Leaflet Of Bilayer Research Articles

Molecular collisions or resonance energy transfer in lipid vesicles? A methodology to tackle this question

In this work, molecular interactions in a lipid membrane are discussed through fluorescence spectroscopy data, both experimentally and theoretically. In particular, the fluorescence quenching mechanisms between the fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) and the potential drug 2-nitrobenzaldehyde-thiosemicarbazone (2-TSC) were studied, both inserted in a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC) model membrane. The fluorescence intensity and the lifetime of Laurdan decrease dramatically in the presence of 2-TSC, in both gel and fluid phases of the DMPC bilayer. It is shown here how to identify the correct quenching mechanism, by conducting a careful analysis of the fluorescence data. The analysis of the bimolecular constant values obtained through the Stern-Volmer equation, considering the collisional mechanism, made clear the incompatibility of the obtained values with estimated diffusion coefficients for Laurdan and 2-TSC inserted into lipid bilayers. On the other hand, using the Förster’s theory of resonance energy transfer (FRET) we obtained results in good agreement with the already known dynamic characteristics of a DMPC bilayer, at its both gel and fluid phases. Through spectroscopy data and computational calculation, Förster distance, energy transfer efficiency and distance distribution were obtained for the donor/acceptor pair Laurdan/2-TSC, at both gel and fluid phases of the bilayer. The distance distribution reflects the occurrence of FRET involving donor/acceptor pairs in the same leaflet of the lipid bilayer and pairs in opposite leaflet, and these results are in good agreement with our previous proposal about the lateral organization and position of Laurdan and 2-TSC molecules in a DMPC bilayer. All these results lead us to conclude that FRET between the donor Laurdan and the acceptor 2-TSC is the mechanism responsible for non-radiative deexcitation of Laurdan. The methodology used here could be extended to other pairs of donor/acceptor molecules, to contribute to the knowledge about their localizations in lipid membranes.

An Aggregation-Induced Emission Optical Highlighter for the Studies of Endoplasmic Reticulum-Lipid Droplet Content Dynamics

An Aggregation-Induced Emission Optical Highlighter for the Studies of Endoplasmic Reticulum-Lipid Droplet Content Dynamics

Model Membrane Systems Used to Study Plasma Membrane Lipid Asymmetry.

It is well known that the lipid distribution in the bilayer leaflets of mammalian plasma membranes (PMs) is not symmetric. Despite this, model membrane studies have largely relied on chemically symmetric model membranes for the study of lipid–lipid and lipid–protein interactions. This is primarily due to the difficulty in preparing stable, asymmetric model membranes that are amenable to biophysical studies. However, in the last 20 years, efforts have been made in producing more biologically faithful model membranes. Here, we review several recently developed experimental and computational techniques for the robust generation of asymmetric model membranes and highlight a new and particularly promising technique to study membrane asymmetry.

Initial Stages of Spontaneous Binding of Folate-Based Vectors to Folate Receptor-α Observed by Unbiased Molecular Dynamics.

Active targeting is a prospective strategy for controlled drug delivery to malignant tumor tissues. One of the approaches relies on recognition of a bioactive ligand by a receptor expressed abundantly on the surface of cancer cell membranes. A promising ligand-receptor pair is folic acid (or its dianionic form, folate) combined with the folate receptor-α (FRα). A number of targeting drug delivery systems based on folate have been suggested, but the mechanism of binding of the ligand or its derivatives to the receptor is not fully known at the molecular level. The current study summarizes the results from unbiased all-atom molecular dynamics simulations at physiological conditions describing the binding of two forms of folate and four of its synthetically available derivatives to FRα. The models (ca. 185,000 atoms) contain one receptor molecule, embedded in the outer leaflet of a lipid bilayer, and one ligand, all immersed in saline. The bilayer represents a human cancer cell membrane and consists of 370 asymmetrically distributed lipid molecules from 35 types. The ability of the vector molecules to bind to the receptor, the position of binding, and the interactions between them are analyzed. Spontaneous binding on the nanosecond scale is observed for all molecules, but its time, position, and persistence depend strongly on the ligand. Only folate, 5-methyltetrahydrofolate, and raltitrexed bind selectively at the active site of the receptor. Two binding poses are observed, one of them (realized by raltitrexed) corresponding qualitatively to that reported for the crystallographic structure of the complex folate-FRα. Pemetrexed adsorbs nonspecifically on the protein surface, while methotrexate and pteroyl ornithine couple much less to the receptor. The molecular simulations reproduce qualitatively correctly the relative binding affinity measured experimentally for five of the ligands. Analysis of the interactions between the ligands and FRα shows that in order to accomplish specific binding to the active site, a combination of hydrogen bonding, π-stacking, and van der Waals and Coulomb attraction should be feasible simultaneously for the vector molecule. The reported results demonstrate that it is possible to observe receptor-ligand binding without applying bias by representing the local environment as close as possible and contain important molecular-level guidelines for the design of folate-based systems for targeted delivery of anticancer drugs.

Physical Characterization of Triolein and Implications for Its Role in Lipid Droplet Biogenesis.

Lipid droplets (LDs) are neutral lipid-storing organelles surrounded by a phospholipid (PL) monolayer. At present, how LDs are formed in the endoplasmic reticulum (ER) bilayer is poorly understood. In this study, we present a revised all-atom (AA) triolein (TG) model, the main constituent of the LD core, and characterize its properties in a bilayer membrane to demonstrate the implications of its behavior in LD biogenesis. In bilayer simulations, TG resides at the surface, adopting PL-like conformations (denoted in this work as SURF-TG). Free energy sampling simulation results estimate the barrier for TG relocating from the bilayer surface to the bilayer center to be ∼2 kcal/mol in the absence of an oil lens. SURF-TG is able to modulate membrane properties by increasing PL ordering, decreasing bending modulus, and creating local negative curvature. The other neutral lipid, dioleoyl-glycerol (DAG), also reduces the membrane bending modulus and populates negative curvature regions. A phenomenological coarse-grained (CG) model is also developed to observe larger-scale SURF-TG-mediated membrane deformation. CG simulations confirm that TG nucleates between the bilayer leaflets at a critical concentration when SURF-TG is evenly distributed. However, when one monolayer contains more SURF-TG, the membrane bends toward the other leaflet, followed by TG nucleation if a concentration is higher than the critical threshold. The central conclusion of this study is that SURF-TG is a negative curvature inducer, as well as a membrane modulator. To this end, a model is proposed in which the accumulation of SURF-TG in the luminal leaflet bends the ER bilayer toward the cytosolic side, followed by TG nucleation.

Species-Specific Urothelial Toxicity With an Anti-HIV Noncatalytic Site Integrase Inhibitor (NCINI) Is Related to Unusual pH-Dependent Physicochemical Changes.

GS-9695 and GS-9822 are next-generation noncatalytic site integrase inhibitors (NCINIs) with significantly improved potency against human immunodeficiency virus compared with previous drugs such as BI-224436. Development stopped due to vacuolation of the bladder urothelium seen in cynomolgus monkey but not in rat; this lesion was absent in equivalent preclinical studies with BI-224436 (tested in dog and rat). Lesions were unlikely to be attributable to target because NCINIs specifically target viral integrase protein and no mammalian homologue is known. Secondary pharmacology studies, mitochondrial toxicity studies, immunophenotyping, and analysis of proteins implicated in cell-cell interactions and/or bladder integrity (E-cadherin, pan-cytokeratin, uroplakins) failed to offer any plausible explanation for the species specificity of the lesion. Because it was characterized by inflammation and disruption of urothelial morphology, we investigated physicochemical changes in the bladder of cynomolgus monkey (urinary pH 5.5-7.4) that might not occur in the bladder of rats (urinary pH 7.3-8.5). In measurements of surface activity, GS-9822 showed an unusual transition from a monolayer to a bilayer at the air/water interface with decreasing pH, attributed to the strong association between drug molecules in adjacent bilayer leaflets and expected to be highly disruptive to the urothelium. Structural analysis of GS-9822 and GS-9695 showed zwitterionic characteristics over the range of pH expected in cynomolgus monkey but not rat urine. This exotic surface behavior is unlikely with BI-224436 since it would transition from neutral to cationic (never zwitterionic) with decreasing pH. These data provide useful insights to guide discovery and development of NCINIs, related compounds, and zwitterions.

Mechanical properties of anionic asymmetric bilayers from atomistic simulations.

Mechanotransduction, the biological response to mechanical stress, is often initiated by activation of mechanosensitive (MS) proteins upon mechanically induced deformations of the cell membrane. A current challenge in fully understanding this process is in predicting how lipid bilayers deform upon the application of mechanical stress. In this context, it is now well established that anionic lipids influence the function of many proteins. Here, we test the hypothesis that anionic lipids could indirectly modulate MS proteins by alteration of the lipid bilayer mechanical properties. Using all-atom molecular dynamics simulations, we computed the bilayer bending rigidity (KC), the area compressibility (KA), and the surface shear viscosity (ηm) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) lipid bilayers with and without phosphatidylserine (PS) or phosphatidylinositol bisphosphate (PIP2) at physiological concentrations in the lower leaflet. Tensionless leaflets were first checked for each asymmetric bilayer model, and a formula for embedding an asymmetric channel in an asymmetric bilayer is proposed. Results from two different sized bilayers show consistently that the addition of 20% surface charge in the lower leaflet of the PC bilayer with PIP2 has minimal impact on its mechanical properties, while PS reduced the bilayer bending rigidity by 22%. As a comparison, supplementing the PIP2-enriched PC membrane with 30% cholesterol, a known rigidifying steroid lipid, produces a significant increase in all three mechanical constants. Analysis of pairwise splay moduli suggests that the effect of anionic lipids on bilayer bending rigidity largely depends on the number of anionic lipid pairs formed during simulations. The potential implication of bilayer bending rigidity is discussed in the framework of MS piezo channels.

An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial␣membrane

Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high‐resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure‐guided mutations demonstrate the importance of both types of protein–lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.

Extreme deformability of insect cell membranes is governed by phospholipid scrambling.

Organization of dynamic cellular structure is crucial for a variety of cellular functions. In this study, we report that Drosophila and Aedes have highly elastic cell membranes with extremely low membrane tension and high resistance to mechanical stress. In contrast to other eukaryotic cells, phospholipids are symmetrically distributed between the bilayer leaflets of the insect plasma membrane, where phospholipid scramblase (XKR) that disrupts the lipid asymmetry is constitutively active. We also demonstrate that XKR-facilitated phospholipid scrambling promotes the deformability of cell membranes by regulating both actin cortex dynamics and mechanical properties of the phospholipid bilayer. Moreover, XKR-mediated construction of elastic cell membranes is essential for hemocyte circulation in the Drosophila cardiovascular system. Deformation of mammalian cells is also enhanced by the expression of Aedes XKR, and thus phospholipid scrambling may contribute to formation of highly deformable cell membranes in a variety of living eukaryotic cells.

TMEM41B acts as an ER scramblase required for lipoprotein biogenesis and lipid homeostasis

How amphipathic phospholipids are shuttled between the membrane bilayer remains an essential but elusive process, particularly at the endoplasmic reticulum (ER). One prominent phospholipid shuttling process concerns the biogenesis of APOB-containing lipoproteins within the ER lumen, which may require bulk trans-bilayer movement of phospholipids from the cytoplasmic leaflet of the ER bilayer. Here, we show that TMEM41B, present in the lipoprotein export machinery, encodes a previously conceptualized ER lipid scramblase mediating trans-bilayer shuttling of bulk phospholipids. Loss of hepatic TMEM41B eliminates plasma lipids, due to complete absence of mature lipoproteins within the ER, but paradoxically also activates lipid production. Mechanistically, scramblase deficiency triggers unique ER morphological changes and unsuppressed activation of SREBPs, which potently promotes lipid synthesis despite stalled secretion. Together, this response induces full-blown nonalcoholic hepatosteatosis in the TMEM41B-deficient mice within weeks. Collectively, our data uncovered a fundamental mechanism safe-guarding ER function and integrity, dysfunction of which disrupts lipid homeostasis.

Membrane Interactions of Virus-like Mesoporous Silica Nanoparticles.

In the present study, we investigated lipid membrane interactions of silica nanoparticles as carriers for the antimicrobial peptide LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES). In doing so, smooth mesoporous nanoparticles were compared to virus-like mesoporous nanoparticles, characterized by a "spiky" external surface, as well as to nonporous silica nanoparticles. For this, we employed a combination of neutron reflectometry, ellipsometry, dynamic light scattering, and ζ-potential measurements for studies of bacteria-mimicking bilayers formed by palmitoyloleoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol. The results show that nanoparticle topography strongly influences membrane binding and destabilization. We found that virus-like particles are able to destabilize such lipid membranes, whereas the corresponding smooth silica nanoparticles are not. This effect of particle spikes becomes further accentuated after loading of such particles with LL-37. Thus, peptide-loaded virus-like nanoparticles displayed more pronounced membrane disruption than either peptide-loaded smooth nanoparticles or free LL-37. The structural basis of this was clarified by neutron reflectometry, demonstrating that the virus-like nanoparticles induce trans-membrane defects and promote incorporation of LL-37 throughout both bilayer leaflets. The relevance of such effects of particle spikes for bacterial membrane rupture was further demonstrated by confocal microscopy and live/dead assays on Escherichia coli bacteria. Taken together, these findings demonstrate that topography influences the interaction of nanoparticles with bacteria-mimicking lipid bilayers, both in the absence and presence of antimicrobial peptides, as well as with bacteria. The results also identify virus-like mesoporous nanoparticles as being of interest in the design of nanoparticles as delivery systems for antimicrobial peptides.

Of membranes and malaria: phospholipid asymmetry in Plasmodium falciparum-infected red blood cells.

Malaria is a vector-borne parasitic disease with a vast impact on human history, and according to the World Health Organisation, Plasmodium parasites still infect over 200 million people per year. Plasmodium falciparum, the deadliest parasite species, has a remarkable ability to undermine the host immune system and cause life-threatening disease during blood infection. The parasite's host cells, red blood cells (RBCs), generally maintain an asymmetric distribution of phospholipids in the two leaflets of the plasma membrane bilayer. Alterations to this asymmetry, particularly the exposure of phosphatidylserine (PS) in the outer leaflet, can be recognised by phagocytes. Because of the importance of innate immune defence numerous studies have investigated PS exposure in RBCs infected with P. falciparum, but have reached different conclusions. Here we review recent advancements in our understanding of the molecular mechanisms which regulate asymmetry in RBCs, and whether infection with the P. falciparum parasite results in changes to PS exposure. On the balance of evidence, it is likely that membrane asymmetry is disrupted in parasitised RBCs, though some methodological issues need addressing. We discuss the potential causes and consequences of altered asymmetry in parasitised RBCs, particularly for in vivo interactions with the immune system, and the role of host-parasite co-evolution. We also examine the potential asymmetric state of parasite membranes and summarise current knowledge on the parasite proteins, which could regulate asymmetry in these membranes. Finally, we highlight unresolved questions at this time and the need for interdisciplinary approaches to uncover the machinery which enables P. falciparum parasites to hide in mature erythrocytes.

Molecular Simulation of Properties of the Long Periodicity Phase in the Stratum Corneum

The stratum corneum (SC) layer of the human skin is the outermost and primary barrier against chemical topical exposure including cosmetics. The ability of a chemical to pass the SC is a key point for risk assessment as well as for drug development. Thus a simulation model of the skin permeability mechanism is of significant translational importance. From neutron diffraction data, the SC peaks comprise a long periodicity phase (LPP) with a repeat distance of 13 nm and a short periodicity phase (SPP) with a repeat distance of 6 nm. The LPP captures long-scale dynamics and can be adequately assigned to a molecular construct termed a sandwich model by Bouwstra, which comprises two bilayers surrounding a fluid interior slab with lipid headgroups pointing in both directions, i.e. a bilayer-slab-bilayer arrangement. A long ceramide-1 (CerEOS) populates only the outer leaflet of the bilayer, penetrating into the inner bilayer leaflet. A systematic study of transdermal solute penetration through the LPP has not yet been performed. Here, we employ microsecond MD simulations in combination with a non-equilibrium pulling methodology to calculate free-energies and permeabilities of transdermal penetration of a set of compounds (e.g. caffeine) through a model of the LPP studied by Groen et al. composed of Ceramide-1 (CerEOS), Ceramide-2 (CerNS), Ceramide-3 (CerNP), Chol, and free-fatty acid (LA), with an equimolar ratio of ceramide, cholesterol and free-fatty acid.

Challenging the Dogma - Cell Plasma Membranes are Asymmetric not only in Phospholipid Composition but also Abundance

The transbilayer distribution of lipids in mammalian plasma membranes (PMs) is functionally important and incompletely understood. It has been generally assumed that the two PM leaflets contain similar number of phospholipids (PLs) due to the constraint that their areas must be matched in a bilayer. Contrary to this widely held assumption, our recent detailed lipidomics analysis of human erythrocytes reveals a large phospholipid imbalance between PM leaflets, with the cytoplasmic leaflet possessing almost 2-fold more PLs as the exoplasmic one. This surprising finding is consistent with several previous observations and challenges our understanding of living membrane organization and structure. To investigate the effects of differential PL abundance on membrane properties, we performed long atomistic molecular dynamics simulations of various PM models with data-driven leaflet compositions and different PL imbalances between the leaflets. The integrity of the bilayer was preserved in part by (re)distribution of the PM's other major component, cholesterol, which shows a strong preference for the exoplasmic leaflet. Detailed leaflet-specific analysis of the physical properties of the membranes revealed systematic changes as the relative number of PLs in the exoplasmic leaflet decreased. These changes were accompanied by differences in the water permeability of the leaflets, their pressure distribution and propensity for hydrophobic defects. To determine which PM model most closely represents the cell PM we calibrated an in vitro reporter of membrane packing to simulations using experimental measurements of model membranes, and compared the simulated models to live cells PMs. Our results were consistent with a model of living PMs with far more cholesterol and fewer PLs in the exoplasmic leaflet. They also reveal a substantial amount of stored tension in the bilayer leaflets, uncovering a host of previously unconsidered physiological implications.

Coupling between Lateral and Transverse Organization in Three-Component Lipid Mixtures Investigated with Asymmetric GUVs Prepared by Hemifusion

Asymmetry is a ubiquitous feature of biological plasma membranes. Asymmetric model membranes allow a means of investigating molecular determinants of coupling between the inner and outer leaflets of the lipid bilayer. Here, we report a partial phase diagram for asymmetric bilayers composed of the ternary mixture DPPC, DOPC, and cholesterol. Symmetric giant unilamellar vesicles (GUVs) of DPPC/DOPC/Cholesterol with compositions along a tie-line in the symmetric composition space were prepared by electroformation.

Stiffening Transition in Asymmetric Lipid Bilayers: The Role of Highly-Ordered Domains and the Effect of Temperature and Size

Cellular membranes consist of a large variety of lipids and proteins, with a composition that generally differs between the two leaflets of the same bilayer. One consequence of this asymmetry is thought to be the emergence of differential stress, i.e., a mismatch in the lateral tension of the two leaflets. This can affect a membrane’s mechanical properties; for instance, it can increase the bending rigidity once the differential stress exceeds a critical threshold. Using coarse-grained molecular dynamics simulations based on the MARTINI model, we show that this effect arises due to the formation of more highly ordered domains in the compressed leaflet. The threshold asymmetry increases with temperature, indicating that the transition to a stiffened regime might be restricted to a limited temperature range above the gel transition. We also show that stiffening occurs more readily for larger membranes with smaller typical curvatures, suggesting that the stiffening transition is easier to observe experimentally than in the small-scale systems accessible to simulation.

Structure of the Yeast Seipin Reveals Determinants for Lipid Droplet Biogenesis

Lipid droplets (LDs) are ubiquitous storage organelles for neutral lipids, mainly triacylglycerols and steryl esters. LDs have a unique structure consisting of a phospholipid monolayer enclosing a neutral lipid core. LDs form at the endoplasmic reticulum (ER) by unclear mechanisms. In the most popular model, LDs assemble at discrete ER domains by the budding of neutral lipid lenses formed in between of the two leaflets of the ER bilayer. Seipin has emerged as a key LD biogenesis factor and mutations are responsible for lipodystrophy in humans.

Cation-Selectivity of a Self-Assembled Peptide Pore in Planar Phospholipid Bilayers using Electrical Impedance Spectroscopy

GALA is a 30-residue, amphipathic peptide that self-assembles into multimeric, transmembrane pores in a pH-dependent fashion. Based on kinetic data and dye release assays, previous studies have reported that in liposomes GALA forms large multimeric pores composed of 8-12 peptides, but no direct structural information is available. In this study, we characterize the pore size and multimeric structure of GALA pores in planar phospholipid bilayers using electrical impedance spectroscopy (EIS) and molecular dynamics (MD) simulations. Specifically, we used a number of alkaline earth cations and the large organic cations, Choline and TEA, to estimate the pore size by measuring concentration-dependent changes in membrane conduction. This data was combined with MD simulations to estimate the multimeric state of GALA pores. We report that in planar tethered lipid bilayers composed of POPC, GALA pores likely consist of six peptide monomers rather than eight to twelve monomers as previously reported in liposomes. We further demonstrate, for the first time, that GALA exhibits cation selectivity, which is based on the free energy of hydration of the ions. We propose that the difference in predicted pore structure between planar bilayers and lipid vesicles exemplifies the importance of phospholipid bilayer structure, which include lipid packing, area per lipid, hydration or charge distribution caused by asymmetry between the inner and outer bilayer leaflets. These bilayer properties could have significant effects on the aggregation of transmembrane helical structures.

Computational Insight Into the Mechanism of SARS-CoV-2 Membrane Fusion.

Membrane fusion, a key step in the early stages of virus propagation, allows the release of the viral genome in the host cell cytoplasm. The process is initiated by fusion peptides that are small, hydrophobic components of viral membrane-embedded glycoproteins and are typically conserved within virus families. Here, we attempted to identify the correct fusion peptide region in the Spike protein of SARS-CoV-2 by all-atom molecular dynamics simulations of dual membrane systems with varied oligomeric units of putative candidate peptides. Of all of the systems tested, only a trimeric unit of a 40-amino-acid region (residues 816-855 of SARS-CoV-2 Spike) was effective in triggering the initial stages of membrane fusion, within 200 ns of simulation time. Association of this trimeric unit with dual membranes resulted in the migration of lipids from the upper leaflet of the lower bilayer toward the lower leaflet of the upper bilayer to create a structural unit reminiscent of a fusion bridge. We submit that residues 816-855 of Spike represent the bona fide fusion peptide of SARS-CoV-2 and that computational methods represent an effective way to identify fusion peptides in viral glycoproteins.

Stiffening transition in asymmetric lipid bilayers: The role of highly ordered domains and the effect of temperature and size.

Cellular membranes consist of a large variety of lipids and proteins, with a composition that generally differs between the two leaflets of the same bilayer. One consequence of this asymmetry is thought to be the emergence of differential stress, i.e., a mismatch in the lateral tension of the two leaflets. This can affect a membrane's mechanical properties; for instance, it can increase the bending rigidity once the differential stress exceeds a critical threshold. Using coarse-grained molecular dynamics simulations based on the MARTINI model, we show that this effect arises due to the formation of more highly ordered domains in the compressed leaflet. The threshold asymmetry increases with temperature, indicating that the transition to a stiffened regime might be restricted to a limited temperature range above the gel transition. We also show that stiffening occurs more readily for larger membranes with smaller typical curvatures, suggesting that the stiffening transition is easier to observe experimentally than in the small-scale systems accessible to simulation.