Negative Spontaneous Curvature Research Articles

Effects of Spontaneous Curvature on Interfacial Adsorption and Collapse of Phospholipid Monolayers.

To function effectively, pulmonary surfactant must adsorb rapidly to the alveolar air/water interface, but avoid collapse from the interface when compressed to low surface tensions. Prior studies show that phospholipids in the cylindrical monolayers of the inverse hexagonal (HII) phase adsorb quickly. The monolayers have negative curvature, defined by the concave shape of the hydrophilic face. Formation of the HII structures, however, involves significant disruption of optimal chain-packing. Samples with significant spontaneous curvature, formed in the absence of applied force, may nonetheless have lamellar structures. The experiments here tested whether planar lamellar bilayers formed by phospholipids with negative spontaneous curvature might adsorb rapidly but collapse slowly. Prior studies have shown that binary mixtures of dioleoyl phosphatidylcholine-dioleoyl phosphatidylethanolamine (DOPC-DOPE) with higher mol fractions of DOPE (XPE) have more negative spontaneous curvature. Samples with higher XPE adsorbed more rapidly but also collapsed more quickly. Over that range of XPE, small angle X-ray scattering showed only lamellar structures. The HII phase was undetectable. The results suggest that the innate tendency of the phospholipids to form curved structures has primary importance for adsorption rather than the presence of HII structures. Planar structures are insufficient to minimize the tendency of spontaneous curvature to promote collapse. These findings are consistent with adsorption and collapse that occur via rate-limiting structures with significant negative curvature.

Homeocurvature adaptation of phospholipids to pressure in deep-sea invertebrates.

Hydrostatic pressure increases with depth in the ocean, but little is known about the molecular bases of biological pressure tolerance. We describe a mode of pressure adaptation in comb jellies (ctenophores) that also constrains these animals' depth range. Structural analysis of deep-sea ctenophore lipids shows that they form a nonbilayer phase at pressures under which the phase is not typically stable. Lipidomics and all-atom simulations identified phospholipids with strong negative spontaneous curvature, including plasmalogens, as a hallmark of deep-adapted membranes that causes this phase behavior. Synthesis of plasmalogens enhanced pressure tolerance in Escherichia coli, whereas low-curvature lipids had the opposite effect. Imaging of ctenophore tissues indicated that the disintegration of deep-sea animals when decompressed could be driven by a phase transition in their phospholipid membranes.

Lipids with negative spontaneous curvature decrease the solubility of the cancer drug paclitaxel in liposomes.

Paclitaxel (PTX) is a hydrophobic small-molecule cancer drug that loads into the membrane (tail) region of lipid carriers such as liposomes and micelles. The development of improved lipid-based carriers of PTX is an important objective to generate chemotherapeutics with fewer side effects. The lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and glyceryl monooleate (GMO) show propensity for fusion with other lipid membranes, which has led to their use in lipid vectors of nucleic acids. We hypothesized that DOPE and GMO could enhance PTX delivery to cells through a similar membrane fusion mechanism. As an important measure of drug carrier performance, we evaluated PTX solubility in cationic liposomes containing GMO or DOPE. Solubility was determined by time-dependent kinetic phase diagrams generated from direct observations of PTX crystal formation using differential-interference-contrast optical microscopy. Remarkably, PTX was much less soluble in these liposomes than in control cationic liposomes containing univalent cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), which are not fusogenic. In particular, PTX was not substantially soluble in GMO-based cationic liposomes. The fusogenicity of DOPE and GMO is related to the negative spontaneous curvature of membranes containing these lipids, which drives formation of nonlamellar self-assembled phases (inverted hexagonal or gyroid cubic). To determine whether PTX solubility is governed by lipid membrane structure or by local intermolecular interactions, we used synchrotron small-angle X-ray scattering. To increase the signal/noise ratio, we used DNA to condense the lipid formulations into lipoplex pellets. The results suggest that local intermolecular interactions are of greater importance and that the negative spontaneous curvature-inducing lipids DOPE and GMO are not suitable components of liposomal carriers for PTX delivery.

Membrane inward/outward budding and transition pathway induced by the asymmetric solutions

The study of membrane morphology transitions is crucial for understanding various biological processes, such as cell division, and intracellular vesicle transport. In this work, we aim to clarify the molecular mechanism underlying the asymmetric solution-induced membrane shape transition in lipid bilayers. Using giant unilamellar vesicles (GUVs) as model membranes, we investigated the interaction of fully hydrated lipid membranes with two distinct sugar solutions—glucose and sucrose. We applied advanced imaging techniques and theoretical analyses to study membrane deformation and its transition pathways. Our experimental observations revealed that asymmetric sugar solutions induce spontaneous curvature in the phospholipid bilayer, resulting in the formation of outward(/inward) budding vesicles over a wide range of parameters. The direction of the curvature can be reversed by exchanging the internal and external solutions, indicating that the sugar-lipid interactions generate a significant positive(/negative) spontaneous curvature. Theoretically, we attempted to determine the parameter range of stable membrane shapes (i.e., phase diagram) using two dimensionless physical parameters—volume-to-area ratio and normalized spontaneous curvature. Obtaining a phase diagram helps identify the transition pathways of GUVs membrane shapes (experimentally, we captured two possible outward budding vesicles and one inward budding vesicle transition pathway). Our findings can be extended to other types of lipid membranes and provide new insights into the deformation of cell-like micron-sized vesicles, such as exosomes and endosomes, which are increasingly used as biomarkers and drug delivery systems. Moreover, lipid vesicles containing anticancer drugs exhibit fewer side effects than non-liposomal anticancer agents and can effectively target solid tumors.

Surfactin molecules with a cone-like structure promote the formation of membrane domains with negative spontaneous curvature and induce membrane invaginations

Surfactin uniquely influences lipid bilayer structure by initially inducing membrane invaginations before solubilization. In this study, we exposed DOPC giant vesicles to various surfactin concentrations at different temperatures and observed surfactin-induced membrane invaginations by using differential interference contrast and confocal laser fluorescence microscopy. These invaginations were stable at room temperature but not at higher temperatures. Surfactin molecules induce membrane nanodomains with negative spontaneous curvature and membrane invaginations despite their intrinsic conical shape and intrinsic positive curvature. Considering the experimentally observed capacity of surfactin to fluidize lipid acyl chains and induce partial dehydration of lipid headgroups, we propose that the resulting surfactin-lipid complexes exhibit a net negative spontaneous curvature. We further conducted 3D numerical Monte Carlo (MC) simulations to investigate the behaviour of vesicles containing negative curvature nanodomains within their membrane at varying temperatures. MC simulations demonstrated strong agreement with experimental results, revealing that invaginations are preferentially formed at low temperatures, while being less pronounced at elevated temperatures. Our findings go beyond the expectations of the Israelachvili molecular shape and packing concepts analysis. These concepts do not take into account the influence of specific interactions between neighboring molecules on the inherent shapes of molecules and their arrangement within curved membrane nanodomains. Our work contributes to a more comprehensive understanding of the complex factors governing vesicle morphology and membrane organization and provides insight into the role of detergent-lipid interactions in modulating vesicle morphology.

Peptide Flexibility and the Hydrophobic Moment are Determinants to Evaluate the Clinical Potential of Magainins.

Using a flexibility prediction algorithm and in silico structural modeling, we have calculated the intrinsic flexibility of several magainin derivatives. In the case of magainin-2 (Mag-2) and magainin H2 (MAG-H2) we have found that MAG-2 is more flexible than its hydrophobic analog, Mag-H2. This affects the degree of bending of both peptides, with a kink around two central residues (R10, R11), whereas, in Mag-H2, W10 stiffens the peptide. Moreover, this increases the hydrophobic moment of Mag-H2, which could explain its propensity to form pores in POPC model membranes, which exhibit near-to-zero spontaneous curvatures. Likewise, the protective effect described in DOPC membranes for this peptide regarding its facilitation in pore formation would be related to the propensity of this lipid to form membranes with negative spontaneous curvature. The flexibility of another magainin analog (MSI-78) is even greater than that of Mag-2. This facilitates the peptide to present a kind of hinge around the central F12 as well as a C-terminal end prone to be disordered. Such characteristics are key to understanding the broad-spectrum antimicrobial actions exhibited by this peptide. These data reinforce the hypothesis on the determinant role of spontaneous membrane curvature, intrinsic peptide flexibility, and specific hydrophobic moment in assessing the bioactivity of membrane-active antimicrobial peptides.

Length matters: Functional flip of the short TatA transmembrane helix

The twin arginine translocase (Tat) exports folded proteins across bacterial membranes. The putative pore-forming or membrane-weakening component (TatAd in B. subtilis) is anchored to the lipid bilayer via an unusually short transmembrane α-helix (TMH), with less than 16 residues. Its tilt angle in different membranes was analyzed under hydrophobic mismatch conditions, using synchrotron radiation circular dichroism and solid-state NMR. Positive mismatch (introduced either by reconstitution in short-chain lipids or by extending the hydrophobic TMH length) increased the helix tilt of the TMH as expected. Negative mismatch (introduced either by reconstitution in long-chain lipids or by shortening the TMH), on the other hand, led to protein aggregation. These data suggest that the TMH of TatA is just about long enough for stable membrane insertion. At the same time, its short length is a crucial factor for successful translocation, as demonstrated here in native membrane vesicles using an in vitro translocation assay. Furthermore, when reconstituted in model membranes with negative spontaneous curvature, the TMH was found to be aligned parallel to the membrane surface. This intrinsic ability of TatA to flip out of the membrane core thus seems to play a key role in its membrane-destabilizing effect during Tat-dependent translocation.

Studying KcsA Channel Clustering Using Single Channel Voltage-Clamp Fluorescence Imaging.

Oligomerization and complex formation play a key role for many membrane proteins and has been described to influence ion channel function in both neurons and the heart. In this study, we observed clustering of single KcsA channels in planar lipid bilayer using single molecule fluorescence, while simultaneously measuring single channel currents. Clustering coincided with cooperative opening of KcsA. We demonstrate that clustering was not caused by direct protein-protein interactions or hydrophobic mismatch with the lipid environment, as suggested earlier, but was mediated via microdomains induced by the channel in the lipid matrix. We found that single channel activity of KcsA requires conically-shaped lipids in the lamellar liquid-crystalline (Lα) phase, and the need for a negative spontaneous curvature seem to lead to the deformations in the membrane that cause the clustering. The method introduced here will be applicable to follow oligomerization of a wide range of membrane proteins.

Modulation of GPCR Rhodopsin Function by Membrane Lipids and Water

G protein‐coupled receptors (GPCRs) are responsible for the transduction of signals across lipid membranes and are the largest family of targets (~35%) for currently approved drugs. They exist as dynamic conformational ensembles with multiple inactive and active conformational substates described by an energy landscape model. Consequently, understanding the modulation of conformational dynamics of GPCRs by soft matter such as membrane lipids and cellular water in the receptor’s environment is critical to understanding GPCR efficacy and selectivity. Here we show how membrane lipids and cellular water play crucial roles in shaping the energy landscape of GPCR activation, thus acting as allosteric modulators. We investigated ways in which the receptor hydration level and lipid bilayer composition influence the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native and POPC recombinant membranes after light activation. Using different polyethylene glycol (PEG) solutions, the osmotic pressure on rhodopsin was varied, while shifting of the metarhodopsin equilibrium was probed using UV‐Visible spectroscopy. Our results show a flood of ~80‐100 water molecules into the rhodopsin interior during photoactivation, forming a solvent‐swollen Meta‐II active state. Dehydrating conditions favor Meta‐I through the efflux of water from the protein interior yet simultaneously increase bilayer thickness, which should favor Meta‐II. Because the osmotic effect on the protein is more significant than the effect of the lipid bilayer, the overall equilibrium is generally shifted to Meta‐I. However, small osmolytes favored the Meta‐II state because they can penetrate the protein core, decreasing the osmotic effect on the protein. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta‐I state in POPC recombinant membranes compared to the native membrane environment. Analysis of transducin C‐terminal peptide‐binding isotherms revealed that the G‐prottein binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. The POPC lipid membrane has zero spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta‐I state. By contrast, the native lipid membrane environment possesses a negative spontaneous curvature that favors the more expanded state of Meta‐II. Our results delineate the crucial role of soft matter in regulating the metarhodopsin equilibrium in a membrane environment.

Influence of lipid bilayer composition on the activity of antimicrobial quaternary ammonium ionenes, the interplay of intrinsic lipid curvature and polymer hydrophobicity, the role of cardiolipin

Incorporation of hydrophobic component into amphiphilic polycations structure is frequently accompanied by an increase of antimicrobial activity. There is, however, a group of relatively hydrophilic polycations containing quaternary ammonium moieties along mainchain, ionenes, which also display strong antimicrobial and limited hemolytic properties. In this work, an influence of a hydrophobic side group length on antimicrobial mechanism of action is investigated in a series of novel amphiphilic ionenes. High antimicrobial activity was found by determination of minimum inhibitory concentration (MIC) and minimum bactericidal, and fungicidal concentration (MBC and MFC) in both growth media and a buffer. Biocompatibility was estimated by hemolytic and mammalian cells viability assays. Mechanistic studies were performed using large unilamellar vesicles (LUVs) with different lipid composition, as simplified models of cell membranes. The investigated ionenes are potent and selective antimicrobial molecules displaying a decrease of antimicrobial activity correlated with increase of hydrophobicity. Studies using LUVs revealed that the cardiolipin is an essential component responsible for the lipid bilayer permeabilization by investigated ionens. In contrast to relatively hydrophilic ionenes, more hydrophobic polymers showed an ability to stabilize membranes composed of lipids with negative spontaneous curvature in a certain range of polymer to lipid ratio. The results substantially contribute to the understanding of antimicrobial activity of the investigated class of polymers.

Membrane Lipids and Cellular Water Modulate the G‐Protein–Coupled Receptor Activation

G-protein-coupled receptors (GPCRs) are a pivotal superfamily of seven-helix transmembrane receptors responsible for the transduction of stimuli across cellular membranes. They are the targets of ~30% of the pharmaceuticals currently in the market. Understanding how the soft membrane matter (membrane Lipids and Cellular Water) influences the structural changes during the activation of GPCRs is crucial to discovering and developing novel GPCR-targeted drugs. Their dynamic conformational ensembles encompassing various inactive and active states can be biased by the lipids and surrounding aqueous environments [1]. By using different polyethylene glycol (PEG) solutions, we explored the effect of osmotic pressure and lipid bilayer composition on the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native membranes and POPC recombinant membranes [2]. Our results show a flood of ~80 water molecules into the rhodopsin interior during photoactivation, forming a solvent-swollen Meta-II active state [3]. Under applied osmotic pressure, the overall equilibrium generally shifted to Meta-I. Dehydrating conditions favor Meta-I through the efflux of water from the protein interior while increasing bilayer thickness and the monolayer spontaneous curvature favor Meta-II. The osmotic effect on the protein is more significant than the effect of the lipid bilayer. However, small osmolytes favored the Meta-II state at lower concentrations because they can penetrate the protein core giving a lower excluded volume, decreasing the osmotic effect on the protein and favoring the Meta-II state. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta-I state in POPC recombinant membranes compared to the native membrane environment. Analysis of transducin C-terminal peptide-binding isotherms revealed that the binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. The POPC lipid membrane has zero-spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta-I state. By contrast, the native lipid membrane environment has a negative spontaneous curvature that favors the more expanded state of Meta-II. Our results delineate the crucial role of soft matter in regulating the metarhodopsin equilibrium in a membrane environment. [1] M.F. Brown (2017) Annu. Rev. Biophys. 46, 379-410. [2] N. Weerasinghe et al. (2018) Biophys. J. 114, 274a. [3] U. Chawla et al. (2020) Angew. Chem. Int. Ed. doi: https://doi.org/10.1002/ange.202003342.

Solvation Drives G-Protein-Coupled Receptor Activation

G-protein–coupled receptors (GPCRs) are membrane proteins that transduce information across lipid bilayers. Their dynamic conformational ensembles encompassing various inactive and active states can be biased by the lipids and surrounding aqueous environments. By using different polyethylene glycol (PEG) solutions, we explored the effect of osmotic pressure and lipid bilayer composition on the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native membranes and POPC recombinant membranes. Our results show a flood of ∼80 water molecules into the rhodopsin interior during photoactivation, forming a solvent-swollen Meta-II active state [1]. Under applied osmotic pressure, the overall equilibrium generally shifted to Meta-I. Dehydrating conditions favor Meta-I through an efflux of water from the protein interior, while increasing bilayer thickness and the monolayer spontaneous curvature favor Meta-II. The osmotic effect on the protein is more significant than the effect of the lipid bilayer. However, small osmolytes favored the Meta-II state at lower concentrations, because they can penetrate the protein core giving a lower excluded volume, decreasing the osmotic effect on the protein and favoring the Meta-II state. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta-I state in POPC recombinant membranes compared to the native membrane environment. Analysis of transducin C-terminal peptide-binding isotherms revealed that the binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. The POPC lipid membrane has zero-spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta-I state. By contrast, the native lipid membrane environment has a negative spontaneous curvature that favors the more expanded state of Meta-II. Our results delineate the crucial role of soft matter in regulating the metarhodopsin equilibrium in a membrane environment. [1] U. Chawla et al. (2020) Angew. Chem. Int. Ed. DOI: https://doi.org/10.1002/ange.202003342.

Simulation of the Influenza Fusion Peptide Pre-Pore Structure

Influenza virus dependent membrane fusion requires facilitation by a fusion peptide. Membrane deformation theory predicts that amphipathic peptide-induced lipidic pores are less stable in membranes containing lipids that generate negative spontaneous curvature, such as cholesterol. To explicitly test predictions of this theory, the membrane permeability of giant unilamellar vesicles (GUV), with and without cholesterol, were investigated experimentally using an influenza fusion peptide analog. The peptide formed stable membrane pores in POPC lipid GUVs, while pore formation was inhibited in 50:50 mol% cholesterol:POPC membranes.

Vesicle deformation and division induced by flip-flops of lipid molecules.

We investigated the deformation of small unilamellar vesicles (SUVs) induced by flip-flops of lipids using coarse-grained molecular dynamics simulations. In the case of single-component SUVs composed of zero spontaneous curvature lipids (ZLs), the flip-flop of ZLs deformed stomatocyte-shaped SUVs into an oblate shape, whereas pear-shaped SUVs were deformed into a prolate shape. These two equilibrium shapes comply with the local minima of elastic energy. In the case of binary vesicles composed of ZLs and negative spontaneous curvature lipids (NLs), the vesicle deformation pathway depended on the initial NL distribution in the bilayer. If the initial difference in the NL concentration between the outer and inner leaflets was small, the flip-flop of ZLs and NLs rapidly deformed pear-shaped SUVs into an equilibrium prolate shape. On the other hand, when NLs were localised in the inner leaflet, the flip-flop of ZLs and NLs deformed pear-shaped SUVs into a limiting shape and then induced vesicle division. Because the flip-flop rate of NLs is much faster than that of ZLs, the total free energy was first relaxed by the flip-flop of NLs and then by that of ZLs. This kinetic effect is responsible for the observed vesicle division induced by flip-flops.

Sphingomyelin Effects in Caveolin-1 Mediated Membrane Curvature.

The caveolin-1 (cav-1) protein is an integral component of caveolae and has been reported to colocalize with cholesterol and sphingomyelin-rich curved membrane domains. Here, we analyze the molecular interactions between cav-1 and sphingomyelin containing bilayers using a series of coarse-grain simulations, focusing on lipid clustering and membrane curvature. We considered a palmitoylated-cav-1 construct interacting with phospholipid/cholesterol membranes with asymmetrically distributed sphingomyelin, varying between 5 and 15% in total. We observe that cav-1 binds to the intracellular leaflet and induces a small positive curvature in the leaflet to which it is bound and an opposing negative curvature in the extracellular leaflet. Both cholesterol and sphingomyelin are observed to cluster in cav-1 bound membranes, mainly in the extracellular leaflet. Due to their negative spontaneous curvature, clustering of cholesterol and sphingomyelin facilitates membrane curvature such that the extent of either cholesterol or sphingomyelin clustering is dependent on the curvature induced. Our results suggest that cav-1 binding induces concentration-dependent curvature effects in sphingomyelin-rich membranes. Overall, our work is an important step in understanding the molecular basis of curvature and lipid clustering in cav-1 bound cellular membranes.

Lipid species affect morphology of endoplasmic reticulum: a sea urchin oocyte model of reversible manipulation

The ER is a large multifunctional organelle of eukaryotic cells. Malfunction of the ER in various disease states, such as atherosclerosis, diabetes, cancer, Alzheimer’s and Parkinson’s and amyotrophic lateral sclerosis, often correlates with alterations in its morphology. The ER exhibits regionally variable membrane morphology that includes, at the extremes, large relatively flat surfaces and interconnected tubular structures highly curved in cross-section. ER morphology is controlled by shaping proteins that associate with membrane lipids. To investigate the role of these lipids, we developed a sea urchin oocyte model, a relatively quiescent cell in which the ER consists mostly of tubules. We altered levels of endogenous diacylglycerol (DAG), phosphatidylethanolamine (PtdEth), and phosphatidylcholine by microinjection of enzymes or lipid delivery by liposomes and evaluated shape changes with 2D and 3D confocal imaging and 3D electron microscopy. Decreases and increases in the levels of lipids such as DAG or PtdEth characterized by negative spontaneous curvature correlated with conversion to sheet structures or tubules, respectively. The effects of endogenous alterations of DAG were reversible upon exogenous delivery of lipids of negative spontaneous curvature. These data suggest that proteins require threshold amounts of such lipids and that localized deficiencies of the lipids could contribute to alterations of ER morphology. The oocyte modeling system should be beneficial to studies directed at understanding requirements of lipid species in interactions leading to alterations of organelle shaping.

Cholesterol: The Plasma Membrane’s Constituent that Chooses Sides

Cholesterol: The Plasma Membrane’s Constituent that Chooses Sides

The Effect of Ions on Membrane Elasticity - Implications for Vesicle Fusion

Ions and charged peptides can have dramatic effects on the properties of membranes. A case in point is calcium and its crucial role in vesicle fusion.[1] Using all-atom molecular dynamics, we simulated calcium mediated vesicle fusion.[2] Aided by a new, local approach to determining membrane elastic properties,[3] we are able to quantify the effect of ions on lipid spontaneous curvature, bending rigidity, and tilt. We probe the effect of ions on membranes of different composition. Our simulations indicate that calcium specifically induces negative spontaneous curvature in those anionic lipid membranes which are susceptible to calcium mediated fusion, concomitantly strongly affecting the Gaussian bending modulus. We further determine the mechanism of membrane curvature induction through ion-headgroup interactions, giving special attention to lipid clustering. In addition to membrane elasticity, we discuss electrostatic effects on osmotic pressures and the hydration force. This systematic approach allows us to fill crucial gaps in our understanding of the mechanism of membrane fusion. Finally, we point out possible implications for synaptic vesicle fusion and endocytosis. [1] E. Neher, R. Schneggenburger, Nature 406, 889-893 (2000) [2] C. Allolio, A. Magarkar, P. Jurkiewicz, K. Baxova, M. Javanainen, R. Šachl, M. Hof, D. Horinek, V. Heinz, R. Rachel, C. Ziegler, M. Cebecauer, A. Schrofel, P. Jungwirth, resubmitted to PNAS (2018) [3] C. Allolio, A. Haluts, D. Harries,Chem. Phys, in press (2018)

Thermodynamically reversible paths of the first fusion intermediate reveal an important role for membrane anchors of fusion proteins

Biological membrane fusion proceeds via an essential topological transition of the two membranes involved. Known players such as certain lipid species and fusion proteins are generally believed to alter the free energy and thus the rate of the fusion reaction. Quantifying these effects by theory poses a major challenge since the essential reaction intermediates are collective, diffusive and of a molecular length scale. We conducted molecular dynamics simulations in conjunction with a state-of-the-art string method to resolve the minimum free-energy path of the first fusion intermediate state, the so-called stalk. We demonstrate that the isolated transmembrane domains (TMDs) of fusion proteins such as SNARE molecules drastically lower the free energy of both the stalk barrier and metastable stalk, which is not trivially explained by molecular shape arguments. We relate this effect to the local thinning of the membrane (negative hydrophobic mismatch) imposed by the TMDs which favors the nearby presence of the highly bent stalk structure or prestalk dimple. The distance between the membranes is the most crucial determinant of the free energy of the stalk, whereas the free-energy barrier changes only slightly. Surprisingly, fusion enhancing lipids, i.e., lipids with a negative spontaneous curvature, such as PE lipids have little effect on the free energy of the stalk barrier, likely because of its single molecular nature. In contrast, the lipid shape plays a crucial role in overcoming the hydration repulsion between two membranes and thus rather lowers the total work required to form a stalk.

Charged giant unilamellar vesicles prepared by electroformation exhibit nanotubes and transbilayer lipid asymmetry

Giant unilamellar vesicles (GUVs) are increasingly used as a versatile research tool to investigate membrane structure, morphology and phase state. In these studies, GUV preparation is typically enhanced by an externally applied electric field, a process called electroformation. We find that upon osmotic deflation, GUVs electroformed from charged and neutral lipids exhibit inward pointing lipid nanotubes, suggesting negative spontaneous curvature of the membrane. By quenching a fluorescent analog of the charged lipid, zeta potential measurements and experiments with the lipid marker annexin A5, we show that electroformed GUVs exhibit an asymmetric lipid distribution across the bilayer leaflets. The asymmetry is lost either after storing electroformed GUVs at room temperature for one day or by applying higher voltages and temperatures during electroformation. GUVs having the same lipid composition but grown via gel-assisted swelling do not show asymmetric lipid distribution. We discuss possible mechanisms for the generation and relaxation of lipid asymmetry, as well as implications for studies using electroformed vesicles. The observed effects allow to control the molecular assembly of lipid bilayer leaflets. Vesicle tubulation as reported here is an example of protein-free reshaping of membranes and is caused by compositional lipid asymmetry between leaflets.