Electrochemical assessment of dielectric damage to phospholipid bilayers by amyloid ß-Oligomers.

Amyloid beta (Aß1-42) oligomers produced in vitro with and without the oligomerization inhibitor hexafluoroisopropanol (HFIP) were studied and compared as agents inflicting damage to the phospholipid bilayers. Tethered lipid membranes (tBLMs) of different compositions were used as model membranes. Dielectric damage of tBLMs by Aß1-42 oligomers was monitored by the electrochemical impedance spectroscopy (EIS). Membranes containing sphingomyelin exhibited the highest susceptibility to Aß1-42 oligomers when assembled in the absence of an inhibitor. The activation barrier of ion translocation through the Aß1-42 oligomer entities in tBLMs was lowest in sphingomyelin membranes (<15 kJ/mol). This is consistent with the formation of water-filled, highly conductive (>50 pS) nanopores in tBLMs by Aß1-42 oligomers assembled without HFIP. Conversely, HFIP-generated Aß1- 42 oligomers exhibited conductance with high activation energies (>38 kJ/mol), suggesting the formation of assemblies with relatively narrow ion pores and the effective conductance in the range < 15 pS. Finally, the EIS data analysis revealed differences in the lateral distribution of Aß1-42 oligomers in tBLMs. The inhibitor-free Aß1-42 oligomers populate the tBLM surface in a random manner, whereas the HFIP-generated Aß1-42 oligomers tend to cluster forming surface areas with markedly different densities of Aß1-42 defects.

Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins.

Integral membrane proteins are localized and/or regulated by lipids present in the surrounding bilayer. While bacteria have relatively simple membranes, there is ample evidence that many bacterial proteins bind to specific lipids, especially the anionic lipid cardiolipin. Here, we apply molecular dynamics simulations to assess lipid binding to 42 different Escherichia coli inner membrane proteins. Our data reveal an asymmetry between the membrane leaflets, with increased anionic lipid binding to the inner leaflet regions of the proteins, particularly for cardiolipin. From our simulations, we identify >700 independent cardiolipin binding sites, allowing us to identify the molecular basis of a prototypical cardiolipin binding site, which we validate against structures of bacterial proteins bound to cardiolipin. This allows us to construct a set of metrics for defining a high-affinity cardiolipin binding site on bacterial membrane proteins, paving the way for a heuristic approach to defining other protein-lipid interactions.

Position-Dependent Diffusion from Biased Simulations and Markov State Model Analysis.

A variety of enhanced statistical and numerical methods are now routinely used to extract important thermodynamic and kinetic information from the vast amount of complex, high-dimensional data obtained from molecular simulations. For the characterization of kinetic properties, Markov state models, in which the long-time statistical dynamics of a system is approximated by a Markov chain on a discrete partition of configuration space, have seen widespread use in recent years. However, obtaining kinetic properties for molecular systems with high energy barriers remains challenging as often enhanced sampling techniques are required with biased simulations to observe the relevant rare events. Particularly, the calculation of diffusion coefficients remains elusive from biased molecular simulation data. Here, we propose a novel method that can calculate multidimensional position-dependent diffusion coefficients equally from either biased or unbiased simulations using the same formalism. Our method builds on Markov state model analysis and the Kramers-Moyal expansion. We demonstrate the validity of our formalism using one- and two-dimensional analytic potentials and also apply it to data from explicit solvent molecular dynamics simulations, including the water-mediated conformations of alanine dipeptide and umbrella sampling simulations of drug transport across a lipid bilayer. Importantly, the developed algorithm presents significant improvement compared to standard methods when the transport of solute across three-dimensional heterogeneous porous media is studied, for example, the prediction of membrane permeation of drug molecules.

The Origin of Lipid Rafts.

The time-averaged lateral organization of the lipids and proteins that make up mammalian cell membranes continues to be the subject of intense interest and debate. Since the introduction of the fluid mosaic model almost 50 years ago, the "lipid raft hypothesis" has emerged as a popular concept that has captured the imagination of a large segment of the biomembrane community. In particular, the notion that lipid rafts play a pivotal role in cellular processes such as signal transduction and membrane protein trafficking is now favored by many investigators. Despite the attractiveness of lipid rafts, their composition, size, lifetime, biological function, and even the very existence remain controversial. The central tenet that underlies this hypothesis is that cholesterol and high-melting lipids have favorable interactions (i.e., they pull together), which lead to transient domains. Recent nearest-neighbor recognition (NNR) studies have expanded the lipid raft hypothesis to include the influence that low-melting lipids have on the organization of lipid membranes. Specifically, it has been found that mimics of cholesterol and high-melting lipids are repelled (i.e., pushed away) by low-melting lipids in fluid bilayers. The picture that has emerged from our NNR studies is that lipid mixing is governed by a balance of these "push and pull" forces, which maximizes the number of hydrocarbon contacts and attractive van der Waals interactions within the membrane. The power of the NNR methodology is that it allows one to probe these push/pull interaction energies that are measured in tens of calories per mole.

FRET from phase-separated vesicles: An analytical solution for a spherical geometry.

Förster resonance energy transfer (FRET) is a powerful tool for investigating heterogeneity in lipid bilayers. In model membrane studies, samples are frequently unilamellar vesicles with diameters of 20-200 nm. It is well-known that FRET efficiency is insensitive to vesicle curvature in uniformly mixed lipid bilayers, and consequently theoretical models for FRET typically assume a planar geometry. Here, we use a spherical harmonic expansion of the acceptor surface density to derive an analytical solution for FRET between donor and acceptor molecules distributed on the surface of a sphere. We find excellent agreement between FRET predicted from the model and FRET calculated from corresponding Monte Carlo simulations, thus validating the model. An extension of the model to the case of a non-uniform acceptor surface density (i.e., a phase-separated vesicle) reveals that FRET efficiency depends on vesicle size when acceptors partition between the coexisting phases, and approaches the efficiency of a uniformly mixed bilayer as the vesicle size decreases. We show that this is an indirect effect of constrained domain size, rather than an intrinsic effect of vesicle curvature. Surprisingly, the theoretical predictions were not borne out in experiments: we did not observe a statistically significant change in FRET efficiency in phase-separated vesicles as a function of vesicle size. We discuss factors that likely mask the vesicle size effect in extruded samples.

Computer simulations of a heterogeneous membrane with enhanced sampling techniques.

Computational determination of the equilibrium state of heterogeneous phospholipid membranes is a significant challenge. We wish to explore the rich phase diagram of these multi-component systems. However, the diffusion and mixing times in membranes are long compared to typical time scales of computer simulations. Here, we evaluate the combination of the enhanced sampling techniques molecular dynamics with alchemical steps and Monte Carlo with molecular dynamics with a coarse-grained model of membranes (Martini) to reduce the number of steps and force evaluations that are needed to reach equilibrium. We illustrate a significant gain compared to straightforward molecular dynamics of the Martini model by factors between 3 and 10. The combination is a useful tool to enhance the study of phase separation and the formation of domains in biological membranes.

Mixed hybrid bilayer lipid membranes on mechanically polished titanium surface.

Mixed self-assembled monolayers of octadecyltrichlorosilane (OTS) and methyltrichlorosilane (MTS) were deposited via simple silanization procedure on a mechanically polished titanium surface. The monolayers act as molecular anchors for mixed hybrid bilayer lipid membranes (mhBLM) which were accomplished via vesicle fusion. A variation of the MTS concentration in silanization solutions significantly affects properties of mhBLMs composed of a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol (Chol). The bilayers become less insulating following an increase of the MTS content. On the other hand, an increase of the MTS concentration provides flexibility of the mhBLM membranes necessary for the functional reconstitution of membrane proteins. The optimal molar ratio of MTS in silanization solution is 40% providing anchors for intact mhBLMs as confirmed by their specific capacitance of 0.86 µF cm-2. We found that the bilayers containing 40% (mol) of cholesterol bind cholesterol dependent pneumolysin (PLY). However, we did not observe functional reconstitution of PLY. While α-hemolysin almost fully disrupts mhBLMs assembled from 100% diphytanoyl. An important advantage of the titanium/OTS/MTS molecular anchor systems is their ability of repetitive regeneration of phospholipid bilayers without losing functional properties as demonstrated in the current study. This creates a possibility for the multiple-use phospholipid membrane biosensors which have a potential of decreasing the cost of such electrochemical/electroanalytical devices.

Transferosomes as nanocarriers for drugs across the skin: Quality by design from lab to industrial scale.

Transferosomes, also known as transfersomes, are ultradeformable vesicles for transdermal applications consisting of a lipid bilayer with phospholipids and an edge activator and an ethanol/aqueous core. Depending on the lipophilicity of the active substance, it can be encapsulated within the core or amongst the lipid bilayer. Compared to liposomes, transferosomes are able to reach intact deeper regions of the skin after topical administration delivering higher concentrations of active substances making them a successful drug delivery carrier for transdermal applications. Most transferosomes contain phosphatidylcholine (C18) as it is the most abundant lipid component of the cell membrane, and hence, it is highly tolerated for the skin, decreasing the risk of undesirable effects, such as hypersensitive reactions. The most common edge activators are surfactants such as sodium deoxycholate, Tween® 80 and Span® 80. Their chain length is optimal for intercalation within the C18 phospholipid bilayer. A wide variety of drugs has been successfully encapsulated within transferosomes such as phytocompounds like sinomenine or apigenin for rheumatoid arthritis and leukaemia respectively, small hydrophobic drugs but also macromolecules like insulin. The main factors to develop optimal transferosomal formulations (with high drug loading and nanometric size) are the optimal ratio between the main components as well as the critical process parameters for their manufacture. Application of quality by design (QbD), specifically design of experiments (DoE), is crucial to understand the interplay among all these factors not only during the preparation at lab scale but also in the scale-up process. Clinical trials of a licensed topical ketoprofen transferosomal gel have shown promising results in the alleviation of symptons in orthreothritis with non-severe skin and subcutaneous tissue disorders. However, the product was withdrawn from the market which probably was related to the higher cost of the medicine linked to the expensive manufacturing process required in the production of transferosomes compared to other conventional gel formulations. This example brings out the need for a careful formulation design to exploit the best properties of this drug delivery system as well as the development of manufacturing processes easily scalable at industrial level.

Comparing an All-Atom and a Coarse-Grained Description of Lipid Bilayers in Terms of Enthalpies and Entropies: From MD Simulations to 2D Lattice Models.

Simulations of lipid bilayers at different levels of detail is of major interest in computational biophysics. With a recently proposed algorithm, by mapping the information from a Molecular Dynamics (MD) simulation of a lipid bilayer on a 2D lattice model, it is possible to gain insight into the enthalpic and entropic contributions, governing the interaction of adjacent lipids and the individual chain entropy, respectively. The contributions are obtained as a function of the lipid chain order parameter. Here we use this approach for the case of pure DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) to compare the all-atom CHARMM36 DPPC lipid with the respective MARTINI coarse-grained DPPC lipid. We individually compare the enthalpy and entropy functions as well as the resulting free energies. This allows us to gain new insight into the comparison of both levels of description. When numerically solving the 2D lattice model via Monte Carlo (MC) simulations, the 2D model displays the gel/liquid transition at the same temperature as the respective MD simulation. The dramatic increase in efficiency of the 2D lattice model as compared to the MD simulation allows us to estimate the equilibrium order parameters of the gel phase, inaccessible by the MD simulations even after 3 µs.

Controlling Anomalous Diffusion in Lipid Membranes.

Diffusion in cell membranes is not just simple two-dimensional Brownian motion but typically depends on the timescale of the observation. The physical origins of this anomalous subdiffusion are unresolved, and model systems capable of quantitative and reproducible control of membrane diffusion have been recognized as a key experimental bottleneck. Here, we control anomalous diffusion using supported lipid bilayers containing lipids derivatized with polyethylene glycol (PEG) headgroups. Bilayers with specific excluded area fractions are formed by control of PEG lipid mole fraction. These bilayers exhibit a switch in diffusive behavior, becoming anomalous as bilayer continuity is disrupted. Using a combination of single-molecule fluorescence and interferometric imaging, we measure the anomalous behavior in this model over four orders of magnitude in time. Diffusion in these bilayers is well described by a power-law dependence of the mean-square displacement with observation time. Anomaleity in this system can be tailored by simply controlling the mole fraction of PEG lipid, producing bilayers with diffusion parameters similar to those observed for anomalous diffusion in biological membranes.

Nanostructured cochleates: a multi-layered platform for cellular transportation of therapeutics.

Among lipid-based nanocarriers, multi-layered cochleates emerge as a novel delivery system because of prevention of oxidation of hydrophobic and hydrophilic drugs, enhancement in permeability, and reduction in dose of drugs. It also improves oral bioavailability and increases the safety of a drug by targeting at a specific site with less side effects. Nanostructured cochleates are used as a carrier for the delivery of water-insoluble or hydrophobic drugs of anticancer, antiviral and anti-inflammatory action. This review article focuses on different methods for preparation of cochleates, mechanism of formation of cochleates, mechanism of action like cochleate undergoes macrophagic endocytosis and release the drug into the systemic circulation by acting on membrane proteins, phospholipids, and receptors. Advanced methods such as calcium-substituted and ß-cyclodextrin-based cochleates, novel techniques include microfluidic and modified trapping method. Cochleates showed enhancement in oral bioavailability of amphotericin B, delivery of factor VII, oral mucosal vaccine adjuvant-delivery system, and delivery of volatile oil. In near future, cochleate will be one of the interesting delivery systems to overcome the stability and encapsulation efficiency issues associated with liposomes. The current limiting factors for commercial preparation of cochleates involve high cost of manufacturing, lack of standardization, and specialized equipments.

How to Determine Lipid Interactions in Membranes from Experiment Through the Ising Model.

The determination and the meaning of interactions in lipid bilayers are discussed and interpreted through the Ising model. Originally developed to understand phase transitions in ferromagnetic systems, the Ising model applies equally well to lipid bilayers. In the case of a membrane, the essence of the Ising model is that each lipid is represented by a site on a lattice and that the interaction of each site with its nearest neighbors is represented by an energy parameter ω. To calculate the thermodynamic properties of the system, such as the enthalpy, the Gibbs energy, and the heat capacity, the partition function is derived. The calculation of the configurational entropy factor in the partition function, however, requires approximations or the use of Monte Carlo (MC) simulations. Those approximations are described. Ultimately, MC simulations are used in combination with experiment to determine the interaction parameters ω in lipid bilayers. Several experimental approaches are described, which can be used to obtain interaction parameters. They include nearest-neighbor recognition, differential scanning calorimetry, and Förster resonance energy transfer. Those approaches are most powerful when used in combination of MC simulations of Ising models. Lipid membranes of different compositions are discussed, which have been studied with these approaches. They include mixtures of cholesterol, saturated (ordered) phospholipids, and unsaturated (disordered) phospholipids. The interactions between those lipid species are examined as a function of molecular properties such as the degree of unsaturation and the acyl chain length. The general rule that emerges is that interactions between different lipids are usually unfavorable. The exception is that cholesterol interacts favorably with saturated (ordered) phospholipids. However, the interaction of cholesterol with unsaturated phospholipids becomes extremely unfavorable as the degree of unsaturation increases.

Net Interactions That Push Cholesterol Away from Unsaturated Phospholipids Are Driven by Enthalpy.

The exchangeable unsaturated phospholipids c1-Phos and c3-Phos, which bear one and three permanent kinks, respectively, in their acyl chains, are mimics of the biologically important, low-melting phosphatidylcholines (PCs) having one and three cis double bonds in their sn-2 chains (i.e., 16:0,18:1 PC and 16:0,18:3 PC, respectively). The net interaction of an exchangeable form of cholesterol (Chol) with c1-Phos and with c3-Phos has been investigated using the nearest-neighbor recognition method. These interactions were found to be unfavorable in both cases having a positive free energy, ω, for replacing like by unlike nearest-neighbor contacts. The values for this free energy (or interaction parameter) were 165 cal/mol between Chol and c1-Phos and 395 cal/mol between Chol and c3-Phos. We now report the temperature dependence of these interactions in liquid-disordered bilayers. Their experimentally determined temperature dependencies, in combination with Monte Carlo simulations, revealed that the interaction parameter ω is dominated in both cases by enthalpy. These findings have important implications for the distribution of lipids in natural membranes and for the formation of lipid rafts.

Spatiotemporal Kinetics of Supported Lipid Bilayer Formation on Glass via Vesicle Adsorption and Rupture.

Supported lipid bilayers (SLBs) represent one of the most popular mimics of the cell membrane. Herein, we have used total internal reflection fluorescence microscopy for in-depth characterization of the vesicle-mediated SLB formation mechanism on a common silica-rich substrate, borosilicate glass. Fluorescently labeling a subset of vesicles allowed us to monitor the adsorption of individual labeled vesicles, resolve the onset of SLB formation from small seeds of SLB patches, and track their growth via SLB-edge-induced autocatalytic rupture of adsorbed vesicles. This made it possible to perform the first quantitative measurement of the SLB front velocity, which is shown to increase up to 1 order of magnitude with time. This effect can be classified as dramatic because in many other physical, chemical, or biological kinetic processes the front velocity is either constant or decreasing with time. The observation was successfully described with a theoretical model and Monte Carlo simulations implying rapid local diffusion of lipids upon vesicle rupture.

A mixed alchemical and equilibrium dynamics to simulate heterogeneous dense fluids: Illustrations for Lennard-Jones mixtures and phospholipid membranes.

An algorithm to efficiently simulate multi-component fluids is proposed and illustrated. The focus is on biological membranes that are heterogeneous and challenging to investigate quantitatively. To achieve rapid equilibration of spatially inhomogeneous fluids, we mix conventional molecular dynamics simulations with alchemical trajectories. The alchemical trajectory switches the positions of randomly selected pairs of molecules and plays the role of an efficient Monte Carlo move. It assists in accomplishing rapid spatial de-correlations. Examples of phase separation and mixing are given in two-dimensional binary Lennard-Jones fluid and a DOPC-POPC membrane. The performance of the algorithm is analyzed, and tools to maximize its efficiency are provided. It is concluded that the algorithm is vastly superior to conventional molecular dynamics for the equilibrium study of biological membranes.

High-Speed Microscopy of Diffusion in Pore-Spanning Lipid Membranes.

Pore-spanning membranes (PSMs) provide a highly attractive model system for investigating fundamental processes in lipid bilayers. We measure and compare lipid diffusion in the supported and suspended regions of PSMs prepared on a microfabricated porous substrate. Although some properties of the suspended regions in PSMs have been characterized using fluorescence studies, it has not been possible to examine the mobility of membrane components on the supported membrane parts. Here, we resolve this issue by employing interferometric scattering microscopy (iSCAT). We study the location-dependent diffusion of DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) lipids (DOPE) labeled with gold nanoparticles in (1,2-dioleoyl-sn-glycero-3-phosphocholine) (DOPC) bilayers prepared on holey silicon nitride substrates that were either (i) oxygen-plasma-treated or (ii) functionalized with gold and 6-mercapto-1-hexanol. For both substrate treatments, diffusion in regions suspended on pores with diameters of 5 µm is found to be free. In the case of functionalization with gold and 6-mercapto-1-hexanol, similar diffusion coefficients are obtained for both the suspended and the supported regions, whereas for oxygen-plasma-treated surfaces, diffusion is almost 4 times slower in the supported parts of the membranes. We attribute this reduced diffusion on the supported parts in the case of oxygen-plasma-treated surfaces to larger membrane-substrate interactions, which lead to a higher membrane tension in the freestanding membrane parts. Furthermore, we find clear indications for a decrease of the diffusion constant in the freestanding regions away from the pore center. We provide a detailed characterization of the diffusion behavior in these membrane systems and discuss future directions.

Quantitative Assessment of Methods Used To Obtain Rate Constants from Molecular Dynamics Simulations-Translocation of Cholesterol across Lipid Bilayers.

Accurately calculating rate constants of macroscopic chemical processes from molecular dynamics simulations is a long-sought but elusive goal. The problem is particularly relevant for processes occurring in biological systems, as is the case for ligand-protein and ligand-membrane interactions. Several formalisms to determine rate constants from easily accessible free-energy profiles [Δ Go( z)] of a molecule along a coordinate of interest have been proposed. However, their applicability for molecular interactions in condensed media has not been critically evaluated or validated. This work presents such evaluation and validation and introduces improved methodology. As a case study, we have characterized quantitatively the rate of translocation of cholesterol across 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayers. Translocation across lipid bilayers is the rate-limiting step in the permeation of most drugs through biomembranes. We use coarse-grained molecular dynamics simulations and different kinetic formalisms to calculate this rate constant. A self-consistent test of the applicability of various available formalisms is provided by comparing their predictions with the translocation rates obtained from actual events observed in long unrestrained simulations. To this effect, a novel procedure was used to obtain the effective rate constant, based on an analysis of time intervals between transitions among different states along the reaction coordinate. While most tested formalisms lead to results in reasonable agreement (within a factor of 5) with this effective rate constant, the most adequate one is based on the explicit relaxation frequencies from the transition state in the forward and backward directions along the reaction coordinate.

Single-lipid tracking on nanoscale membrane buds: The effects of curvature on lipid diffusion and sorting.

Nanoscale membrane curvature in cells is critical for endocytosis/exocytosis and membrane trafficking. However, the biophysical ramifications of nanoscale membrane curvature on the behavior of lipids remain poorly understood. Here, we created an experimental model system of membrane curvature at a physiologically-relevant scale and obtained nanoscopic information on single-lipid distributions and dynamics. Supported lipid bilayers were created over 50 and 70 nm radius nanoparticles to create membrane buds. Single-molecule localization microscopy was performed with diverse mixtures of fluorescent and non-fluorescent lipids. Variations in lipid acyl tales length, saturation, head-group, and fluorescent labeling strategy were tested while maintaining a single fluid lipid phase throughout the membrane. Monte Carlo simulations were used to fit our experimental results and quantify the effects of curvature on the lipid diffusion and sorting. Whereas varying the composition of the non-fluorescent lipids yielded minimal changes to the curvature effects, the labeling strategy of the fluorescent lipids yielded highly varying effects of curvature. Most conditions yield single-population Brownian diffusion throughout the membrane; however, curvature-induced lipid sorting, slowing, and aggregation were observed in some conditions. Head-group labeled lipids such as DPPE-Texas Red and POPE-Rhodamine diffused >2.4× slower on the curved vs. the planar membranes; tail-labeled lipids such as NBD-PPC, TopFluor-PPC, and TopFluor-PIP2, as well as DiIC12 and DiIC18 displayed no significant changes in diffusion due to the membrane curvature. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.

Effects of ß-Sitosteryl Sulfate on the Hydration Behavior of Dipalmitoylphosphatidylcholine.

We investigated the hydration behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers containing sodium ß-sitosteryl sulfate (PSO4). PSO4 was found to enhance hydration in the headgroup region of DPPC bilayers. Therefore, with the incorporation of PSO4 into DPPC membranes, the amount of water required to reach the fully hydrated state was enhanced as indicated by the constant values of the main phase transition temperature (Tm) and the bilayer repeat distance (d). For example, with the addition of 20 mol% of PSO4, the saturation point was shifted to ~70 wt% water compared to ~40 wt% for pure DPPC and 47 wt% for DPPC-cholesterol. The effectiveness of PSO4 in fluidizing the membrane and enhancing its hydration state can be useful in the pharmaceutical and cosmetic industries.

Lipid-protein interaction induced domains: Kinetics and conformational changes in multicomponent vesicles.

The spatio-temporal organization of proteins and the associated morphological changes in membranes are of importance in cell signaling. Several mechanisms that promote the aggregation of proteins at low cell surface concentrations have been investigated in the past. We show, using Monte Carlo simulations, that the affinity of proteins for specific lipids can hasten their aggregation kinetics. The lipid membrane is modeled as a dynamically triangulated surface with the proteins defined as in-plane fields at the vertices. We show that, even at low protein concentrations, strong lipid-protein interactions can result in large protein clusters indicating a route to lipid mediated signal amplification. At high protein concentrations, the domains form buds similar to that seen in lipid-lipid interaction induced phase separation. Protein interaction induced domain budding is suppressed when proteins act as anisotropic inclusions and exhibit nematic orientational order. The kinetics of protein clustering and resulting conformational changes are shown to be significantly different for the isotropic and anisotropic curvature inducing proteins.