Model Lipid Membranes Research Articles

Interactions of Novel Phosphonium Dye with Lipid Bilayers: A Molecular Dynamics Study

In the present work the 100-ns molecular dynamics simulations (MD) were performed in the CHARMM36m force field using the GROMACS package to estimate the bilayer location and mechanisms of the interaction between the novel phosphonium dye TDV and the model lipid membranes composed of the phosphatidylcholine (PC) and its mixtures with cholesterol (Chol) or/and anionic phospholipid cardiolipin (CL). Varying the dye initial position relative to the membrane midplane, the dye relative orientation and the charge state of the TDV molecule it was found that the one charge form of TDV, which was initially translated to a distance of 20 Å from the membrane midplane along the bilayer normal, readily penetrates deeper into the membrane interior and remains within the lipid bilayer during the entire simulation time. It was revealed that the probe partitioning into the model membranes was accompanied by the reorientation of TDV molecule from perpendicular to nearly parallel to the membrane surface. The analysis of the MD simulation results showed that the lipid bilayer partitioning and location of the one charge form of TDV depend on the membrane composition. The dye binds more rapidly to the neat PC bilayer than to CL- and Chol-containing model membranes. It was found that in the neat PC and CL-containing membranes the one charge TDV resides at the level of carbonyl groups of lipids (the distances ~ 1.1 nm, 1.2 nm and 1.3 nm from the bilayer center for the PC, CL10 and CL20 lipid membranes, respectively), whereas in the Chol-containing membranes the probe is located at the level of glycerol moiety (~ 1.5 nm and 1.6 nm for the Chol30 and CL10/Chol30 lipid membranes, respectively). It was demonstrated that the dye partitioning into the lipid bilayer does not affect the membrane structural properties.

Effect of Protocatechuic Acid Ethyl Ester on Biomembrane Models: Multilamellar Vesicles and Monolayers.

The interactions of drugs with cell membranes are of primary importance for several processes involved in drugs activity. However, these interactions are very difficult to study due to the complexity of biological membranes. Lipid model membranes have been developed and used to gain insight into drug–membrane interactions. In this study, the interaction of protocatechuic acid ethyl ester, showing radical-scavenging activity, antimicrobial, antitumor and anti-inflammatory effects, with model membranes constituted by multilamellar vesicles and monolayers made of DMPC and DSPC, has been studied. Differential scanning calorimetry and Langmuir–Blodgett techniques have been used. Protocatechuic acid ethyl ester interacted both with MLV and monolayers. However, a stronger interaction of the drug with DMPC-based model membranes has been obtained. The finding of this study could help to understand the protocatechuic acid ethyl ester action mechanism.

The interaction and orientation of Peptide KL[formula omitted] in model membranes

We report on the orientation and location of synthetic pulmonary surfactant peptide KL4, (KLLLL)4K, in model lipid membranes. The partitioning depths of selectively deuterated leucine residues within KL4 were determined in DPPC:POPG (4:1) and POPC:POPG (4:1) bilayers by oriented neutron diffraction. These measurements were combined with an NMR-generated model of the peptide structure to determine the orientation and partitioning of the peptide at the lipid–water interface. The results demonstrate KL4 adopting an orientation that interacts with a single membrane leaflet. These observations are consistent with past 2H NMR and EPR studies (Antharam et al., 2009; Turner et al., 2014).

Deciphering Ethanol-Driven Swelling, Rupturing, Aggregation, and Fusion of Lipid Vesicles Using Coarse-Grained Molecular Dynamics Simulations.

Traditionally, liquid ethanol is known to enhance the permeability of lipid membranes and causes vesicle aggregation and fusion. However, how the amphiphilic ethanol molecules perturb the lipid vesicles to facilitate their aggregation or fusion has not been addressed at any level of molecular simulations. Herein, not only have we developed a coarse-grained (CG) model for liquid ethanol, its aqueous mixture, and hydrated lipid membranes for molecular dynamics (MD) simulations, but also utilized it to delineate the aggregation and fusion of lipid vesicles using CG-MD simulations with multimillion particles. We have systematically parametrized the force-field for pure ethanol and its interactions with hydrated POPC and POPE model lipid membranes. In this process, we have successfully reproduced the bulk ethanol structure and concentration-dependent density of aqueous ethanol. To quantify the interaction of ethanol with lipid membranes, we have reproduced the transfer free energy of the ethanol molecule across the hydrated bilayers, and the concentration-dependent distribution of ethanol molecules across the lipid bilayers. After having acceptable force-field parameters for ethanol-membrane interactions, we have checked the effect of ethanol toward the vesicles comprising POPC lipids. We observe a rapid increase in the size of the POPC lipid vesicles with increasing amounts of ethanol up to 30 mol %. We unambiguously observe swelling and decrease in the thickness of the POPC vesicles with increasing amounts of ethanol up to 30 mol %, beyond which the vesicles begin to lose their integrity and rupture at higher mol % of ethanol. The fusion study of two vesicles demonstrates that fused vesicles can be obtained from 20 to 30 mol % of ethanol provided that they are brought closer than a critical distance at a particular mol %. The multivesicle simulations show that along with the increase in the sizes of vesicles the propensity of vesicle aggregation increases as the mol % of ethanol increases.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs).

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Calmodulin interactions with model lipid membranes: the interplay of Ca2+and lipid composition

Interactions of ionic proteins and lipids are believed to be crucial for the structure and function of membrane receptors and ion-channels. Calmodulin (CaM) is a highly conserved, multifunctional calcium-modulated protein, expressed in all eukaryotic cells. It binds to a wide variety of target proteins, influencing their function. Moreover, CaM binds up to four calcium ions through four EF-hand motifs, consisting of helix-loop-helix domains. This is known to lead to a conformational change that allows the binding with other target proteins, essential in many biological processes and driven by hydrophobic interactions.

Interactions of antibiotic organic molecules with lipid bilayers

Triterpenoid saponins are plant compounds with important medical values; their biological function is closely related to their interactions with lipid membranes. For example, saponins can serve as antibacterial by destroying the integrity of bacterial membranes; saponins are also known to interact and modulate the composition and size of lipid domains in membranes. This study focuses on the interaction between four kinds of triterpenoid saponins and a lipid membrane model using a ternary mixture of POPE/POPG/DPPG (80:15:5 mol%).

Effect of procaine hydrochoride on lipid bilayers

We are interested in the influence of natural osmolytes, e.g. Urea and TMAO (Trimethylamine N-oxide) on the fluidity and stability of lipid model membranes. Here we extend our interest to Procaine Hydrochoride. We used a combination of simultaneous SAXS/WAXS/DSC measurements, aiming to identify the structures, their dimensions and the phase transition temperatures. Thermal scans show morphologies similar to aqueous solution of the pure lipids, although with different dimensions and transition temperatures.

Antimicrobial peptides derived from the SPLUNC1 protein perturb bacterial and eukaryotic lipid model membranes

SPLUNC1 (short palate lung and nasal epithelial clone 1) is a multifunctional host defense protein found in the human respiratory tract that possesses antimicrobial properties. Through modification of the 4th alpha helical motif of the protein, five novel antimicrobial peptides (AMPs) were designed with different lengths, charges, hydrophobicities (H) and hydrophobic moments (μH). The goal of this study was to compare the biological activities of these AMPs by means of testing against paired clinical isolates of the Gram-negative (G(-)) bacteria Klebsiella pneumoniae, obtained from twelve patients before and after colistin treatments (half susceptible and half resistant to colistin).

The effect of dopamine on model lipid membrane structure

Dopamine is a neurotransmitter released by neurons to send signals to other nerve cells. Previous work on the interaction of dopamine with model lipid bilayers made using solution-state NMR spectroscopy has shown that dopamine interacts with phosphatidylcholine (PC) and phosphatidylserine (PS) lipid headgroups primarily through the aromatic side opposite to the hydroxyl groups [1]. Here we use low-angle x-ray scattering (LAXS), solid-state deuterium NMR, as well as electron paramagnetic resonance (EPR) to determine the effect of dopamine on the physical properties of lipid membranes.

Comparing α-Synuclein Fibrils Formed in the Absence and Presence of a Model Lipid Membrane: A Small and Wide-Angle X-Ray Scattering Study

Amyloid fibrils are associated with a number of different neurodegenerative diseases. Detailed knowledge of the fibril structure will be of importance in the search of therapy and may guide experiments to understand amyloid formation. In this paper we investigate the morphology of α-synuclein amyloid fibrils, associated with Parkinson’s disease, formed under different conditions. In particular, we study, by means of small and wide-angle X-ray scattering, whether the presence of model lipid membranes affect the overall structure of the fibrils formed, motivated by the fact that amyloid fibrils in vivo are formed in a highly lipid-rich environment. Comparing fibrils formed in the presence of lipid with fibrils formed in their absence, show that the presence of lipids has no detectable effect on the fibril cross-section radius and that the characteristic β-strand repeat distance of 4.7 Å of the extended intermolecular β-sheets remains unaffected. We also show that the observed fibril radius is consistent with a fibril structure composed of two protofilaments. This indicates overall that the particular fibril structure, with their stacks of two-dimensionally folded α-synuclein molecules, represent a deep free energy minimum, not largely affected by the co-aggregation with lipids.

A [formula omitted] finite element method for axisymmetric lipid membranes in the presence of the Gaussian energy

The classical model of lipid membranes is expressed in terms of a curvature-dependent energy involving the mean and the Gaussian curvatures of membranes. It is customary to ignore the Gaussian energy as it takes a constant value for topologically equivalent closed membranes. In this work, we study the effect of the Gaussian energy on configurations of axisymmetric lipid membranes by using a phase field model. As the weak form of the governing equations involves the second-order derivatives of the phase field function, we employ C1 Argyris finite element to approximate solutions. The numerical results of this paper suggest that including the Gaussian energy in the existing mathematical model may be necessary to capture the topology changes of membranes accurately.

Probing Liquid-Ordered and Disordered Phases in Lipid Model Membranes: A Combined Theoretical and Spectroscopic Study of a Fluorescent Molecular Rotor

An integrated theoretical/experimental strategy has been applied to the study of environmental effects on the spectroscopic parameters of 4-(diphenylamino)phtalonitrile (DPAP), a fluorescent molecular rotor. The computational part starts from the development of an effective force field for the first excited electronic state of DPAP and proceeds through molecular dynamics simulations in solvents of different polarities toward the evaluation of Stokes shifts by quantum mechanics/molecular mechanics (QM/MM) approaches. The trends of the computed results closely parallel the available experimental results thus giving confidence to the interpretation of new experimental studies of the photophysics of DPAP in lipid bilayers. In this context, results show unambiguously that both flexible dihedral angles and global rotations are significantly retarded in a cholesterol/DPPC lipid matrix with respect to the DOPC matrix, thus confirming the sensitivity of DPAP to probe different environments and, therefore, its applicability as a probe for detecting different structures and levels of plasma membrane organization.

Dynamics of Amyloid Formation from Simplified Representation to Atomistic Simulations.

Amyloid fibril formation is an intrinsic property of short peptides, non-disease proteins, and proteins associated with neurodegenerative diseases. Aggregates of the Aβ and tau proteins, the α-synuclein protein, and the prion protein are observed in the brain of Alzheimer's, Parkinson's, and prion disease patients, respectively. Due to the transient short-range and long-range interactions of all species and their high aggregation propensities, the conformational ensemble of these devastating proteins, the exception being for the monomeric prion protein, remains elusive by standard structural biology methods in bulk solution and in lipid membranes. To overcome these limitations, an increasing number of simulations using different sampling methods and protein models have been performed. In this chapter, we first review our main contributions to the field of amyloid protein simulations aimed at understanding the early aggregation steps of short linear amyloid peptides, the conformational ensemble of the Aβ40/42 dimers in bulk solution, and the stability of Aβ aggregates in lipid membrane models. Then we focus on our studies on the interactions of amyloid peptides/inhibitors to prevent aggregation, and long amyloid sequences, including new results on a monomeric tau construct.

Selectivity of mTOR-Phosphatidic Acid Interactions Is Driven by Acyl Chain Structure and Cholesterol.

The need to gain insights into the molecular details of peripheral membrane proteins’ specificity towards phosphatidic acid (PA) is undeniable. The variety of PA species classified in terms of acyl chain length and saturation translates into a complicated, enigmatic network of functional effects that exert a critical influence on cell physiology. As a consequence, numerous studies on the importance of phosphatidic acid in human diseases have been conducted in recent years. One of the key proteins in this context is mTOR, considered to be the most important cellular sensor of essential nutrients while regulating cell proliferation, and which also appears to require PA to build stable and active complexes. Here, we investigated the specific recognition of three physiologically important PA species by the mTOR FRB domain in the presence or absence of cholesterol in targeted membranes. Using a broad range of methods based on model lipid membrane systems, we elucidated how the length and saturation of PA acyl chains influence specific binding of the mTOR FRB domain to the membrane. We also discovered that cholesterol exerts a strong modulatory effect on PA-FRB recognition. Our data provide insight into the molecular details of some physiological effects reported previously and reveal novel mechanisms of fine-tuning the signaling cascades dependent on PA.

Insights into molecular mechanism of action of citrus flavonoids hesperidin and naringin on lipid bilayers using spectroscopic, calorimetric, microscopic and theoretical studies

Hesperidin and naringin are predominant flavonoids in citrus fruits and exert various biological activities. However, mechanism of these activities has not been completely investigated at cellular and molecular levels. For this reason, we aim to systematically elucidate molecular interactions between citrus flavonoids, hesperidin and naringin, and model lipid membranes composed of dimyristoylphosphatidylcholine (DMPC) using combinatorial experimental biophysical techniques, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, differential scanning calorimetry (DSC), atomic force microscopy (AFM) and field emission scanning electron microscopy (FE-SEM) as a function of citrus flavonoid concentrations, and computational study. ATR-FTIR showed that both flavonoids significantly increase the fluidity of DMPC membranes with the increase in the bandwidth values of the CH2 antisymmetric stretching vibration. While hesperidin exhibits weakly disordering effect in hydrophobic region, naringin has an ordering effect in this region. Both flavonoids induce alterations in the arrangement of polar heads of lipids. DSC revealed that naringin significantly influences the cooperativity and thermotropic phase behavior of DMPC compared to hesperidin. Furthermore, microscopic observations are in cooperation with the spectroscopic and calorimetric results indicating that naringin is more effective than hesperidin in terms of inducing changes in the membrane organization. Density functional theory (DFT) study demonstrated that both DMPC − Hesperidin and DMPC − Naringin systems form strong hydrogen bonding via PO2− moieties. Methodologies showed that small structural differences can influence the membrane bilayers distinctly. Consequently, this study implies that variations in structural and dynamical properties of lipid membranes can be critical for processes involving the inhibition of lipid peroxidation and antioxidant mechanism.

Interactions of Novel Phosphonium Dye with Lipid Bilayers: A Fluorescence Study

The phosphonium-based optical probes attract ever growing interest due to their excellent chemical and photophysical stability, high aqueous solubility, long wavelength absorption and emission, large extinction coefficient, high fluorescence quantum yield, low cytotoxicity, etc. The present study was focused on assessing the ability of the novel phosphonium dye TDV to monitor the changes in physicochemical properties of the model lipid membranes. To this end, the fluorescence spectral properties of TDV have been explored in lipid bilayers composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with cholesterol (Chol) or/and anionic phospholipid cardiolipin (CL). It was observed that in the buffer solution TDV possesses one well-defined fluorescence peak with the emission maximum at 533 nm. The dye transfer from the aqueous to lipid phase was followed by the enhancement of the fluorescence intensity coupled with a red shift of the emission maximum up to 67 nm, depending on the liposome composition. The quantitative information about the dye partitioning into lipid phase of the model membranes was obtained through approximating the experimental dependencies of the fluorescence intensity increase vs lipid concentration by the partition model. Analysis of the partition coefficients showed that TDV has a rather high lipid-associating ability and displays sensitivity to the changes in physicochemical properties of the model lipid membranes. The addition of CL, Chol or both lipids to the PC bilayer gives rise to the increase of the TDV partition coefficients compared to the neat PC membranes. The enhancement of the phosphonium dye partitioning in the CL and Chol-containing lipid bilayers has been attributed to the cardiolopin- and cholesterol-induced changes in the structure and physicochemical characteristics of the polar membrane region.

Effect of glycyrrhizic acid on phospholipid membranes in media with different pH.

Glycyrrhizic acid (GA) is the active ingredient in licorice root, which exhibits a wide range of biological activities, including anti-inflammatory and antiviral activities. In particular, the virus-inhibiting effect of GA on SARS-associated coronavirus was demonstrated. In addition, GA was found to be capable of increasing bioaccessibility of other drugs when used together. All these effects can be based on the ability of GA to incorporate into cell membranes and change their physical and functional properties. One of the possible mechanisms of the antiviral action of GA against COVID-19 is also considered to be the prevention of fusion of the virus envelope with the plasma membrane of the host cell. The interaction of GA with model lipid membranes was studied by the NMR method. Different factors influencing the incorporation of the GA molecule into the lipid bilayer (phospholipid structure, pH of the medium) were examined.

Correlating biological activity to thermo-structural analysis of the interaction of CTX with synthetic models of macrophage membranes

The important pharmacological actions of Crotoxin (CTX) on macrophages, the main toxin in the venom of Crotalus durissus terrificus, and its important participation in the control of different pathophysiological processes, have been demonstrated. The biological activities performed by macrophages are related to signaling mediated by receptors expressed on the membrane surface of these cells or opening and closing of ion channels, generation of membrane curvature and pore formation. In the present work, the interaction of the CTX complex with the cell membrane of macrophages is studied, both using biological cells and synthetic lipid membranes to monitor structural alterations induced by the protein. Here we show that CTX can penetrate THP-1 cells and induce pores only in anionic lipid model membranes, suggesting that a possible access pathway for CTX to the cell is via lipids with anionic polar heads. Considering that the selectivity of the lipid composition varies in different tissues and organs of the human body, the thermostructural studies presented here are extremely important to open new investigations on the biological activities of CTX in different biological systems.