Lipid Diffusion Coefficients Research Articles

Diffusion of Proteins and Lipids in Membranes Corrected for Finite-Size Effects

Translational diffusion coefficients are primary reporters on the dynamics in lipid membranes. However, diffusion coefficients of lipids and membrane proteins calculated in molecular dynamics (MD) simulations suffer from severe finite-size effects. We show that these finite-size effects can be quantified in terms of hydrodynamic theory and develop accurate correction formulas. Using large-scale coarse-grained MD simulations of up to 132 million particles, we assess the effects of box size and shape on translational diffusion in lipid membrane simulations. For the flat periodic boxes commonly used for membrane simulations, we find that the diffusion coefficients of lipids and membrane proteins increase logarithmically (and therefore without bound) with the box width. Only if the box size is increased also normal to the membrane surface, the diffusion coefficient converges. This dependence on both the size and the shape of the simulation box is captured quantitatively by a hydrodynamic theory. The corresponding correction formulas make it possible to perform meaningful comparisons of diffusion coefficients from simulations with different box sizes, and facilitate the comparison to experiment.

Mimicking Cell Pinocytosis: Lipid Vesicles Engulfment of Oil-in-Water Droplets

Pinocytosis, a type of endocytosis, is a process by which extracellular liquid droplets are taken up by cells. It involves droplet adhesion and engulfment by the membrane. We develop a synthetic system that reproduces the essential steps of pinocytosis at the level of a single interacting pair - the membrane and the droplet. The system consists of an engulfing giant unilamellar vesicle (GUV) and an engulfed polymer-stabilized oil-in-water emulsion droplet. The interaction between the GUV and the droplet is mediated by magnesium ions and is reversed by EDTA or Triton X-100. Adhesion energy |W|, measured from the contact angle between a GUV adhering to a droplet, either freestanding or manipulated with micropipettes, is affected by the magnesium concentration. In the weak adhesion regime (|W|∼0.4 mJ/m2, ≤ 0.5 mM Mg2+), GUVs adhere to the droplets and deform. Interaction is reversed by retracting the pipette-held GUV. In the strong adhesion regime (γ∼1.2 mJ/m2, 0.5 > mM Mg2+), the GUV completely engulfs the droplet and the interaction is irreversible. The droplet becomes wrapped by a single bilayer in a process that increases membrane tension and leads to vesicle poration. Both adhesion and engulfment lead to a decrease in lipid diffusion coefficient from 7 μm2/s on free-standing bilayers to 1 μm2/s on the droplet-supported bilayer. Interestingly, the contact line of the membrane-droplet adhesion area is often connected to many lipid nanotubes. The developed synthetic pinocytosis system of GUV-droplet pairs allows detailed investigation of the endocytosis processes of natural cells but with reduced complexity. Future surface modifications of the membrane/droplet interaction have the potential to mimic specific pathogen-recognition interactions. This work is part of the MaxSynBio consortium which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society.

Archaeal-Inspired Lipids Exhibit Low Membrane Permeability due to Entropic Effects

Many archaea are able to withstand extremes of temperature, pressure, pH and/or levels of salt. One unique feature of these extremophiles that gives them this capability is that their membranes are made from covalently linked, tetraether lipids that form a monolayer rather than a traditional bilayer. Here we present experimental and computational results on a series of synthetic archaeal-inspired lipids, looking at their biophysical properties and rates of permeation. Apart from a number of interesting mechanical and material properties, we find that tethered lipids exhibit a much stronger dependence on the entropy of activation for small molecules to cross the membrane. In relating our measured permeation rates with properties of the membrane, we find that the order parameters for the lipid tails describe the changes in entropy, and the combination of area per lipid, membrane thickness and short-range diffusion coefficient can predict our permeation results. Interestingly, we also see that the rates of water penetration into the core of the membrane provide an excellent empirical measure that matches our experimental permeation rates.

Effect of Sodium and Chloride Binding on a Lecithin Bilayer. A Molecular Dynamics Study.

The effect of ion binding on the structural, mechanical, dynamic and electrostatic properties of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer in a 0.5 M aqueous NaCl solution is investigated using classical atomistic molecular dynamics simulation with different force-field descriptions for ion-ion and ion-lipid interactions. Most importantly, the repulsive Lennard–Jones parameters for the latter were modified, such that approximately similar binding of cations and anions to the lipid membrane is achieved. This was done to qualitatively improve the apparent ion-lipid binding constants obtained from simulations with the original force field (Berger lipids and GROMOS87 ions in combination with the SPC water model) in comparison to experimental data. Furthermore, various parameters characterizing membrane structure, elasticity, order and dynamics are analyzed. It is found that ion binding as observed in simulations involving the modified in comparison to the original force-field description leads to: (i) a smaller salt-induced change in the area per lipid, which is in closer agreement with the experiment; (ii) a decrease in the area compressibility and bilayer thickness to values comparable to a bilayer in pure water; (iii) lipid deuterium order parameters and lipid diffusion coefficients on nanosecond timescales that are very similar to the values for a membrane in pure water. In general, salt effects on the structural properties of a POPC bilayer in an aqueous sodium-chloride solution appear to be reproduced reasonably well by the new force-field description. An analysis of membrane-membrane disjoining pressure suggests that the smaller salt-induced change in area per lipid induced by the new force-field description is not due to the alteration of membrane-associated net charge, but must rather be understood as a consequence of ion-specific effects on the arrangement of lipid molecules.

3D Probed Lipid Dynamics in Small Unilamellar Vesicles.

Single-molecule fluorescence correlation spectroscopy overcomes the resolution barrier of optical microscopy (10≈-20 nm) and is utilized to look into lipid dynamics in small unilamellar vesicles (SUVs; diameter < 100 nm). The fluorescence trajectories of lipid-like tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) in the membrane bilayers are acquired at a single-molecule level. The autocorrelation analysis yields the kinetic information on lipid organization, oxygen transport, and lateral diffusion in SUVs' membrane. First, the isomerization feasibility may be restricted by the addition of cholesterols, which form structure conjugation with DiD chromophore. Second, the oxygen transport is prevented from the ultrasmall cluster and cholesterol-rich regions, whereas it can pass through the membrane region with liquid-disordered phase (Ld ) and defects. Third, by analyzing 2D spectra correlating the lipid diffusion coefficient and triplet-state lifetime, the heterogeneity in lipid bilayer can be precisely visualized such as lipid domain with different phases, the defects of lipid packing, and DiD-induced "bouquet" ultrasmall clusters.

Time dependence of the enhancement effect of chemical enhancers: Molecular mechanisms of enhancing kinetics

Chemical enhancers are widely used for facilitating drug penetration in transdermal drug delivery system (TDDS). However, there is a lack of knowledge about how the enhancement effect changes over time. In this study, on the basis of kinetic parameters of enhancement effect, molecular details of the dynamic enhancement process was described and a new hypothesis of the recovery mechanism of the skin barrier function was proposed. Using pretreated skin and flurbiprofen patch, the effects of Azone (AZ) and menthyl decanoate (MT-10) were evaluated with in vitro permeation experiment and further confirmed by confocal laser scanning microscopy (CLSM) and TEWL. The results showed that the enhancement ratio (ER) increased firstly, then reached a plateau and finally decreased. The enhancement effect of MT-10 was slower (Tonset, MT-10>Tonset, AZ), weaker (ERmax, MT-10<ERmax, AZ) and shorter (Teff, MT-10<Teff, AZ) than that of AZ. According to the results of CLSM, ATR-FTIR and molecular dynamic simulation, the dynamic enhancement effect was caused by the variation of the diffusion coefficient of intercellular lipid in the stratum corneum (SC), which was dependent on the affinity between enhancers and lipid. Moreover, the skin barrier function recovered although a large amount of enhancers still existed in the SC. Additionally, according to the results of ATR-FTIR, molecular docking and skin retention study, the dynamic effect of AZ on the skin protein only induced skin irritation but showed no influence on drug penetration, so did the amount of the enhancer. In conclusion, dynamic enhancement effect was caused by the dynamic effect of the enhancer on the SC intercellular lipid, and the skin barrier function recovered by accepting the enhancer as a new component of the lipid bilayer.

Divergent Diffusion Coefficients in Simulations of Fluids and Lipid Membranes.

We investigate the dependence of single-particle diffusion coefficients on the size and shape of the simulation box in molecular dynamics simulations of fluids and lipid membranes. We find that the diffusion coefficients of lipids and a carbon nanotube embedded in a lipid membrane diverge with the logarithm of the box width. For a neat Lennard-Jones fluid in flat rectangular boxes, diffusion becomes anisotropic, diverging logarithmically in all three directions with increasing box width. In elongated boxes, the diffusion coefficients normal to the long axis diverge linearly with the height-to-width ratio. For both lipid membranes and neat fluids, this behavior is predicted quantitatively by hydrodynamic theory. Mean-square displacements in the neat fluid exhibit intermediate regimes of anomalous diffusion, with t ln t and t(3/2) components in flat and elongated boxes, respectively. For membranes, the large finite-size effects, and the apparent inability to determine a well-defined lipid diffusion coefficient from simulation, rationalize difficulties in comparing simulation results to each other and to those from experiments.

Point-to-Plane Nonhomogeneous Electric-Field-Induced Simultaneous Formation of Giant Unilamellar Vesicles (GUVs) and Lipid Tubes.

It is well-known that homogeneous electric fields can be used to generate giant unilamellar vesicles (GUVs). Herein we report an interesting phenomenon of formation of GUVs and lipid tubes simultaneously using a nonhomogeneous electric field generated by point-to-plane electrodes. The underlying mechanism was analyzed using finite element analysis. The two forces play main roles, that is, the pulling force (F) to drag GUVs into lipid tubes induced by fluid flow, and the critical force (Fc) to prevent GUVs from deforming into lipid tubes induced by electric fields. In the center area underneath the needle electrode, the GUVs were found because F is less than Fc in that region, whereas in the edge area the lipid tubes were obtained because F is larger than Fc. The diffusion coefficient of lipid in the tubes was found to be 4.45 μm(2) s(-1) using a fluorescence recovery after photobleaching (FRAP) technique. The method demonstrated here is superior to conventional GUV or lipid tube fabrication methods, and has great potential in cell mimic or hollow material fabrication using GUVs and tubes as templates.

The Role of the Membrane in the Structure and Biophysical Robustness of the Dengue Virion Envelope

SummaryThe dengue virion is surrounded by an envelope of membrane proteins surrounding a lipid bilayer. We have combined the cryoelectron microscopy structures of the membrane proteins (PDB: 3J27) with a lipid bilayer whose composition is based on lipidomics data for insect cell membranes, to obtain a near-atomic resolution computational model of the envelope of the dengue virion. A coarse-grained molecular dynamics simulation on the microsecond timescale enables analysis of key biophysical properties of the dengue outer envelope. Properties analyzed include area per lipid values (for a spherical virion with a mixed lipid composition), bilayer thickness, and lipid diffusion coefficients. Despite the absence of cholesterol from the lipid bilayer, the virion exhibits biophysical robustness (slow lipid diffusion alongside stable bilayer thickness, virion diameter, and shape) that matches the cholesterol-rich membrane of influenza A, with similarly anomalous diffusion of lipids. Biophysical robustness of the envelope may confer resilience to environmental perturbations.

Alzheimer's peptide amyloid-β, fragment 22-40, perturbs lipid dynamics.

The peptide amyloid-β (Aβ) interacts with membranes of cells in the human brain and is associated with Alzheimer's disease (AD). The intercalation of Aβ in membranes alters membrane properties, including the structure and lipid dynamics. Any change in the membrane lipid dynamics will affect essential membrane processes, such as energy conversion, signal transduction and amyloid precursor protein (APP) processing, and may result in the observed neurotoxicity associated with the disease. The influence of this peptide on membrane dynamics was studied with quasi-elastic neutron scattering, a technique which allows a wide range of observation times from picoseconds to nanoseconds, over nanometer length scales. The effect of the membrane integral neurotoxic peptide amyloid-β, residues 22-40, on the in- and out-of-plane lipid dynamics was observed in an oriented DMPC/DMPS bilayer at 15 °C, in its gel phase, and at 30 °C, near the phase transition temperature of the lipids. Near the phase-transition temperature, a 1.5 mol% of peptide causes up to a twofold decrease in the lipid diffusion coefficients. In the gel-phase, this effect is reversed, with amyloid-β(22-40) increasing the lipid diffusion coefficients. The observed changes in lipid diffusion are relevant to protein-protein interactions, which are strongly influenced by the diffusion of membrane components. The effect of the amyloid-β peptide fragment on the diffusion of membrane lipids will provide insight into the membrane's role in AD.

Fluidity evaluation of cell membrane model formed on graphene oxide with single particle tracking using quantum dot

The lipid bilayer is the fundamental structure of plasma membranes, and artificial lipid bilayer membranes are used as model systems of cell membranes. Recently we reported the formation of a supported lipid bilayer (SLB) on graphene oxide (GO) by the vesicle fusion method. In this study, we conjugated a quantum dot (Qdot) on the SLB surface as a fluorescence probe brighter than dye-labeled lipid molecules, to qualitatively evaluate the fluidity of the SLB on GO by the single particle tracking method. We obtained the diffusion coefficient of the Qdot-conjugated lipids in the SLB on GO. We also performed the Qdot conjugation on the SLB containing a lipid conjugated with polyethylene glycol, to prevent the nonspecific adsorption of Qdots. The difference in the diffusion coefficients between the SLBs on the GO and the bare SiO2 regions was evaluated from the trajectory of single Qdot-conjugated lipid diffusing between the two regions.

Charged particles interacting with a mixed supported lipid bilayer as a biomimetic pulmonary surfactant.

This study shows the interactions of charged particles with mixed supported lipid bilayers (SLB) as biomimetic pulmonary surfactants. We tested two types of charged particles: positively charged and negatively charged particles. Two parameters were measured: adsorption density of particles on the SLB and the diffusion coefficient of lipids by FRAPP techniques as a measure of interaction strength between particles and lipids. We found that positively charged particles do not adsorb on the bilayer, probably due to the electrostatic repulsion between positively charged parts of the lipid head and the positive groups on the particle surface, therefore no variation in diffusion coefficient of lipid molecules was observed. On the contrary, the negatively charged particles, driven by electrostatic interactions are adsorbed onto the supported bilayer. The adsorption of negatively charged particles increases with the zeta-potential of the particle. Consecutively, the diffusion coefficient of lipids is reduced probably due to binding onto the lipid heads which slows down their Brownian motion. The results are directly relevant for understanding the interactions of particulate matter with pulmonary structures which could lead to pulmonary surfactant inhibition or deficiency causing severe respiratory distress or pathologies.

Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli.

SummaryFluidity is essential for many biological membrane functions. The basis for understanding membrane structure remains the classic Singer-Nicolson model, in which proteins are embedded within a fluid lipid bilayer and able to diffuse laterally within a sea of lipid. Here we report lipid and protein diffusion in the plasma membrane of live cells of the bacterium Escherichia coli, using Fluorescence Recovery after Photobleaching (FRAP) and Total Internal Reflection Fluorescence (TIRF) microscopy to measure lateral diffusion coefficients. Lipid and protein mobility within the membrane were probed by visualizing an artificial fluorescent lipid and a simple model membrane protein consisting of a single membrane-spanning alpha-helix with a Green Fluorescent Protein (GFP) tag on the cytoplasmic side. The effective viscosity of the lipid bilayer is strongly temperature-dependent, as indicated by changes in the lipid diffusion coefficient. Surprisingly, the mobility of the model protein was unaffected by changes in the effective viscosity of the bulk lipid, and TIRF microscopy indicates that it clusters in segregated, mobile domains. We suggest that this segregation profoundly influences the physical behaviour of the protein in the membrane, with strong implications for bacterial membrane function and bacterial physiology.

Correlated Lateral Diffusion of Lipids

Recent years have witnessed a proliferation of publications concerning the lateral diffusion of membrane lipids. The reported diffusion coefficient (D) values appear to depend on systems under study and on techniques of investigation. There is no clear consensus regarding their dynamics that would explain the different D values as well as the relation between mobility and the radius of the diffusing object.Here we focus on the lateral diffusion of lipids, which has been less scrutinized than that of membrane proteins. Using fluorescence recovery after photobleaching, we have obtained lateral diffusion coefficients of lipids inserted into bilayers of giant unilamellar vesicles. We demonstrate that lipids diffuse with various diffusion coefficients ranging from D = 3.7 ± 0.4 to 13.9 ± 0.6 μm2/s, and that these values depend on the type of lipid. Interestingly, a transmembrane peptide, having nearly the same radius as lipids and whose hydrophobic thickness matches that of the bilayer, exhibits a D value of 9.6 ± 0.4 μm2/s. Since the lipids and the transmembrane peptide possess a similar diameter and lipids do not span membranes, the peptide diffusion coefficient is expected to be smaller than that of lipids, which is obviously not the case. Our systematic study suggests that the slower diffusion of lipids (as compared to that of the transmembrane peptide) is caused by the formation of dynamic lipid nano-patches that diffuse like a single object with an increased radius. These nano-patches seem to form more spontaneously when lipids are saturated. Consequently, one should be cautious when comparing diffusion coefficient of transmembrane proteins and lipids. Transmembrane peptides should be used as a reference instead of lipids.

Correlation Functions Provide a Universal Framework for Quantitative Analysis of Localization-Based Super-Resolution Microscopy Images

Super-resolution images reconstructed from single molecule coordinates reveal cellular structures close to the macromolecular scale. However, a widely applicable analysis tool that can extract a quantitative description and biophysical parameters from these images is still unavailable. Here, we propose a universal analysis concept for coordinate-based super-resolution images using correlation functions. We demonstrate how this analysis can quantify diffusion, localization precision, distance and co-localization. Moreover, we developed a framework for averaging multiple images by non-linear transformations to reveal structures with much higher resolution.First, we show that the commonly used pair-distance histogram is mathematically equivalent to pixel-based correlation analysis. Therefore, many strategies of pixel-based image correlation spectroscopy can also be applied to coordinate-based super-resolution images. In particular, the computational complexity of pair-distance calculations can be decreased by pixel-binning the coordinate image and employing the Fast Fourier Transform algorithm.Second, we demonstrate that the pair-distance histogram of molecule coordinates from consecutive frames allows measuring the diffusion coefficient of membrane lipids in live cells with high spatial resolution. Our analysis can handle extremely high fluorophore densities, in contrast to conventional single-particle tracking methods. The same frame-pair histogram can analogously be used to generate a map of molecule localization precision for super-resolution images, which is still elusive information.Third, we show how coordinate-based correlation functions can be used for image alignment involving transformations beyond translation. For example, we rotationally aligned multiple images of the nuclear pore complex from yeast, which exposes the octagonal complex with high precision.Finally, we propose a new correlation function, namely the point-set distance histogram, which allowed us to quantify both co-localization and distance of Clathrin to proteins of the Golgi apparatus, even though the super-resolution images were highly corrupted by background and two-color crosstalk.

Structure and properties of tethered bilayer lipid membranes with unsaturated anchor molecules.

The self-assembled monolayers (SAMs) of new lipidic anchor molecule HC18 [Z-20-(Z-octadec-9-enyloxy)-3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol] and mixed HC18/β-mercaptoethanol (βME) SAMs were studied by spectroscopic ellipsometry, contact angle measurements, reflection-absorption infrared spectroscopy, and electrochemical impedance spectroscopy (EIS) and were evaluated in tethered bilayer lipid membranes (tBLMs). Our data indicate that HC18, containing a double bond in the alkyl segments, forms highly disordered SAMs up to anchor/βME molar fraction ratios of 80/20 and result in tBLMs that exhibit higher lipid diffusion coefficients relative to those of previous anchor compounds with saturated alkyl chains, as determined by fluorescence correlation spectroscopy. EIS data shows the HC18 tBLMs, completed by rapid solvent exchange or vesicle fusion, form more easily than with saturated lipidic anchors, exhibit excellent electrical insulating properties indicating low defect densities, and readily incorporate the pore-forming toxin α-hemolysin. Neutron reflectivity measurements on HC18 tBLMs confirm the formation of complete tBLMs, even at low tether compositions and high ionic lipid compositions. Our data indicate that HC18 results in tBLMs with improved physical properties for the incorporation of integral membrane proteins (IMPs) and that 80% HC18 tBLMs appear to be optimal for practical applications such as biosensors where high electrical insulation and IMP/peptide reconstitution are imperative.

Effect of Ionic Strength on Dynamics of Supported Phosphatidylcholine Lipid Bilayer Revealed by FRAPP and Langmuir–Blodgett Transfer Ratios

To determine how lipid bilayer/support interactions are affected by ionic strength, we carried out lipid diffusion coefficient measurements by fluorescence recovery after patterned photobleaching (FRAPP) and transfer ratio measurements using a Langmuir balance on supported bilayers of phosphatidylcholine lipids. The main effect of increasing ionic strength is shown to be enhanced diffusion of the lipids due to a decrease in the electrostatic interaction between the bilayer and the support. We experimentally confirm that the two main parameters governing bilayer behavior are electrostatic interaction and bilayer/support distance. Both these parameters can therefore be used to vary the potential that acts on the bilayer. Additionally, our findings show that FRAPP is an extremely sensitive tool to study interaction effects: here, variations in diffusion coefficient as well as the presence or absence of leaflet decoupling.

Reduced Lateral Mobility of Lipids and Proteins in Crowded Membranes

Coarse-grained molecular dynamics simulations of the E. coli outer membrane proteins FhuA, LamB, NanC, OmpA and OmpF in a POPE/POPG (3∶1) bilayer were performed to characterise the diffusive nature of each component of the membrane. At small observation times (<10 ns) particle vibrations dominate phospholipid diffusion elevating the calculated values from the longer time-scale bulk value (>50 ns) of 8.5×10−7 cm2 s−1. The phospholipid diffusion around each protein was found to vary based on distance from protein. An asymmetry in the diffusion of annular lipids in the inner and outer leaflets was observed and correlated with an asymmetry in charged residues in the vicinity of the inner and outer leaflet head-groups. Protein rotational and translational diffusion were also found to vary with observation time and were inversely correlated with the radius of gyration of the protein in the plane of the bilayer. As the concentration of protein within the bilayer was increased, the overall mobility of the membrane decreased reflected in reduced lipid diffusion coefficients for both lipid and protein components. The increase in protein concentration also resulted in a decrease in the anomalous diffusion exponent α of the lipid. Formation of extended clusters and networks of proteins led to compartmentalisation of lipids in extreme cases.

Lateral Membrane Diffusion Corralled

Lateral Membrane Diffusion Corralled

ITO/poly(aniline)/sol-gel glass: An optically transparent, pH-responsive substrate for supported lipid bilayers.

Described here is fabrication of a pH-sensitive, optically transparent transducer composed of a planar indium-tin oxide (ITO) electrode overcoated with a a poly(aniline) (PANI) thin film and a porous sol-gel layer. Adsorption of the PANI film renders the ITO electrode sensitive to pH, whereas the sol-gel spin-coated layer makes the upper surface compatible with fusion of phospholipid vesicles to form a planar supported lipid bilayer (PSLB). The response to changes in the pH of the buffer contacting the sol-gel/PANI/ITO electrode is pseudo-Nernstian with a slope of 52 mV/pH over a pH range of 4-9. Vesicle fusion forms a laterally continuous PSLB on the upper sol-gel surface that is fluid with a lateral lipid diffusion coefficient of 2.2 μm2/s measured by fluorescence recovery after photobleaching. Due to its lateral continuity and lack of defects, the PSLB blocks the pH response of the underlying electrode to changes in the pH of the overlying buffer. This architecture is simpler to fabricate than previously reported ITO electrodes derivatized for PSLB formation, and should be useful for optical monitoring of proton transport across supported membranes derivatized with ionophores and ion channels.