayer bending rigidity by 22%. As a comparison, supplementing the PIP2-enriched PC membrane with 30% cholesterol, a known rigidifying steroid lipid, produces a significant increase in all three mechanical constants. Analysis of pairwise splay moduli suggests that the effect of anionic lipids on bilayer bending rigidity largely depends on the number of anionic lipid pairs formed during simulations. The potential implication of bilayer bending rigidity is discussed in the framework of MS piezo channels.

Complexes of Biopolymers with Essential Lipids: Relationships between the Structure and Functional Properties

Nanosized and water-soluble complexes have been formed between biopolymers (a covalent conjugate of sodium caseinate with maltodextrin) and essential lipids. These lipids were liposomes of soy phosphatidylcholine (PC: 59% omega-6 linoleic acid (LA); 7% omega-3 alpha-linolenic acid (ALA)) enriched by omega-3 ALA as a constituent of triglycerides of linseed oil (LO: 55% ALA; 18% LA). They were prepared without and with such plant antioxidant as an essential oil of clove bud (EOC). The basic relationships have been revealed between functional properties and the structural (size, molar mass, architecture, charge, density, structural arrangement of PC bilayers) as well as the thermodynamic (thermodynamic affinity for a solvent)parameters of the complexes. To measure these parameters we used a combination of the precise physico-chemical methods. They were the followings: a laser light scattering in static, dynamic and electrophoretic modes; an electron spin resonance spectroscopy; and an atomic force microscopy. The studied functional properties of the complexes included their water-solubility and protective ability against auto oxidation of the essential lipids encapsulated into the complexes.

Understanding Interactions of Curcumin with Lipid Bilayers: A Coarse-Grained Molecular Dynamics Study

The interactions of curcumin with various lipid bilayers (POPC, DOPC, oxidized POPC, and oxidized DOPC) and model biomembranes (symmetric bacteria and yeast plasma membranes, as well as asymmetric mammalian plasma membrane) are investigated. A nonlinear thinning effect of curcumin with respect to its concentration is demonstrated in PC membranes and in the yeast. Curcumin induces asymmetry to the symmetric yeast membranes but reduces the degree of asymmetry of the mammalian plasma membranes when the molecule is placed facing the outer leaflets. The molecule is found to diffuse through oxidized PC bilayers, POPC bilayers at a curcumin to lipid ratio C/L = 1/5, yeast membranes at C/L = 1/100, and the mammalian plasma membranes at C/L = 1/5 and when the molecule placed facing the outer leaflets. The results of this work demonstrate that the lipid type, the lipid distribution, and curcumin amount play a critical role in defining the interactions of curcumin with the lipids and their transport behavior through the bilayers.

Activity and characterization of a pH-sensitive antimicrobial peptide

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. Previous work showed that when Histidine was incorporated into the peptide C18G it lost antimicrobial activity. The role of pH on activity and biophysical properties of the peptide was investigated to explain this phenomenon. Minimal inhibitory concentration (MIC) results demonstrated that decreased media pH increased antimicrobial activity. Trichloroethanol (TCE) quenching and red-edge excitation spectroscopy (REES) showed a clear pH dependence on peptide aggregation in solution. Trp fluorescence was used to monitor binding to lipid vesicles and demonstrated the peptide binds to anionic bilayers at all pH values tested, however, binding to zwitterionic bilayers was enhanced at pH 7 and 8 (above the His pKa). Dual Quencher Analysis (DQA) confirmed the peptide inserted more deeply in PC:PG and PE:PG membranes, but could insert into PC bilayers at pH conditions above the His pKa. Bacterial membrane permeabilization assays which showed enhanced membrane permeabilization at pH 5 and 6 but vesicle leakage assays indicate enhanced permeabilization of PC and PC:PG bilayers at neutral pH. The results indicate the ionization of the His side chain affects the aggregation state of the peptide in solution and the conformation the peptide adopts when bound to bilayers, but there are likely more subtle influences of lipid composition and properties that impact the ability of the peptide to form pores in membranes.

Membrane States of Saturated Glycerophospholipids: A Thermodynamic Study of Bilayer Phase Transitions.

Bilayer membranes formed by phospholipids vary in their membrane states by undergoing phase transitions in response to various external environmental factors. Pressure is one of these important environmental factors, but there are very few studies on the effects of pressure on phospholipid bilayer membranes. It is possible to deepen our understanding of the membrane states of phospholipid bilayer membranes by combining information regarding temperature- and/or ligand-responsivity with that regarding pressure-responsivity. In this review, we thermodynamically characterize the bilayer phase transitions of three kinds of saturated glycerophospholipids, each with a different polar head group (phosphatidyl-ethanolamine (PE), -choline (PC) or -glycerol (PG)), and explain their various membrane states depending on temperature and pressure. Both temperature- and pressure-responsivity reveal inherent features of these bilayer membranes: the metastability of the gel phase for PE bilayer membranes, the polymorphism of the gel phases for PC bilayer membranes and morphological changes in bilayer aggregates for PG bilayer membranes.

Submolecular Imaging of Parallel Offset π–π Stacking in Nonplanar Phthalocyanine Bilayers

In aromatic systems, π–π interaction plays a central role in determining the stacking geometry and binding strength of molecules and thus a detailed microscopic understanding is highly desirable. Herein, by using scanning tunneling microscopy with submolecular resolution complemented with first-principles calculations based on density functional theory, we report the atomic-scale imaging of π–π interaction in nonplanar phthalocyanine (Pc) bilayers on different substrates, including graphite and Au(111) with weak interaction and Cu(111) with strong binding. We reveal that nonplanar Pc of the second layer on all substrates exhibits an in-plane rotation angle of 15° with a parallel offset of 1.19 A, which minimizes π–π repulsion. Interestingly, on Cu(111), it is found that the inequivalent charge distribution along with the alternating orientation of Pc molecules in the first layer creates a preferable anchoring site for Pc of the second layer, leading to the assembly of the √2 × √2R45° superstructure, consi...

Peptide-functionalized supported lipid bilayers to construct cell membrane mimicking interfaces.

Supported lipid bilayers (SLB) functionalized with bioactive molecules can be effectively used to study the interaction of cells with different molecules for fundamental research or to develop biosynthetic systems for various biomedical applications. In this study, RGD and Osteocalcin mimetic (OSN) peptides were used as model molecules for functionalization of otherwise passive SLBs to evaluate cell-surface interactions via real-time monitoring in quartz crystal microbalance with dissipation. Similar platforms were also used in cell culture environment. It was seen that low density of mobile RGD peptides on SLB platforms preserved their biological activity and promoted cell adhesion more efficiently than high number of immobile, physisorbed peptides. Even though nonspecific protein and cell attachment was promoted, cells did not spread well on OSN-coated control surfaces. The stability of SLBs produced with different lipids were evaluated in various medium conditions. Enrichment with different lipids increased the stability of SLB to pure PC bilayer.

Salt-induced effects on natural and inverse DPPC lipid membranes: Molecular dynamics simulation

Molecular dynamics (MD) simulations of a dipalmitoylphosphatidylcholine (DPPC) bilayer and its neutral inverse-phosphocholine equivalent (DPCPe) were performed to find salt-induced effects on their surface structure and the nature of ion-lipid interactions. We found that the area per lipid is not considerably affected by the inversion, but the deuterium order parameter of carbon atoms in the region of carbonyl carbons changes dramatically. MD simulations indicate that Ca2+ ions can bind to the surface of both DPPC and DPCPe membranes, but K+ ions do not bind to them. In the case of Na+, however, the ions can bind to natural lipids but not to the inverse ones. Also, our results demonstrate that the hydration level of CPe bilayers is substantially lower than PC bilayers and the averaged orientation of water dipoles in the region of CPe headgroups is effectively inverted compared to PC lipids. This might be important in the interaction of the bilayer with its biological environment. Furthermore, it was found for the CPe bilayers that the enhanced peaks of the electrostatic potential profiles shift further away from the bilayer center relative to those of PC bilayers. This behavior makes the penetration of cations into the bilayer more difficult and possibly explains the experimentally observed enhanced release rates of anionic compounds in the CPe membrane.

Making Soft Magnetically-Orientable Membranes: An Alternative to Bicelles

Model lipid membranes have long been employed in a variety of biophysical techniques to circumvent the complexity of natural membranes when studying their constituents or interactions. The development of magnetically-oriented bilayers, such as DMPC/DHPC bicelles, has been notably important in solid-state NMR where they facilitate the study of membrane interactions, or provide valuable information on the structure and orientation of membrane peptides. Bicelles can be prepared through PC bilayer stabilisation by a variety of detergents, providing that physicochemical requirements are met by adjusting detergent to lipid ratio, concentration, and temperature. In this work we propose an alternative to bicelles, obtained by softening PC liposomes with a gentle surfactant. We show that polysorbate 80 (Tween 80) mixed to DMPC and DPPC bilayers leads to elongated oriented structures which maintain their alignment over a wide range of lipid-to-detergent molar ratios (2<q<13) and temperatures (∼25-80oC), as demonstrated by 31P and 2H solid-state NMR. The orientation quality of these “elastosomes”, determined by spectral simulations, is comparable to traditional bicelles, although they display a different morphology. Moreover, we show that the magnetic orientation can also be flipped by addition of lanthanides, and that transmembrane peptides can be studied. The Tween-based approach for oriented model membranes preparation is promising, and it should in principle be applicable to membrane bilayers of higher complexity - including native membranes - thus bridging the gap between model and intact membranes.

Exchange of Gramicidin between Lipid Bilayers: Implications for the Mechanism of Channel Formation

The canonical mechanism of gramicidin (gA) channel formation is transmembrane dimerization of nonconducting subunits that reside in opposite bilayer leaflets. The channels do not open and close; they appear and disappear due to subunit association and dissociation. Many different types of experiments support this monomer ↔ dimer mechanism. Recently, however, this mechanism was challenged, based on experiments with lipid vesicle-incorporated gA under conditions where vesicle fusion could be controlled. In these experiments, sustained channel activity was observed long after fusion had been terminated, which led to the proposal that gA single-channel current transitions result from closed-open transitions in long-lived bilayer-spanning dimers. This proposal is at odds with 40 years of experiments, but involves the key assumption that gA monomers do not exchange between bilayers. We tested the possibility of peptide exchange between bilayers using three different types of experiments. First, we demonstrated the exchange of gA between 1,2-dierucoyl-sn-glycero-3-phosphocholine (DC22:1PC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DC18:1PC) lipid vesicles using a fluorescence assay for gA channel activity. Second, we added gA-free DC22:1PC vesicles to both sides of planar DC18:1PC bilayers preincubated with gA, which reduced channel activity up to 10-fold. Third, we added gA-containing DC22:1PC vesicles to one or both sides of DC18:1PC planar bilayers, which produced much higher channel activity when the gA-containing vesicles were added to both sides of the bilayer, as compared to one side only. All three types of experiments show that gA subunits can exchange between lipid bilayers. The exchange of subunits between bilayers thus is firmly established, which becomes a crucial consideration with respect to the mechanism of channel formation.

The mitochondrial VDAC of bean seeds recruits phosphatidylethanolamine lipids for its proper functioning

The voltage-dependent anion-selective channel (VDAC) is the main pathway for inorganic ions and metabolites through the mitochondrial outer membrane. Studies recently demonstrated that membrane lipids regulate its function. It remains, however, unclear how this regulation takes place. In this study, we show that phospholipids are key regulators of Phaseolus VDAC function and, furthermore, that the salt concentration modulates this regulation. Both selectivity and voltage dependence of Phaseolus VDAC are very sensitive to a change in the lipid polar head from PC to PE. Interestingly enough, this dependence is observed only at low salt concentration. Furthermore, significant changes in VDAC functional properties also occur with the gradual methylation of the PE group pointing to the role of subtle chemical variations in the lipid head group. The dependence of PcVDAC gating upon the introduction of a small mole fraction of PE in a PC bilayer has prompted us to propose the existence of a specific interaction site for PE on the outer surface of PcVDAC.Eventually, comparative modeling and molecular dynamics simulations suggest a potential mechanism to get insight into the anion selectivity enhancement of PcVDAC observed in PE relative to PC.

Extension of CAVS coarse-grained model to phospholipid membranes: The importance of electrostatics.

It is evident from experiment that electrostatic potential (or dipole potential) is positive inside PC or PE lipid bilayers in the absence of ions. MARTINI coarse-grained (CG) model, which has been widely used in simulating physical properties of lipid bilayers, fails to reproduce the positive value for the dipole potential in the membrane interior. Although the total dipole potential can be correctly described by the BMW/MARTINI model, the contribution from the ester dipoles, playing a nontrivial role in the electrostatic potential across lipid membranes, is neglected by this hybrid approach. In the ELBA CG model, the role of the ester dipoles is considered, but it is overweighed because various atomistic models have consistently shown that water is actually the leading contributor of dipole potential. Here, we present a CG approach by combining the BMW-like water model (namely CAVS model) with the ELBA-like lipid model proposed in this work. Our CG model was designed not only to correctly reproduce the positive values for the dipole potential inside PC and PE lipid bilayers but also to properly balance the individual contributions from the ester dipoles and water, surmounting the limitations of current CG models in the calculations of dipole potential. © 2017 Wiley Periodicals, Inc.

Scalable Synthesis and Purification of Acetylated Phosphatidyl Choline Headgroup.

The acetylated headgroup of the most abundant mammalian phospholipid, 1,2-diacetyl-3-sn-phosphatidyl choline (DAcPC), has several important applications in research. For instance, it can be dissolved in the same amount of water as in the fluid PC bilayer, to create a surrogate of a PC headgroup stratum, for studying solvation of small molecules and the influence of their structure on the process. In contrast to PC derivatives with longer acyl chains, DAcPC does not self-aggregate, rendering the aqueous solution homogeneous and suitable for simplified analyses of interactions of molecules with the headgroups. Several studies have been published where DAcPC was used in a crudely purified form. Here we describe a one-step preparation of DAcPC from commercially available bulk chemicals and purification of the product by crystallization and washing. The process gives a good yield and is easily scalable. The availability of enantiopure, crystalline DAcPC could open the door to more extensive biochemical, pharmacological, and nutritional studies of this interesting chemical.

Stability of Charged Membranes: The Role of Pore Edge Tension

Biological membranes contain a significant fraction of anionic lipids. These lipids are known to influence membrane material properties such as the bending rigidity and lysis tension. Here, we investigate the stability of giant unilamellar vesicles (GUVs) composed of mixtures of zwitterionic (PC) and anionic (PG) phospholipids when perturbed by electric pulses. Strong DC pulses cause electroporation of cells, a phenomenon that is widely used to render the cellular membrane transiently permeable. In GUVs composed of PC, electric pulses induce opening of macropores of few micrometers in diameter, which then reseal within tens of milliseconds. Strikingly, pulses applied to GUVs containing large fractions of PG can cause vesicle bursting: The created macropores never reseal and the membrane rearranges into a network of nanotubes. The probability for vesicle bursting is highly correlated with the fraction of PG in the membrane and becomes dominant above 25-30 mol% PG. The likelihood of the bursting phenomenon increases with the degree of chain unsaturation and decreases in the presence of calcium, which has a strong affinity for the phosphate group. Charged GUVs, for which the macropores reseal, usually remain permeable minutes after the pulse, showing that membrane integrity is not completely restored. Pore closure dynamics is governed by the pore edge tension, which reflects the energy penalty for reorganizing the lipids at the pore rim. We assessed this parameter from the dynamics of macropore closure for PC:PG membranes and found a three-fold decrease for membranes containing 20-50 mol% PG (20±12 pN) when compared to pure PC bilayers (60±26 pN). Interestingly, when pores are induced by the presence of a detergent, the same bursting phenomenon is observed, showing that this is a universal property of PG membranes rather than an electric-field effect.

Improved prediction of bilayer and monolayer properties using a refined BMW-MARTINI force field

Coarse-grained (CG) models allow enlarging the size and time scales that are reachable by atomistic molecular dynamics simulations. A CG force field (FF) for lipids and amino acids that possesses a polarizable water model has been developed following the MARTINI parametrization strategy, the BMW-MARTINI [1]. We tested the BMW-MARTINI FF capability to describe some structural and thermodynamical properties of lipid monolayers and bilayers. We found that, since the surface tension values of oil/water interfaces calculated with the model are not correct, compression isotherms of lipid monolayers present artifacts. Also, this FF predicts DPPC and DAPC bilayers to remain in the Lα phase at temperatures as low as 283K, contrary to the expected from their experimental Tm values. Finally, simulations at constant temperature of bilayers of saturated lipids belonging to PC homologous, showed an increase in the mean molecular area (Mma) upon increasing the chain length, inversely to the experimental observation.We refined BMW-MARTINI FF by modifying as few parameters as possible in order to bring simulated and experimental measurements closer. We have also modified structural parameters of the lipid geometry that do not have direct influence in global properties of the bilayer membranes or monolayers, but serve to approach the obtained CG geometry to atomistic reference values. The refined FF is able to better reproduce phase transition temperatures and Mma for saturated PC bilayers than BMW-MARTINI and MARTINI FF. Finally, the simulated surface pressure-Mma isotherms of PC monolayers resemble the experimental ones and eliminate serious artifacts of previous models.

The Water Permeability and Pore Entrance Structure of Aquaporin-4 Depend on Lipid Bilayer Thickness

Aquaporin-4 (AQP4), the primary water channel in glial cells of the mammalian brain, plays a critical role in water transport in the central nervous system. Previous experiments have shown that the water permeability of AQP4 depends on the cholesterol content in the lipid bilayer, but it was not clear whether changes in permeability were due to direct cholesterol-AQP4 interactions or to indirect effects caused by cholesterol-induced changes in bilayer elasticity or bilayer thickness. To determine the effects resulting only from bilayer thickness, here we use a combination of experiments and simulations to analyze AQP4 in cholesterol-free phospholipid bilayers with similar elastic properties but different hydrocarbon core thicknesses previously determined by x-ray diffraction. The channel (unit) water permeabilities of AQP4 measured by osmotic-gradient experiments were 3.5 ± 0.2 × 10−13 cm3/s (mean ± SE), 3.0 ± 0.3 × 10−13 cm3/s, 2.5 ± 0.2 × 10−13 cm3/s, and 0.9 ± 0.1 × 10−13 cm3/s in bilayers containing (C22:1)(C22:1)PC, (C20:1)(C20:1)PC, (C16:0)(C18:1)PC, and (C13:0)(C13:0)PC, respectively. Channel permeabilities obtained by molecular dynamics (MD) simulations were 3.3 ± 0.1 × 10−13 cm3/s and 2.5 ± 0.1 × 10−13 cm3/s in (C22:1)(C22:1)PC and (C14:0)(C14:0)PC bilayers, respectively. Both the osmotic-gradient and MD-simulation results indicated that AQP4 channel permeability decreased with decreasing bilayer hydrocarbon thickness. The MD simulations also suggested structural modifications in AQP4 in response to changes in bilayer thickness. Although the simulations showed no appreciable changes to the radius of the pore located in the hydrocarbon region of the bilayers, the simulations indicated that there were changes in both pore length and α-helix organization near the cytoplasmic vestibule of the channel. These structural changes, caused by mismatch between the hydrophobic length of AQP4 and the bilayer hydrocarbon thickness, could explain the observed differences in water permeability with changes in bilayer thickness.

Does Lipid Composition Regulate Anthrax Toxin Uptake?

Biological roles of lipids are not limited toproviding structural stability to bilayer membranes. Lipids have numerous functions, e.g. acting in cellular signaling and protein regulation. Cells adjust bilayer biophysical parameters by controlling lipid content and gradient of the plasma and intracellular organelle membranes. Thus, membrane composition changes along the endocytic pathway, with early endosomal membranes resembling plasma membranes, and late endosomal membranes showing dramatic increase in anionic lysobisphosphatidic acid level.Here we investigate the role of the lipid environmentin the anthrax toxin uptake. The anthrax toxin, formed by three proteins (binding protective antigen, PA, and enzymatic lethal factor, LF and edema factor, EF), follows a multistep internalization process which involves PA binding to its cellular receptors, proteolytic cleavage to PA63, oligomerization to form prepores, and LF and EF binding followed by endocytosis. The acidic endosomal environment causes conformational changes in PA63 leading to its insertion into the early endosomal membrane and ion channel formation. The delivery of LF to cytoplasm was suggested to take place later in the endocytic pathway via the back-fusion of intraluminal vesicles of late endosomes. Given the variations in membrane lipid composition of the early endosome, the intraluminal vesicles and the late endosome, it becomes important to understand if these changes influence the PA63 insertion and structural dynamics, LF and EF binding and translocation.Using bilayer lipid membrane technique, we mimic the endosomal membrane composition probing its properties in-vitro. We show that at pH 5.5, PA63 insertion rate increases dramatically in PS compared with PC bilayers, whereas LF binds ten times less effectively to PA63 in PS compared to PC. When the solution pH is raised from 5 to 6.75, this difference is diminished.

Rational modification of a dendrimeric peptide with antimicrobial activity: consequences on membrane-binding and biological properties.

Peptide-based antibiotics might help containing the rising tide of antimicrobial resistance. We developed SB056, a semi-synthetic peptide with a dimeric dendrimer scaffold, active against both Gram-negative and Gram-positive bacteria. Being the mechanism of SB056 attributed to disruption of bacterial membranes, we enhanced the amphiphilic profile of the original, empirically derived sequence [WKKIRVRLSA-NH2] by interchanging the first two residues [KWKIRVRLSA-NH2], and explored the effects of this modification on the interaction of peptide, both in linear and dimeric forms, with model membranes and on antimicrobial activity. Results obtained against Escherichia coli and Staphylococcus aureus planktonic strains, with or without salts at physiological concentrations, confirmed the added value of dendrimeric structure over the linear one, especially at physiological ionic strength, and the impact of the higher amphipathicity obtained through sequence modification on enhancing peptide performances. SB056 peptides also displayed intriguing antibiofilm properties. Staphylococcus epidermidis was the most susceptible strain in sessile form, notably to optimized linear analog lin-SB056-1 and the wild-type dendrimer den-SB056. Membrane affinity of all peptides increased with the percentage of negatively charged lipids and was less influenced by the presence of salt in the case of dendrimeric peptides. The analog lin-SB056-1 displayed the highest overall affinity, even for zwitterionic PC bilayers. Thus, in addition to electrostatics, distribution of charged/polar and hydrophobic residues along the sequence might have a significant role in driving peptide-lipid interaction. Supporting this view, dendrimeric analog den-SB056-1 retained greater membrane affinity in the presence of salt than den-SB056, despite the fact that they bear exactly the same net positive charge.