Affinity For Lipid Bilayers Research Articles

Artificial Water Channels—Progress Innovations and Prospects

Artificial Water Channels—Progress Innovations and Prospects

Investigating Antimicrobial Peptide-Membrane Interactions Using Fast Photochemical Oxidation of Peptides in Nanodiscs.

Antimicrobial peptides (AMPs) are an important part of the innate immune system and demonstrate promising applications in the fight against antibiotic-resistant infections due to their unique mechanism of targeting bacterial membranes. However, it is challenging to study the interactions of these peptides within lipid bilayers, making it difficult to understand their mechanisms of toxicity and selectivity. Here, we used fast photochemical oxidation of peptides, an irreversible footprinting technique that labels solvent accessible residues, and native charge detection-mass spectrometry to study AMP-lipid interactions with different lipid bilayer nanodiscs. We observed differences in the oxidation of two peptides, indolicidin and LL-37, in three distinct lipid environments, which reveal their affinity for lipid bilayers. Our findings suggest that indolicidin interacts with lipid head groups via a simple charge-driven mechanism, but LL-37 is more specific for Escherichia coli nanodiscs. These results provide complementary information on the potential modes of action and lipid selectivity of AMPs.

Amino acids change solute affinity for lipid bilayers

Time-resolved fluorescence and differential scanning calorimetry (DSC) were used to examine how two amino acids, L-phenylalanine (L-PA) and N-acetyl-DL-tryptophan (NAT), affect the temperature-dependent membrane affinity of two structurally similar coumarin solutes for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles. The 7-aminocoumarin solutes, coumarin 151 (C151) and coumarin 152 (C152), differ in their substitution at amine position—C151 is a primary amine, and C152 is a tertiary amine—and both solutes show different tendencies to associate with lipid bilayers consistent with differences in their respective log-P-values. Adding L-PA to the DPPC vesicle solution did not change C151’s propensity to remain freely solvated in aqueous solution, but C152 showed a greater tendency to partition into the hydrophobic bilayer interior at temperatures below DPPC’s gel-liquid crystalline transition temperature (Tgel-lc). This finding is consistent with L-PA’s ability to enhance membrane permeability by disrupting chain-chain interactions. Adding NAT to DPPC-vesicle-containing solutions changed C151 and C152 affinity for the DPPC membranes in unexpected ways. DSC data show that NAT interacts strongly with the lipid bilayer, lowering Tgel-lc by up to 2°C at concentrations of 10 mM. These effects disappear when either C151 or C152 is added to solution at concentrations below 10 μM, and Tgel-lc returns to a value consistent with unperturbed DPPC bilayers. Together with DSC results, fluorescence data imply that NAT promotes coumarin adsorption to the vesicle bilayer surface. NAT’s effects diminish above Tgel-lc and imply that unlike L-PA, NAT does not penetrate into the bilayer but instead remains adsorbed to the bilayer’s exterior. Taken in their entirety, these discoveries suggest that amino acids—and by inference, polypeptides and proteins—change solute affinity for lipid bilayers with specific effects that depend on individualized amino-acid-lipid-bilayer interactions.

Structure of Fungal α Mating Pheromone in Membrane Mimetics Suggests a Possible Role for Regulation at the Water-Membrane Interface.

Fusarium oxysporum is a highly destructive plant pathogen and an emerging pathogen of humans. Like other ascomycete fungi, F. oxysporum secretes α-pheromone, a small peptide that functions both as a chemoattractant and as a quorum-sensing signal. Three of the ten amino acid residues of α-pheromone are tryptophan, an amino acid whose sidechain has high affinity for lipid bilayers, suggesting a possible interaction with biological membranes. Here we tested the effect of different lipid environments on α-pheromone structure and function. Using spectroscopic and calorimetric approaches, we show that this peptide interacts with negatively charged model phospholipid vesicles. Fluorescence emission spectroscopy and nuclear magnetic resonance (NMR) measurements revealed a key role of the positively charged groups and Trp residues. Furthermore, NMR-based calculation of the 3D structure in the presence of micelles, formed by lipid surfactants, suggests that α-pheromone can establish an intramolecular disulfide bond between the two cysteine residues during interaction with membranes, but not in the absence of lipid mimetics. Remarkably, this oxidized version of α-pheromone lacks biological activity as a chemoattractant and quorum-sensing molecule. These results suggest the presence of a previously unidentified redox regulated control of α-pheromone activity at the surface of the plasma membrane that could influence the interaction with its cognate G-protein coupled receptor.

Manipulation of a spider peptide toxin alters its affinity for lipid bilayers and potency and selectivity for voltage-gated sodium channel subtype 1.7

Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels, and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analogue (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (NaV1.7), a channel targeted in pain management. Here, we show that replacements of the positively-charged residues that contribute to the activity of the peptide can improve gHwTx-IV's potency and selectivity for NaV1.7. Using HwTx-IV, gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human NaV1.7 and their affinity for the lipid bilayer. [R26A]gHwTx-IV consistently displayed the most improved potency and selectivity for NaV1.7, examined alongside off-target NaVs, compared with HwTx-IV and gHwTx-IV. The lipid affinity of each of the three novel analogues was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at NaV1.7 and affinity for lipid bilayers. In a murine NaV1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable with that of native HwTx-IV. In summary, this study reports the development of an HwTx-IV analogue with improved in vitro selectivity for the pain target NaV1.7 and with an in vivo efficacy similar to that of native HwTx-IV.

Gating modifier toxins isolated from spider venom: Modulation of voltage-gated sodium channels and the role of lipid membranes

Gating modifier toxins (GMTs) are venom-derived peptides isolated from spiders and other venomous creatures and modulate activity of disease-relevant voltage-gated ion channels and are therefore being pursued as therapeutic leads. The amphipathic surface profile of GMTs has prompted the proposal that some GMTs simultaneously bind to the cell membrane and voltage-gated ion channels in a trimolecular complex. Here, we examined whether there is a relationship among spider GMT amphipathicity, membrane binding, and potency or selectivity for voltage-gated sodium (NaV) channels. We used NMR spectroscopy and in silico calculations to examine the structures and physicochemical properties of a panel of nine GMTs and deployed surface plasmon resonance to measure GMT affinity for lipids putatively found in proximity to NaV channels. Electrophysiology was used to quantify GMT activity on NaV1.7, an ion channel linked to chronic pain. Selectivity of the peptides was further examined against a panel of NaV channel subtypes. We show that GMTs adsorb to the outer leaflet of anionic lipid bilayers through electrostatic interactions. We did not observe a direct correlation between GMT amphipathicity and affinity for lipid bilayers. Furthermore, GMT-lipid bilayer interactions did not correlate with potency or selectivity for NaVs. We therefore propose that increased membrane binding is unlikely to improve subtype selectivity and that the conserved amphipathic GMT surface profile is an adaptation that facilitates simultaneous modulation of multiple NaVs.

A biophysical approach to daunorubicin interaction with model membranes: relevance for the drug's biological activity

Daunorubicin is extensively used in chemotherapy for diverse types of cancer. Over the years, evidence has suggested that the mechanisms by which daunorubicin causes cytotoxic effects are also associated with interactions at the membrane level. The aim of the present work was to study the interplay between daunorubicin and mimetic membrane models composed of different ratios of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), sphingomyelin (SM) and cholesterol (Chol). Several biophysical parameters were assessed using liposomes as mimetic model membranes. Thereby, the ability of daunorubicin to partition into lipid bilayers, its apparent location within the membrane and its effect on membrane fluidity were investigated. The results showed that daunorubicin has higher affinity for lipid bilayers composed of DMPC, followed by DMPC : SM, DMPC : Chol and lastly by DMPC : SM : Chol. The addition of SM or Chol into DMPC membranes not only increases the complexity of the model membrane but also decreases its fluidity, which, in turn, reduces the amount of anticancer drug that can partition into these mimetic models. Fluorescence quenching studies suggest a broad distribution of the drug across the bilayer thickness, with a preferential location in the phospholipid tails. The gathered data support that daunorubicin permeates all types of membranes to different degrees, interacts with phospholipids through electrostatic and hydrophobic bonds and causes alterations in the biophysical properties of the bilayers, namely in membrane fluidity. In fact, a decrease in membrane fluidity can be observed in the acyl region of the phospholipids. Ultimately, such outcomes can be correlated with daunorubicin's biological action, where membrane structure and lipid composition have an important role. In fact, the results indicate that the intercalation of daunorubicin between the phospholipids can also take place in rigid domains, such as rafts that are known to be involved in different receptor processes, which are important for cellular function.

Structure-Activity Relationship Studies Reveal that the Spider Toxin Protx-II has Unusual Membrane-Binding Properties and Inhibits NAV1.7 Channel at the Membrane Surface

ProTx-II, a peptide toxin isolated from spider venom, has large potential as a pain therapeutic. This disulfide-rich peptide inhibits the membrane voltage-gated sodium channel Nav 1.7, a recognized therapeutic target to treat pain, through binding to the voltage-sensor domains of the channel inserted in the membrane. The exact peptide-channel binding site and the importance of the membrane in its inhibition activity remain unknown. In the current study we examined the structure and the membrane-binding properties of ProTx-II, and of a series of analogues, using NMR spectroscopy, surface plasmon resonance and fluorescence spectroscopy methodologies. Our studies reveal that ProTx-II has an amphipathic surface and high affinity for lipid bilayers with preference for negatively-charged lipids. Interestingly, despite of each of the four Trp residues being essential for its membrane-binding properties, the peptide has a shallow location in the bilayer. Furthermore, contrary to other membrane-active peptides, replacement of Lys with Arg residues decreases the membrane-binding properties of ProTx-II. Our studies also suggest that sphingomyelinase D, an enzyme present in spider venoms that converts the neutral sphingomyelin into a negatively-charged lipid, increases the ability of ProTx-II to bind to membranes that mimic raft-like domains where the ion channels are located. Overall these results suggest: 1) ProTx-II interacts with Nav 1.7 voltage sensor domain at the membrane surface; 2) sphingomyelinase D in spider venoms increases the concentration of positively-charged toxins in the channel vicinity by converting the neutral raft-domains into negatively-charged surfaces.

Not only oxidation of cardiolipin affects the affinity of cytochrome C for lipid bilayers.

Fluorescence quenching of lipid-bound pyrene was used to assess the binding of cytochrome c (cyt c) to liposomes that mimic the inner mitochondrial membrane (IMM) POPC/DOPE/TOCL, with the conditions that it did or did not contain oxidized phosphatidylcholine molecules, i.e., 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), or a mixture of two hydroperoxide isomers derived from POPC (POPCOX). The binding isotherms reveal two dissociation constants, K(D)(1) and K(D)(2), representing, respectively, the low- and high-affinity states of the membrane. These dissociation constants probably are due to the lipid reorganization promoted by cyt c, as observed in giant unilamellar vesicles that contain fluorescent cardiolipin (CL). The presence of PazePC, which has a nonreactive carboxylic group, increased the K(D)(1) and K(D)(2) values 1.2- and 4.5-fold, respectively. The presence of POPCOX which has a reactive peroxide group, decreased the K(D)(1) value 1.5-fold, increased the K(D)(2) value 10-fold, and significantly reduced the salt-induced detachment of cyt c. MALDI-TOF spectrometry analysis of cyt c incubated with liposomes containing POPCox demonstrated a mass increase corresponding to the formation of nonenal adducts as hydrophobic anchors. Electronic absorption spectroscopy, circular dichroism, and magnetic circular dichroism demonstrated that all of the lipids studied promoted changes in the cyt c coordination sphere. Therefore, in the presence of CL, the oxidation of zwitterionic lipids also promotes changes in the cyt c structure and in the affinity for lipid bilayers.

Structure- Function Relationship Studies of Piscidin 1 Bound to Model Tumor Cell Membranes

Piscidins, which are antimicrobial peptides (AMPs) produced in the mast cells of hybrid striped bass, are found in immune-related organs as well as non-immune tissues, including neuronal cell bodies. While they are unstructured in water, they form amphipathic, cationic and alpha-helical structure in the presence of lipid bilayers at physiological pH. The biologically most active piscidin, piscidin 1 (P1), has broad spectrum antimicrobial activity against various bacteria, fungi, and viruses. Recently, it was found to be selective against HT1080 and HeLa cells as compared to healthy cells. This research investigates the factors that imparts P1 this selectivity against cancer cells. We have been particularly interested in special chemical properties of cancer cells, such as their low extracellular pH, presence of phosphatidylserine (PS) in the outer membrane leaflet, and elevated cholesterol level. We hypothesize that the positive charges on the histidine residues of P1 at the low extracellular pH of cancer cells allow P1 to align parallel to the surface and disrupt the membrane via the carpet-like mechanism of action.Lipid membranes have been made using 2:2:1 phosphatidylcholine/phoshatidylserine/cholesterol to mimic tumor cells. Circular dichroism experiments were performed using large unilamellar vesicles to investigate the effect of pH, cholesterol, and lipid charge on the peptide's helical content and therefore affinity for lipid bilayers. Using 2H and 15N solid-state NMR, we have investigated the fluidity of the lipid bilayers and bilayer orientation of P1, respectively. These results are contrasted with those obtained on healthy mammalian cell mimics. The principles learned from these studies could help design peptides with enhanced activity against cancer cells.

Importance of the Cell Membrane on the Mechanism of Action of Cyclotides

Their distinctive structures, diverse range of bioactivities, and potential for pharmaceutical or agricultural applications make cyclotides an intriguing family of cyclic peptides. Together with the physiological role in plant host defense, cyclotides possess antimicrobial, anticancer, and anti-HIV activities. In all of the reported activities, cell membranes seem to be the primary target for cyclotide binding. This article examines recent literature on cyclotide-membrane studies and highlights the hypothesis that the activity of cyclotides is dependent on their affinity for lipid bilayers and enhanced by the presence of specific lipids, i.e., phospholipids containing phosphatidylethanolamine headgroups. There is growing evidence that the lipid composition of target cell membranes dictates the amount of cyclotides bound to the cell and the extent of their activity. After membrane targeting and insertion in the bilayer core, cyclotides induce disruption of membranes by a pore formation mechanism. This proposed mechanism of action is supported by biophysical studies with model membranes and by studies on natural biological membranes of known lipid compositions.

On the role of anionic lipids in charged protein interactions with membranes

We investigate the role of anionic lipids in the binding to, and subsequent movement of charged protein groups in lipid membranes, to help understand the role of membrane composition in all membrane-active protein sequences. We demonstrate a small effect of phosphatidylglycerol (PG) lipids on the ability of an arginine (Arg) side chain to bind to, and cross a lipid membrane, despite possessing a neutralizing charge. We observe similar membrane deformations in lipid bilayers composed of phosphatidylcholine (PC) and PC/PG mixtures, with comparable numbers of water and lipid head groups pulled into the bilayer hydrocarbon core, and prohibitively large ~20kcal/mol barriers for Arg transfer across each bilayer, dropping by just 2–3kcal/mol due to the binding of PG lipids. We explore the causes of this small effect of introducing PG lipids and offer an explanation in terms of the limited membrane interaction for the choline groups of PC lipids bound to the translocating ion. Our calculations reveal a surprising lack of preference for Arg binding to PG lipids themselves, but a small increase in interfacial binding affinity for lipid bilayers containing PG lipids. These results help to explain the nature of competitive lipid binding to charged protein sequences, with implications for a wide range of membrane binding domains and cell perturbing peptides.

Effect of Fengycin, a Lipopeptide Produced by Bacillus subtilis, on Model Biomembranes

Fengycin is a biologically active lipopeptide produced by several Bacillus subtilis strains. The lipopeptide is known to develop antifungal activity against filamentous fungi and to have hemolytic activity 40-fold lower than that of surfactin, another lipopeptide produced by B. subtilis. The aim of this work is to use complementary biophysical techniques to reveal the mechanism of membrane perturbation by fengycin. These include: 1), the Langmuir trough technique in combination with Brewster angle microscopy to study the lipopeptide penetration into monolayers; 2), ellipsometry to investigate the adsorption of fengycin onto supported lipid bilayers; 3), differential scanning calorimetry to determine the thermotropic properties of lipid bilayers in the presence of fengycin; and 4), cryogenic transmission electron microscopy, which provides information on the structural organization of the lipid/lipopeptide system. From these experiments, the mechanism of fengycin action appears to be based on a two-state transition controlled by the lipopeptide concentration. One state is the monomeric, not deeply anchored and nonperturbing lipopeptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity. The mechanism, thus, appears to be driven mainly by the physicochemical properties of the lipopeptide, i.e., its amphiphilic character and affinity for lipid bilayers.

Binding of bovine seminal plasma protein BSP-A1/-A2 to model membranes: Lipid specificity and effect of the temperature

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins. The affinity of the protein BSP-A1/-A2 for lipid membranes composed of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), and POPC containing 30% (mol/mol) 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) or cholesterol, has been investigated by the isothermal titration calorimetry (ITC). This study confirms the association of these proteins to lipid bilayers, and provides a direct characterization of this exothermic process, at 37 °C. The measurements indicate that the protein affinity for lipid bilayers is modulated by the lipid composition, the lipid/protein ratio, and the temperature. The saturation lipid/protein ratio was increased in the presence of cholesterol and, to a lesser extent, of phosphatidylethanolamine, suggesting that it is modulated by the lipid acyl chain order. For all the investigated systems, the binding of BSP-A1/-A2 could not be modeled using a simple partitioning of the proteins between the aqueous and lipid phases. The existence of "binding sites", and lipid phase separations is discussed. The decrease of temperature, from 37 to 10 °C, converts the exothermic association of the proteins to the POPC bilayers to an endothermic process. A complementary 1-D and 2-D infrared spectroscopy study excludes the thermal denaturation of BSP-A1/-A2 as a contributor in the temperature dependence of the protein affinity for lipid bilayers. The reported findings suggest that changes in the affinity of BSP-A1/-A2 for lipid bilayers could be involved in modulating the association of these proteins to sperm membranes as a function of space and time; this would consequently modulate the extent of lipid extraction, including cholesterol, at a given place and given time.

Effects of external factors on the interaction of tea catechins with lipid bilayers.

Green tea contains a high concentration of such catechins as (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Their biological activities have been evaluated by in vitro experiments using cultured cells or bacteria, but the order of activity of the various catechins differed with the study. We have been studying the interaction of tea catechins with lipid bilayers, and clarified that the number of hydroxyl groups on the B-ring, the presence of the galloyl moiety, and the stereochemical structure of each catechin govern their affinity for lipid bilayers. We investigated in this present study the effects of various external factors on the affinity of tea catechins for lipid bilayers by using liposomes as model membranes. The amount of tea catechins incorporated into the lipid bilayers increased with increasing salt concentration in an aqueous medium and decreased with increasing negative electric charge of the lipid bilayers. Furthermore, the amount of EGCg or ECg incorporated into the lipid bilayers increased with increasing EC concentration. These results reveal that the salt concentration in an aqueous medium, the electric charge of the membrane, and the presence of other catechins governed the affinity of tea catechins for the lipid bilayers.

Steric effects on interaction of tea catechins with lipid bilayers.

Interaction of tea catechins with lipid bilayers has been investigated with liposome systems. Tea catechins are classified into cis-type and trans-type from the configuration of the two hydrogens at the 2 and 3 positions on the C-ring. The amount of trans-type catechins incorporated into liposomes was less than that of the respective cis-type catechins. Furthermore, the order of the partition coefficients of catechins in an n-octanol/PBS system is the same as that of the amount incorporated into liposomes. These results indicate that in addition to the number of hydroxyl groups on the B-ring and the presence of the galloyl moiety, the stereochemical structure of the C-ring also governs the hydrophobicity and the affinity for lipid bilayers. Trans-type catechins with the galloyl moiety were located on the surface of the lipid bilayer, as well as cis-type catechins with the galloyl moiety, and perturbed the membrane structure. These different stereochemical structures should influence the affinity for lipid bilayers, the alteration of membrane structures, and the difference in the order of the biological activities.

Common properties of fusion peptides from diverse systems.

Although membrane fusion occurs ubiquitously and continuously in alleukaroytic cells, little is known about the mechanism that governs lipidbilayer fusion associated with any intracellular fusion reactions. Recentstudies of the fusion of enveloped viruses with host cell membranes havehelped to define the fusion process. The identification and characterizationof key proteins involved in fusion reactions have mainly driven recent advancesin our understanding of membrane fusion. The most important denominator amongthe fusion proteins is the fusion peptide. In this review, work done in thelast few years on the molecular mechanism of viral membrane fusion will behighlighted, focusing in particular on the role of the fusion peptide and themodification of the lipid bilayer structure. Much of what is known regardingthe molecular mechanism of viral membrane fusion has been gained using liposomesas model systems in which the molecular components of the membrane and the environmentare strictly controlled. Many amphilphilic peptides have a high affinity forlipid bilayers, but only a few sequences are able to induce membrane fusion. Thepresence of α-helical structure in at least part of the fusion peptideis strongly correlated with activity whereas, β-structure tends to beless prevalent, associated with non-native experimental conditions, and morerelated to vesicle aggregation than fusion. The specific angle of insertionof the peptides into the membrane plane is also found to be an importantcharacteristic for the fusion process. A shallow penetration, extending onlyto the central aliphatic core region, is likely responsible for the destabilization ofthe lipids required for coalescence of the apposing membranes and fusion.

Interactions between HIV-1 gp41 Core and Detergents and Their Implications for Membrane Fusion

The gp41 envelope protein mediates entry of human immunodeficiency virus type 1 (HIV-1) into the cell by promoting membrane fusion. The crystal structure of a gp41 ectodomain core in its fusion-active state is a six-helix bundle in which a N-terminal trimeric coiled coil is surrounded by three C-terminal outer helices in an antiparallel orientation. Here we demonstrate that the N34(L6)C28 model of the gp41 core is stabilized by interaction with the ionic detergent sodium dodecyl sulfate (SDS) or the nonionic detergent n-octyl-beta-D-glucopyranoside (betaOG). The high resolution x-ray structures of N34(L6)C28 crystallized from two different detergent micellar media reveal a six-helix bundle conformation very similar to that of the molecule in water. Moreover, N34(L6)C28 adopts a highly alpha-helical conformation in lipid vesicles. Taken together, these results suggest that the six-helix bundle of the gp41 core displays substantial affinity for lipid bilayers rather than unfolding in the membrane environment. This characteristic may be important for formation of the fusion-active gp41 core structure and close apposition of the viral and cellular membranes for fusion.

Affinity of polyphenols for lipid bilayers.

Interaction of tea catechins with lipid bilayers has been investigated with liposome systems. Epicatechin gallate had the highest affinity for lipid bilayers, followed by epigallocatechin gallate, epicatechin, and epigallocatechin. Epicatechin gallate and epigallocatechin gallate in the surface of lipid bilayer perturbed the membrane structure.

Interaction of tea catechins with lipid bilayers investigated with liposome systems.

Interaction of tea catechins with lipid bilayers was investigated with liposome systems, which enabled us to separate liposomes from the external medium by centrifugation. We found that epicatechin gallate had the highest affinity for lipid bilayers, followed by epigallocatechin gallate, epicatechin, and epigallocatechin. Epicatechin gallate and epigallocatechin gallate in the surface of lipid bilayer perturbed the membrane structure.