Phosphatidylcholine Bilayers Research Articles

Interaction of meloxicam derivatives with phosphatidylcholine bilayers: A calorimetric study.

The drug interactions with the lipid membranes are crucial in many biochemical processes. Phospholipid model membranes are often used to assess such interactions. Our team has been researching new compounds with anti-inflammatory and analgesic effects for many years. Such compounds are derivatives of the well-known non-steroidal anti-inflammatory drug (NSAID) - meloxicam (MLX). Their biological target is cyclooxygenase (COX) - a membrane protein. The NSAIDs are mainly taken orally; therefore, drug-membrane interaction is a preliminary stage in the body. The purpose of the present work was to investigate the ability of 2 new MLX derivatives (compound PR51 and PR52) to interact with model membranes, in comparison to known NSAIDs medicine - MLX. The differential scanning calorimetry (DSC) method was used to study those interactions. As a model membrane, bilayers obtained from a phospholipid (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)) were used. Calorimetric measurements were performed using a differential scanning calorimeter DSC 214 Polyma equipped with an intracooler IC70. All examined compounds decreased the main transition temperature of DPPC in a concentration-dependent manner. The addition of studied compounds to DPPC also resulted in broadening of the transition peaks. Moreover, all examined compounds decreased the enthalpy of the DPPC main phase transition. For all DPPC gel-liquid crystalline phase transition parameters, the most pronounced effects were found for PR51 compound. We have shown that the above interactions depend on the chemical structure of individual compound. All studied compounds alter biophysical properties of phospholipid bilayer.

A molecular mechanism for how pressure induces interdigitation of phospholipid bilayer membranes

The phase transition from the ripple gel phase to the interdigitated gel phase of bilayers of phosphatidylcholines (PCs) with two saturated long-chain fatty acids under high pressure was investigated by pressure-scanning microscopy, fluorometry, and dynamic light scattering (DLS) measurements. Microscopic observation for giant vesicles (GVs) of distearoyl-PC (DSPC) under high pressure showed that spherical GVs transforms significantly into warped and distorted spherical ones instantaneously at the pressure-induced interdigitation. The fluorescence intensities of amphiphilic probe Prodan and hydrophobic probe Laurdan in the dipalmitoyl-PC (DPPC) bilayer steeply decreased and increased, respectively, at the interdigitation, suggesting that the conformational change of the polar head group of DPPC molecule in the bilayer transiently occurred at the interdigitation. Further, it was found from the high-pressure DLS measurements that the size of the vesicle particles of the DPPC and DSPC transiently increases near the interdigitation pressure, whereas the chemically induced interdigitation by adding ethanol to the DSPC bilayer membrane under atmospheric pressure produce no such change in the particle size. Taking account of the critical packing parameter of the PC molecule, the above experimental results would lead us to the conclusion that the pressure-induced interdigitation is attributable to the increase in repulsive interaction between the polar head groups of the PC molecules resulting from the orientational change of the head group from a parallel alignment to a perpendicular one with respect to the bilayer surface by applying pressure, namely the transient state: it occurs when the repulsive interaction exceeds a threshold value for the balance between the repulsive interaction and the attractive interaction among the hydrophobic acyl chains.

Masks As a New Hotspot for Antibiotic Resistance Gene Spread: Reveal the Contribution of Atmospheric Pollutants and Potential Risks.

The consumption of disposable surgical masks (DSMs) considerably increased during the coronavirus pandemic in 2019. Herein, we explored the spread of antibiotic resistance genes (ARGs) and the potential risks of antibiotic resistant bacteria (ARB) on DSMs. At environmentally relevant concentrations, the conjugate transfer frequency (CTF) of ARGs increased by 1.34-2.37 folds by 20 μg/m3 of atmospheric water-soluble inorganic ions (WSIIs), and it increased by 2.62-2.86 folds by 80 ng/m3 of polycyclic aromatic hydrocarbons (PAHs). Total suspended particulates (TSP) further promoted the CTF in combination with WSIIs or PAHs. Under WSII and PAH exposure, gene expression levels related to oxidative stress, cell membrane, and the adenosine triphosphate (ATP) were upregulated. WSIIs predominantly induced cellular contact, while PAHs triggered ATP formation and membrane damage. Molecular dynamics simulations showed that WSIIs and PAHs reduced membrane lipid fluidity and increased membrane permeability through interactions with the phosphatidylcholine bilayer. DSM filtering performance decreased, and the CTF of ARGs increased with the wearing time. The gut simulator test showed that ARB disrupted the human gut microbial community and increased total ARG abundance but did not change the ARG abundance carried by ARB themselves. A mathematical model showed that long-term WSII and PAH exposure accelerated ARG dissemination in DSMs.

Unravelling the interactions between small molecules and liposomal bilayers via molecular dynamics and thermodynamic modelling

Lipid-based drug delivery systems hold immense promise in addressing critical medical needs, from cancer and neurodegenerative diseases to infectious diseases. By encapsulating active pharmaceutical ingredients – ranging from small molecule drugs to proteins and nucleic acids – these nanocarriers enhance treatment efficacy and safety. However, their commercial success faces hurdles, such as the lack of a systematic design approach and the issues related to scalability and reproducibility.This work aims to provide insights into the drug-phospholipid interaction by combining molecular dynamic simulations and thermodynamic modelling techniques. In particular, we have made a connection between the structural properties of the drug-phospholipid system and the physicochemical performance of the drug-loaded liposomal nanoformulations. We have considered two prototypical drugs, felodipine (FEL) and naproxen (NPX), and one model hydrogenated soy phosphatidylcholine (HSPC) bilayer membrane. Molecular dynamic simulations revealed which regions within the phospholipid bilayers are most and least favoured by the drug molecules. NPX tends to reside at the water-phospholipid interface and is characterized by a lower free energy barrier for bilayer membrane permeation. Meanwhile, FEL prefers to sit within the hydrophobic tails of the phospholipids and is characterized by a higher free energy barrier for membrane permeation. Flory-Huggins thermodynamic modelling, small angle X-ray scattering, dynamic light scattering, TEM, and drug release studies of these liposomal nanoformulations confirmed this drug-phospholipid structural difference. The naproxen-phospholipid system has a lower free energy barrier for permeation, higher drug miscibility with the bilayer, larger liposomal nanoparticle size, and faster drug release in the aqueous medium than felodipine. We suggest that this combination of molecular dynamics and thermodynamics approach may offer a new tool for designing and developing lipid-based nanocarriers for unmet medical applications.

Biomimetic nanoplatforms constructed from dialkylaminostyryl hetarene dyes and phospholipids exhibiting selective fluorescent response to specific proteins

The present work explores the specificity of supramolecular assemblies comprising dialkylaminostyrylhetarene dye molecules incorporated into phosphatidylcholine (PC) or phosphatidylserine (PS) aggregates. In PS-based assemblies, the dyes demonstrate a concentration-dependent fluorescent response, distinguishing anionic proteins such as bovine serum albumin (BSA) and pepsin from lysozyme (LYZ) in aqueous solutions. Conversely, no significant response is observed when the dyes are incorporated into the well-organized bilayers of neutral PC. The fluorescent response arises from the binding of dyes to proteins, leading to the detachment of dye molecules from the assemblies, rather than from the binding of proteins to the assemblies, although the latter process is facilitated by electrostatic attraction. Thus, both the poor ordering of PS molecules and the interfacial arrangement of the dyes are prerequisites for the fluorescent response of dye-PS aggregates. The structure of the dyes significantly impacts the spectral features of dye-PS and dye-protein assemblies. An optimal dye structure has been identified for the recognition of BSA, with a limit of detection (LOD) of 10.8 nM.

Exogenous polyserine fibrils change membrane properties of phosphatidylcholine-liposome and red blood cells

The causative genes for neurodegenerative polyglutamine (polyQ) diseases produce homopolymeric polyglutamine (polyQ), polyserine (polyS), polyalanine (polyA), polycysteine (polyC), and polyleucine (polyL) sequences by repeat-associated non-AUG (RAN) translation. The cytotoxicity of the intracellular polyQ and RAN products has been extensively investigated. However, little is known about the toxicity of the extracellular polyQ and RAN products on the membranes of viable cells. Because polyQ aggregates induce a deflated morphology of a model membrane, we hypothesized that extracellular polyQ and RAN products might affect the membrane properties of viable cells. In this study, we demonstrated that exogenous polyS fibrils but not polyS or polyQ non-fibril aggregates altered the thermal phase transition behavior of a model membrane composed of a phosphatidylcholine bilayer using differential scanning calorimetry. PolyS fibrils induced morphological changes in viable red blood cells (RBCs). However, both polyS and polyQ non-fibril aggregates had no effects on RBCs. These results highlight the possibility that extracellular fibrils generated from RAN products may alter the properties of neuronal cell membranes, which may contribute to changes in the brain pathology.

Glycerophospholipid polyunsaturation modulates resveratrol action on biomimetic membranes

The phytoalexin resveratrol has received increasing attention for its potential to prevent oxidative damages in human organism. To shed further light on molecular mechanisms of its interaction with lipid membranes we study resveratrol influence on the organisation and mechanical properties of biomimetic lipid systems composed of synthetic phosphatidylcholines with mixed aliphatic chains and different degree of unsaturation at sn-2 position (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC, and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, PDPC). High-sensitivity isothermal titration calorimetric measurements reveal stronger spontaneous resveratrol association to polyunsaturated phosphatidylcholine bilayers compared to the monounsaturated ones resulting from hydrophobic interactions, conformational changes of the interacting species and desolvation of molecular surfaces. The latter is supported by the results from Laurdan spectroscopy of large unilamellar vesicles providing data on hydration at the glycerol backbones of glycerophospholipides. Higher degree of lipid order is reported for POPC membranes compared to PDPC. While resveratrol mostly enhances the hydration of PDPC membranes, increasing POPC dehydration is reported upon treatment with the polyphenol. Dehydration of the polyunsaturated lipid bilayers is measured only at the highest phytoalexin content studied (resveratrol/lipid 0.5 mol/mol) and is less pronounced than the effect reported for POPC membranes. The polyphenol effect on membrane mechanics is probed by thermal shape fluctuation analysis of quasispherical giant unilamellar vesicles. Markedly different trend of the bending elasticity with increasing resveratrol concentration is reported for the two types of phospholipid bilayers studied. POPC membranes become more rigid in the presence of resveratrol, whereas PDPC-containing bilayers exhibit softening at lower concentrations of the polyphenol followed by a slight growth without bilayer stiffening even at the highest resveratrol content explored. The new data on the structural organization and membrane properties of resveratrol-treated phosphatidylcholine membranes may underpin the development of future liposomal applications of the polyphenol in medicinal chemistry.

Liposome Encapsulation of the Palmitoyl-KTTKS Peptide: Structural and Functional Characterization.

In this study, the amphiphilic N-palmitoyl-KTTKS peptide was integrated in the bilayer of egg-derived phosphatidylcholine (PC) vesicles using two different preparation methods, namely thin-film evaporation (TLE) and reverse-phase evaporation (REV). Both the REV and TLE methods allowed for the formation of homogeneous liposome dispersions (PdI < 0.20) with mean hydrodynamic diameters of <100 nm and <200 nm, respectively, a net negative surface charge and a percentage of structured phospholipids higher than 90%. The inclusion of the amphiphilic N-palmitoyl-KTTKS peptide within phospholipid-based vesicles could improve peptide stability and skin delivery. Therefore, the obtained liposomes were evaluated via experiments assessing the synthesis of collagen and the ECM in 3T3-NIH fibroblasts. The obtained results showed that, when delivered with PC liposomes, pal-KTTKS stimulated collagen production more than free pentapeptide and 1 mM ascorbic acid, used as a positive control.

Thermodynamic study on hydrated bilayers of ether-linked phosphatidylcholines with terminal perfluorobutyl group

Novel terminally perfluorobutyl group-containing ether-linked phosphatidylcholines with different alkyl chain lengths (di-O-F4-Cn-PCs, n = 14,16 and 18) were developed as possible materials for stable liposomes aiming at applications of structural and functional analyses of membrane proteins. Differential scanning calorimetric investigations of the thermotropic transition of hydrated di-O-F4-Cn-PC bilayers demonstrated that the transition temperature of every di-O-F4-Cn-PC decreases by ~20 °C compared to their corresponding non-fluorinated PCs, di-O-Cn-PCs. With the elongation of the hydrophobic chain, on the other hand, the transition enthalpy (ΔH) and entropy (ΔS) increased in a linear manner. Comparison of ΔH and ΔS values against the net hydrocarbon chain length between di-O-F4-Cn-PCs and di-O-Cn-PCs strongly suggests that in the thermotropic transition of the di-O-F4-Cn-PC membrane, the perfluorobutyl segments undergo very limited structural changes; therefore, the hydrocarbon segments are mainly responsible for the phase transition.

LASER INDUCED CONFORMATIONAL CHANGES OF LIPIDS WITHIN MULTILAMELLAR VESICLES

"The biomedical potential of destabilizing liposomes through photoinduction relies on the use of near-infrared light, which offers inherent therapeutic advantages. Researchers have explored the effects of infrared laser light on dipalmitoyl phosphatidylcholine (DPPC) multilamellar vesicles, specifically investigating the interaction between the laser and zwitterionic dipalmitoyl phosphatidylcholine multilamellar vesicles using Fourier transform infrared spectroscopy and differential scanning calorimetry measurements. The results revealed that laser irradiation increased the number of gauche conformers and led to conformational changes within the acyl chains of the phospholipids. The transition temperature of lyophilized vesicles was also shifted to a lower temperature after laser irradiation, indicating that the laser had a dipalmitoyl phosphatidylcholine multilamellar significant effect on the acyl chains in the dipalmitoyl phosphatidylcholine bilayers and decreased the transition cooperativity of lipid acyl chains. These findings could aid in the development of more effective liposomal drug delivery systems by enhancing our understanding of the interaction between laser and lipid bilayers."

Photomanipulation of Minimal Synthetic Cells: Area Increase, Softening, and Interleaflet Coupling of Membrane Models Doped with Azobenzene-Lipid Photoswitches.

Light can effectively interrogate biological systems in a reversible and physiologically compatible manner with high spatiotemporal precision. Understanding the biophysics of photo-induced processes in bio-systems is crucial for achieving relevant clinical applications. Employing membranes doped with the photolipid azobenzene-phosphatidylcholine (azo-PC), a holistic picture of light-triggered changes in membrane kinetics, morphology, and material properties obtained from correlative studies on cell-sized vesicles, Langmuir monolayers, supported lipid bilayers, and molecular dynamics simulations is provided. Light-induced membrane area increases as high as ≈25% and a ten-fold decrease in the membrane bending rigidity is observed upon trans-to-cis azo-PC isomerization associated with membrane leaflet coupling and molecular curvature changes. Vesicle electrodeformation measurements and atomic force microscopy reveal that trans azo-PC bilayers are thicker than palmitoyl-oleoyl phosphatidylcholine (POPC) bilayers but have higher specific membrane capacitance and dielectric constant suggesting an increased ability to store electric charges across the membrane. Lastly, incubating POPC vesicles with azo-PC solutions results in the insertion of azo-PC in the membrane enabling them to become photoresponsive. All these results demonstrate that light can be used to finely manipulate the shape, mechanical and electric properties of photolipid-doped minimal cell models, and liposomal drug carriers, thus, presenting a promising therapeutic alternative for the repair of cellular disorders.

L-Phenylalanine Partitioning Mechanisms in Model Biological Membranes.

Time-resolved fluorescence spectroscopy in combination with differential scanning calorimetry (DSC) was used to study the chemical interactions that occur when l-phenylalanine is introduced to solutions containing phosphatidylcholine vesicles. Studies reported in this work address open questions about l-Phe's affinity for lipid vesicle bilayers, the effects of l-Phe partitioning on bilayer properties, l-Phe's solvation within a lipid bilayer, and the amount of l-Phe within that local solvation environment. DSC data show that l-Phe reduces the amount of heat necessary to melt saturated phosphatidylcholine bilayers from their gel to liquid-crystalline state but does not change the transition temperature (Tgel-lc). Time-resolved emission shows only a single l-Phe lifetime at low temperatures corresponding to l-Phe remaining solvated in aqueous solution. At temperatures close to Tgel-lc, a second, shorter lifetime appears that is assigned to l-Phe already embedded within the membrane that becomes hydrated as water starts to permeate the lipid bilayer. This new lifetime is attributed to a conformationally restricted rotamer in the bilayer's polar headgroup region and accounts for up to 30% of the emission amplitude. Results reported for dipalmitoylphosphatidylcholine (DPPC, 16:0) lipid vesicles prove to be general, with similar effects observed for dimyristoylphosphatidylcholine (DMPC, 14:0) and distearoylphosphatidylcholine (DSPC, 18:0) vesicles. Taken together, these results create a complete and compelling picture of how l-Phe associates with model biological membranes. Furthermore, this approach to examining amino acid partitioning into membranes and the resulting solvation forces points to new strategies for studying the structure and chemistry of membrane-soluble peptides and selected membrane proteins.

Fine-Tuning and Enhancement of pH-Dependent Membrane Permeation of Cyclic Peptides by Utilizing Noncanonical Amino Acids with Extended Side Chains.

The development of cyclic peptides that exhibit pH-sensitive membrane permeation is a promising strategy for tissue-selective drug delivery. We investigated the pH-dependent interactions of designed cyclic peptides bearing noncanonical amino acids of long acidic side chains with lipid membranes, including surface binding, insertion, and translocation across the membrane. As the length of the side chain of acidic amino acid increased, the binding affinity of the peptides to phosphatidylcholine bilayer surfaces decreased, while the pH for the 50% insertion of the peptides into the bilayers increased. The pH for membrane permeation of the peptides increased with the side chain length, resulting in specific membrane permeation at pH ∼6.5. The longer side chain of acidic amino acids improved the maximum rate of membrane permeation at low pH, where both entropic and enthalpic contributions affected the permeation. Our peptide also showed intracellular delivery of cargo molecules into living cells in a pH-dependent manner.

Calorimetric, volumetric and structural studies of the interaction between chlorogenic acid and dipalmitoylphosphatidylcholine bilayers

Chlorogenic acid (CGA) is the main component of coffee and an antioxidant. CGA has been reported to bear various good health effects. At the same time, it has been found that the addition of CGA induces an undesirable deformation of red blood cells. This fact suggests that CGA may bind to the proteins or/and membrane lipids of red blood cells. This study aimed to examine how CGA binds the bilayers of phosphatidylcholine (PC), one of red blood cells' primary lipids. To this end, we investigated the effect of CGA on the phase behavior and the structure of dipalmitoyl-PC (DPPC) bilayers in the form of multi-lamellar vesicles. Calorimetry and dilatometry measurements showed that the DPPC chain melting transition cooperativity decreases as increasing CGA concentrations. In addition, X-ray diffraction results showed that the lamellar repeat periodicity becomes disordered, and the periodicity disappears completely at high CGA concentrations. Together with these findings, it can be inferred that the CGA molecules do not penetrate inside the DPPC bilayers but bind to their surface in a negatively charged form.

Phthalates Promote Dissemination of Antibiotic Resistance Genes: An Overlooked Environmental Risk.

Plastics-microorganism interactions have aroused growing environmental and ecological concerns. However, previous studies concentrated mainly on the direct interactions and paid little attention to the ecotoxicology effects of phthalates (PAEs), a common plastic additive that is continuously released and accumulates in the environment. Here, we provide insights into the impacts of PAEs on the dissemination of antibiotic resistance genes (ARGs) among environmental microorganisms. Dimethyl phthalate (DMP, a model PAE) at environmentally relevant concentrations (2-50 μg/L) significantly boosted the plasmid-mediated conjugation transfer of ARGs among intrageneric, intergeneric, and wastewater microbiota by up to 3.82, 4.96, and 4.77 times, respectively. The experimental and molecular dynamics simulation results unveil a strong interaction between the DMP molecules and phosphatidylcholine bilayer of the cell membrane, which lowers the membrane lipid fluidity and increases the membrane permeability to favor transfer of ARGs. In addition, the increased reactive oxygen species generation and conjugation-associated gene overexpression under DMP stress also contribute to the increased gene transfer. This study provides fundamental knowledge of the PAE-bacteria interactions to broaden our understanding of the environmental and ecological risks of plastics, especially in niches with colonized microbes, and to guide the control of ARG environmental spreading.

Spectral Characteristics and Functional Responses of Phospholipid Bilayers in the Terahertz Band.

Understanding the vibrational information encoded within the terahertz (THz) spectrum of biomolecules is critical for guiding the exploration of its functional responses to specific THz radiation wavelengths. This study investigated several important phospholipid components of biological membranes-distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylcholine (DPPC), sphingosine phosphorylcholine (SPH), and lecithin bilayer-using THz time-domain spectroscopy. We observed similar spectral patterns for DPPC, SPH, and the lecithin bilayer, all of which contain the choline group as the hydrophilic head. Notably, the spectrum of DSPE, which has an ethanolamine head group, was different. Interestingly, density functional theory calculations confirmed that the absorption peak common to DSPE and DPPC at approximately 3.0 THz originated from a collective vibration of their similar hydrophobic tails. Accordingly, the cell membrane fluidity of RAW264.7 macrophages with irradiation at 3.1 THz was significantly enhanced, leading to improved phagocytosis. Our results highlight the importance of the spectral characteristics of the phospholipid bilayers when studying their functional responses in the THz band and suggest that irradiation at 3.1 THz is a potential non-invasive strategy to increase the fluidity of phospholipid bilayers for biomedical applications such as immune activation or drug administration.

Miscibility of Phosphatidylcholines in Bilayers: Effect of Acyl Chain Unsaturation

The miscibility of phospholipids in a hydrated bilayer is an issue of fundamental importance for understanding the organization of biological membranes. Despite research on lipid miscibility, its molecular basis remains poorly understood. In this study, all-atom MD simulations complemented by Langmuir monolayer and DSC experiments have been performed to investigate the molecular organization and properties of lipid bilayers composed of phosphatidylcholines with saturated (palmitoyl, DPPC) and unsaturated (oleoyl, DOPC) acyl chains. The experimental results showed that the DOPC/DPPC bilayers are systems exhibiting a very limited miscibility (strongly positive values of excess free energy of mixing) at temperatures below the DPPC phase transition. The excess free energy of mixing is divided into an entropic component, related to the ordering of the acyl chains, and an enthalpic component, resulting from the mainly electrostatic interactions between the headgroups of lipids. MD simulations showed that the electrostatic interactions for lipid like-pairs are much stronger than that for mixed pairs and temperature has only a slight influence on these interactions. On the contrary, the entropic component increases strongly with increasing temperature, due to the freeing of rotation of acyl chains. Therefore, the miscibility of phospholipids with different saturations of acyl chains is an entropy-driven process.

Interaction of guanidinium and ammonium cations with phosphatidylcholine and phosphatidylserine lipid bilayers – Calorimetric, spectroscopic and molecular dynamics simulations study

The ability of arginine-rich peptides to cross the lipid bilayer and enter cytoplasm, unlike their lysine-based analogues, is intensively studied in the context of cell-penetrating peptides. Although the experiments have not yet reconstructed their internalization mechanism, the computational studies have shown that the type or charge of lipid polar groups is one of the crucial factors in their translocation. In order to gain more detailed insight into the interaction of guanidinium (Gdm+) and ammonium (NH4+) cations, as important building blocks in arginine and lysine amino acids, with lipid bilayers, we conducted the experimental and computational study that tackles this phenomenon. The adsorption of Gdm+ and NH4+ on lipid bilayers prepared from a zwitterionic (DPPC) and an anionic (DPPS) lipid was examined by thermoanalytic and spectroscopic techniques. Using temperature-dependent UV–Vis spectroscopy and DSC calorimetry we determined the impact of Gdm+ and NH4+ on the thermotropic properties of lipid bilayers. FTIR data, along with molecular dynamics simulations, unraveled the molecular-level details on the nature of their interactions, showing the proton transfer between NH4+ and DPPS, but not between Gdm+ and DPPS. The findings originated from this work imply that Gdm+ and NH4+ form qualitatively different interactions with lipids of different charge which is reflected in the physico-chemical interactions that arginine-and lysine-based peptides establish at a complex and chemically heterogeneous environment such as the biological membrane.

Untangling the rivalry between cholesterol and antimicrobial peptides.

Antimicrobial peptides (AMPs) are promising candidates for next generation antimicrobial therapeutics. In mammalian cell membranes cholesterol plays a key regulatory function in antibiotic drug resistance and the immune response. However, the mechanism of action and the selectivity of AMPs towards bacterial membranes are not yet well understood. We hypothesized that differences in biophysical properties of mammalian versus bacterial membranes give rise to differences in peptide-membrane interactions which underlie the AMP mechanism and selectivity. Solid-state 2H NMR spectroscopy was employed to study di-monounsaturated phosphatidylcholine and phosphatidylethanolaminelipids (DOPC versus DOPE) bilayers in the presence of the antimicrobial peptide LL37 (2−4 mol%) and cholesterol (20−30% mol) as model systems mimicking mammalian and bacterial membranes. The lipid systems were probed with POPC-d31 (10 mol%) where the residual quadrupolar couplings yielded segmental order parameters (SCD) for the individual acyl segments. The LL37-peptide decreased the quadrupolar splittings indicating it increased the area per lipid, while cholesterol showed the opposite. The mean-torque model was used to calculate the key bilayer properties of area per lipid and bilayer thickness [2]. The model-free functional dependence of spin-lattice relaxation rates on SCD (square-law plots) indicated greater bending rigidity with cholesterol for both DOPC and DOPE membranes. By contrast LL37 decreased the bending rigidity of the membranes. Additionally all-atom molecular dynamics simulations for bilayers comprising POPE/DMPG/cardiolipin (90:5:5) and DMPC/cholesterol (90:10) were conducted with LL37 peptides on the bilayer surface to capture the molecular-level interactions. We observed partial insertion of the LL37-peptides in the POPE system, while the peptides were repelled from the membrane in the DMPC/cholesterol system. These observations reveal the surprising rivalry between cholesterol and AMPs which underlies AMP mechanism and selectivity.

Why do polyarginines adsorb at neutral phospholipid bilayers and polylysines do not? An insight from density functional theory calculations and molecular dynamics simulations.

Adsorption of cell-penetrating peptides (CPPs) at cellular membranes is the first and necessary step for their subsequent translocation across cellular membranes into the cytosol. It has been experimentally shown that CPPs rich in arginine (Arg) amino acid penetrate across phospholipid bilayers more effectively than their lysine (Lys) rich counterparts. In this work, we aim to understand the differences in the first translocation step, adsorption of Arg9 and Lys9 peptides at fully hydrated neutral phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipid bilayers and evaluate in detail the energetics of the process using molecular dynamics (MD) simulations and free energy calculations of adsorption of the single peptide. We show that the adsorption of Arg9 is energetically feasible, with the free energy of adsorption being ∼-5.0 kcal mol-1 at PC and ∼-5.5 kcal mol-1 at PE bilayers. In contrast, adsorption of Lys9 is not observed at PC bilayers, and their adsorption at PE bilayers is very weak, being ∼-0.5 kcal mol-1. We show by energy decomposition and analysis of peptide hydration along the membrane that significantly stronger electrostatic interactions of Arg9 with lipid phosphate groups, together with the greater loss of peptide hydration (and in turn stronger hydrophobic interactions) along the membrane translocation path, are the main driving factors governing the adsorption of Arg-rich peptides at neutral lipid bilayers in contrast to Lys-rich peptides. Finally, we also compare the energetics in lipid/bilayer systems with the density functional theory (DFT) calculations of the corresponding model systems in the continuum water model and reveal the energetic differences in different environments.