Negative Intrinsic Curvature Research Articles

Novel insight into the lipid network of plasma extracellular vesicles reveal sex-based differences in the lipidomic profile of alcohol use disorder patients

BackgroundAlcohol use disorder (AUD) is one of the most common psychiatric disorders, with the consumption of alcohol considered a leading cause of preventable deaths worldwide. Lipids play a crucial functional role in cell membranes; however, we know little about the role of lipids in extracellular vesicles (EVs) as regulatory molecules and disease biomarkers.MethodsWe employed a sensitive lipidomic strategy to characterize lipid species from the plasma EVs of AUD patients to evaluate functional roles and enzymatic activity networks to improve the knowledge of lipid metabolism after alcohol consumption. We analyzed plasma EV lipids from AUD females and males and healthy individuals to highlight lipids with differential abundance and biologically interpreted lipidomics data using LINEX2, which evaluates enzymatic dysregulation using an enrichment algorithm.ResultsOur results show, for the first time, that AUD females exhibited more significant substrate-product changes in lysophosphatidylcholine/phosphatidylcholine lipids and phospholipase/acyltransferase activity, which are potentially linked to cancer progression and neuroinflammation. Conversely, AUD males suffer from dysregulated ceramide and sphingomyelin lipids involving sphingomyelinase, sphingomyelin phosphodiesterase, and sphingomyelin synthase activity, which relates to hepatotoxicity. Notably, the analysis of plasma EVs from AUD females and males demonstrates enrichment of lipid ontology terms associated with “negative intrinsic curvature” and “positive intrinsic curvature”, respectively.ConclusionsOur methodological developments support an improved understanding of lipid metabolism and regulatory mechanisms, which contribute to the identification of novel lipid targets and the discovery of sex-specific clinical biomarkers in AUD.

Predicting lipid sorting in curved membranes.

Most biological membranes are curved, and both lipids and proteins play a role in generating curvature. For any given membrane shape and composition, it is not trivial to determine whether lipids are laterally distributed in a homogeneous or inhomogeneous way, and whether the inter-leaflet distribution is symmetric or not. Here we present a simple computational tool that allows to predict the preference of any lipid type for membranes with positive vs. negative curvature, for any given value of curvature. The tool is based on molecular dynamics simulations of tubular membranes with hydrophilic pores. The pores allow spontaneous, barrierless flip-flop of most lipids, while also preventing differences in pressure between the inner and outer water compartments and minimizing membrane asymmetric stresses. Specifically, we provide scripts to build and analyze the simulations. We test the tool by performing simulations on simple binary lipid mixtures, and we show that, as expected, lipids with negative intrinsic curvature distribute to the tubule inner leaflet, the more so when the radius of the tubular membrane is small. Compared to other existing computational methods, relying on membrane buckles and tethers, our method is based on spontaneous inter-leaflet transport of lipids, and therefore allows to explore lipid distribution in asymmetric membranes. The method can easily be adapted to work with any molecular dynamics code and any force field.

Glycerophospholipid Analysis of Optic Nerve Regeneration Models Indicate Potential Membrane Order Changes Associated with the Lipidomic Shifts.

Purpose: Optic nerve (ON) injury causes irreversible degeneration, leading to vision loss that cannot be restored with available therapeutics. Current therapies slow further degeneration but do not promote regeneration. New regenerative factors have been discovered that are successful in vivo. However, the mechanisms of efficient long-distance regeneration are still unknown. Membrane expansion by lipid insertion is an essential regenerative process, so lipid profiles for regenerating axons can provide insight into growth mechanisms. This article's analysis aims to add to the increasingly available ON regeneration lipid profiles and relate it to membrane order/properties. Methods: In this study, we present an analysis of glycerophospholipids, one of the largest axonal lipid groups, from three mammalian ON regeneration lipid profiles: Wnt3a, Zymosan + CPT-cAMP, and Phosphatase/Tensin homolog knockout (PTENKO) at 7 and 14 days post crush (dpc). Significant lipid classes, species, and ontological properties were crossreferenced between treatments and analyzed using Metaboanalyst 5.0 and Lipid Ontology (LION). Membrane order changes associated with significant lipid classes were evaluated by C-Laurdan dye and exogenous lipids provided to a neuroblastoma cell line. Results and Conclusions: At 7 dpc, ONs show increased lysoglycerophospholipids and decreased phosphatidylethanolamines (PEs)/negative intrinsic curvature lipids. At 14 dpc, regenerative treatments show divergence: Wnt3a displays higher lysoglycerophospholipid content, while Zymosan and PTENKO decrease lysoglycerophospholipids and increase phosphatidylcholine (PC)-related species. Membrane order imaging indicates lysoglycerophospholipids decreases membrane order while PE and PC had no significant membrane order effects. Understanding these changes will allow therapeutic development targeting lipid metabolic pathways that can be used for vision loss treatments.

Membrane Lipid Reshaping Underlies Oxidative Stress Sensing by the Mitochondrial Proteins UCP1 and ANT1.

Oxidative stress and ROS are important players in the pathogenesis of numerous diseases. In addition to directly altering proteins, ROS also affects lipids with negative intrinsic curvature such as phosphatidylethanolamine (PE), producing PE adducts and lysolipids. The formation of PE adducts potentiates the protonophoric activity of mitochondrial uncoupling proteins, but the molecular mechanism remains unclear. Here, we linked the ROS-mediated change in lipid shape to the mechanical properties of the membrane and the function of uncoupling protein 1 (UCP1) and adenine nucleotide translocase 1 (ANT1). We show that the increase in the protonophoric activity of both proteins occurs due to the decrease in bending modulus in lipid bilayers in the presence of lysophosphatidylcholines (OPC and MPC) and PE adducts. Moreover, MD simulations showed that modified PEs and lysolipids change the lateral pressure profile of the membrane in the same direction and by the similar amplitude, indicating that modified PEs act as lipids with positive intrinsic curvature. Both results indicate that oxidative stress decreases stored curvature elastic stress (SCES) in the lipid bilayer membrane. We demonstrated that UCP1 and ANT1 sense SCES and proposed a novel regulatory mechanism for the function of these proteins. The new findings should draw the attention of the scientific community to this important and unexplored area of redox biochemistry.

Advances in Molecular Understanding of α-Helical Membrane-Active Peptides.

Biological membranes separate the interior of cells or cellular compartments from their outer environments. This barrier function of membranes can be disrupted by membrane-active peptides, some of which can spontaneously penetrate through the membranes or open leaky transmembrane pores. However, the origin of their activity/toxicity is not sufficiently understood for the development of more potent peptides. To this day, there are no design rules that would be generally valid, and the role of individual amino acids tends to be sequence-specific.In this Account, we describe recent progress in understanding the design principles that govern the activity of membrane-active peptides. We focus on α-helical amphiphilic peptides and their ability to (1) translocate across phospholipid bilayers, (2) form transmembrane pores, or (3) act synergistically, i.e., to produce a significantly more potent effect in a mixture than the individual components.We refined the description of peptide translocation using computer simulations and demonstrated the effect of selected residues. Our simulations showed the necessity to explicitly include charged residues in the translocation description to correctly sample the membrane perturbations they can cause. Using this description, we calculated the translocation of helical peptides with and without the kink induced by the proline/glycine residue. The presence of the kink had no effect on the translocation barrier, but it decreased the peptide affinity to the membrane and reduced the peptide stability inside the membrane. Interestingly, the effects were mainly caused by the peptide's increased polarity, not the higher flexibility of the kink.Flexibility plays a crucial role in pore formation and affects distinct pore structures in different ways. The presence of a kink destabilizes barrel-stave pores, because the kink prevents the tight packing of peptides in the bundle, which is characteristic of the barrel-stave structure. In contrast, the kink facilitates the formation of toroidal pores, where the peptides are only loosely arranged and do not need to closely assemble. The exact position of the kink in the sequence further determines the preferred arrangement of peptides in the pore, i.e., an hourglass or U-shaped structure. In addition, we demonstrated that two self-associated (via termini) helical peptides could mimic the behavior of peptides with a helix-kink-helix motif.Finally, we review the recent findings on the peptide synergism of the archetypal mixture of Magainin 2 and PGLa peptides. We focused on a bacterial plasma membrane mimic that contains negatively charged lipids and lipids with negative intrinsic curvature. We showed that the synergistic action of peptides was highly dependent on the lipid composition. When the lipid composition and peptide/lipid ratios were changed, the systems exhibited more complex behavior than just the previously reported pore formation. We observed membrane adhesion, fusion, and even the formation of the sponge phase in this regime. Furthermore, enhanced adhesion/partitioning to the membrane was reported to be caused by lipid-induced peptide aggregation.In conclusion, the provided molecular insight into the complex behavior of membrane-active peptides provides clues for the design and modification of antimicrobial peptides or toxins.

Membrane curvature induces cardiolipin sorting

Cardiolipin is a cone-shaped lipid predominantly localized in curved membrane sites of bacteria and in the mitochondrial cristae. This specific localization has been argued to be geometry-driven, since the CL’s conical shape relaxes curvature frustration. Although previous evidence suggests a coupling between CL concentration and membrane shape in vivo, no precise experimental data are available for curvature-based CL sorting in vitro. Here, we test this hypothesis in experiments that isolate the effects of membrane curvature in lipid-bilayer nanotubes. CL sorting is observed with increasing tube curvature, reaching a maximum at optimal CL concentrations, a fact compatible with self-associative clustering. Observations are compatible with a model of membrane elasticity including van der Waals entropy, from which a negative intrinsic curvature of −1.1 nm−1 is predicted for CL. The results contribute to understanding the physicochemical interplay between membrane curvature and composition, providing key insights into mitochondrial and bacterial membrane organization and dynamics.

Nonlamellar-Phase-Promoting Colipids Enhance Segregation of Palmitoyl Ceramide in Fluid Bilayers

Ceramide is an important intermediate in sphingolipid homeostasis. We examined how colipids, with negative intrinsic curvature and which may induce curvature stress in the bilayers, affected the segregation of palmitoyl ceramide (PCer). Such colipids include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and tetra-linoleoyl cardiolipin (CL). In 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers, PCer formed ordered, gel-like domains at concentrations above 10 mol% at 23°C, as evidenced by the change in the average lifetime of the trans-parinaric acid emission. When POPE or DOPE were included in the DOPC bilayer (at 20:80 or 40:60 POPE or DOPE to DOPC, by mol), the lateral segregation of PCer was facilitated in a concentration-dependent manner, and less PCer was required for the formation of the ordered ceramide-rich domains. Inclusion of CL in the DOPE bilayer (at 10:90 or 20:80 CL to PC, by mol) also caused a similar facilitation of the lateral segregation of PCer. The PCer-rich domains formed in the presence of POPE, DOPE, or CL in DOPC bilayers were slightly more thermostable (by 2–10°C) when compared to PCer-rich domains in DOPC-only bilayers. Nonlamellar phases were not present in bilayers in which the effects of POPE or DOPE on PCer segregation were the largest, as verified by 31P NMR. When palmitoyl sphingomyelin was added to the different bilayer compositions at 5 mol%, relative to the phospholipids, PCer segregated into gel domains at lower concentrations (2–3 mol% PCer), and the effect of POPE on PCer segregation was eliminated. We suggest that the effects of POPE, DOPE, and CL on PCer segregation was in part influenced by their effects on membrane curvature stress and in part because of unfavorable interactions with PCer due to their unsaturated acyl chains. These lipids are abundant in mitochondrial membranes and are likely to affect functional properties of saturated ceramides in them.

Lipid flip-flop vs. lateral diffusion in the relaxation of hemifusion diaphragms

Molecular dynamics simulations of a solvent-free coarse-grained lipid model are used to characterize the mechanisms by which lipid-bilayer hemifusion diaphragm (HD) intermediates relax, across a range of global compositions of negative intrinsic curvature (NIC) lipids and neutral-curvature lipids. At low concentrations of NIC lipids, rapid fission produces a double bilayer end state through a lateral diffusion-based mechanism enabled by spontaneous rim-pore defects. At moderately higher NIC lipid concentrations, rim pores are absent and stable leaflet three-junctions persist, revealing an HD relaxation mechanism entirely reliant on lipid flip-flop, and end states that are either stable fusion pores or stable HD's. These fusogenic systems exhibit dynamics highly dependent on NIC lipid concentration via an underlying sensitivity of flip-flop rates for neutral lipids on NIC lipid concentration. This work illustrates that HD dynamics may be altered through regulation of lipid composition in the immediate three-junction region. This work further highlights the potential role of flippases in biological fusion and the importance of lipid composition on fusion dynamics.

The Nonbilayer Lipid MGDG and the Major Light-Harvesting Complex (LHCII) Promote Membrane Stacking in Supported Lipid Bilayers.

The thylakoid membrane of algae and land plants is characterized by its intricate architecture, comprising tightly appressed membrane stacks termed grana. The contributions of individual components to grana stack formation are not yet fully elucidated. As an in vitro model, we use supported lipid bilayers made of thylakoid lipid mixtures to study the effect of major light-harvesting complex (LHCII), different lipids, and ions on membrane stacking, seen as elevated structures forming on top of the planar membrane surface in the presence of LHCII protein. These structures were examined by confocal laser scanning microscopy, atomic force microscopy, and fluorescence recovery after photobleaching, revealing multilamellar LHCII-membrane stacks composed of connected lipid bilayers. Both native-like and non-native interactions between the LHCII complexes may contribute to membrane appression in the supported bilayers. However, applying in vivo-like salt conditions to uncharged glycolipid membranes drastically increased the level of stack formation due to enforced LHCII-LHCII interactions, which is in line with recent crystallographic and cryo-electron microscopic data [Wan, T., et al. (2014) Mol. Plant 7, 916-919; Albanese, P., et al. (2017) Sci. Rep. 7, 10067-10083]. Furthermore, we observed the nonbilayer lipid MGDG to strongly promote membrane stacking, pointing to the long-term proposed function of MGDG in stabilizing the inner membrane leaflet of highly curved margins in the periphery of each grana disc because of its negative intrinsic curvature [Murphy, D. J. (1982) FEBS Lett. 150, 19-26].

Architecture of Lipid Droplets in Endoplasmic Reticulum Is Determined by Phospholipid Intrinsic Curvature

Lipid droplets (LDs) store fats and play critical roles in lipid and energy homeostasis. They form between the leaflets of the endoplasmic reticulum (ER) membrane and consist of a neutral lipid core wrapped in a phospholipid monolayer with proteins. Two types of ER-LD architecture are thought to exist and be essential for LD functioning. Maturing LDs either emerge from the ER into the cytoplasm, remaining attached to the ER by a narrow membrane neck, or stay embedded in the ER and are surrounded by ER membrane. Here, we identify a lipid-based mechanism that controls which of these two architectures is favored. Theoretical modeling indicated that the intrinsic molecular curvatures of ER phospholipids can determine whether LDs remain embedded in or emerge from the ER; lipids with negative intrinsic curvature such as diacylglycerol (DAG) and phosphatidylethanolamine favor LD embedding, while those withpositive intrinsic curvature, like lysolipids, support LD emergence. This prediction was verified by altering the lipid composition of the ER in S.cerevisiae using mutants and the addition of exogenous lipids. We found that fat-storage-inducing transmembrane protein 2 (FIT2) homologs become enriched at sites of LD generation when biogenesis is induced. DAG accumulates at sites of LD biogenesis, and FIT2 proteins may promote LD emergence from the ER by reducing DAG levels at these sites. Altogether, our findings suggest that cells regulate LD integration in the ER by modulating ER lipid composition, particularly at sites of LD biogenesis and that FIT2 proteins may play a central role in this process.

Rate of hemifusion diaphragm dissipation and ability to form three-junction bound HD determined by lipid composition.

Though the hemifusion diaphragm (HD) is widely accepted as an intermediate in bilayer membrane fusion, lipid contributions toward HD stability and dynamics are still not fully understood. In this paper, we study large, binary, protein-free HD systems at varying compositions of negative intrinsic curvature (NIC) lipids using molecular dynamics (MD) simulations of a solvent-free coarse-grained lipid model. Under MD, initially created HDs are found to relax to three major end states depending on the composition and lipid intrinsic curvature. Low compositions of NIC lipids or weak intrinsic curvature result in double-bilayer end states, and moderate compositions of moderate to strong NIC lipids result in metastable fusion pores. Importantly, high compositions of moderate NIC lipids result in a metastable HD that persists beyond μs time scales. NIC lipids stabilize the HD by filling the junction core around the HD. Sorting of NIC lipids toward the three-junction region occurs in fused-endpoint systems, but no significant sorting was seen in systems that end in a double bilayer indicating that high line tension at the triple junction drives HD dissipation faster than sorting can enrich that junction enough to lower that line tension. The appearance of three end states dependent on the NIC lipid composition highlights the necessity of NIC lipids for non-leaky fusion.

Lipid Modulation of LC3/GABARAP-Mediated Autophagosomal Elongation

Autophagy, an important catabolic pathway involved in a broad spectrum of human diseases, implies the formation of double-membrane structures called autophagosomes (AP) which engulf material to be degraded in lytic compartments. How AP form, especially how the membrane expands and eventually closes upon itself is an area of intense research. Ubiquitin-like ATG8 has been related to both membrane expansion and membrane fusion, but the underlying molecular mechanisms are poorly understood. Here we used two minimal reconstituted systems (enzymatic and chemical conjugation) to compare the ability of human ATG8 homologues (LC3, GABARAP and GATE-16) to mediate membrane fusion. We found that both enzymatically- and chemically-lipidated forms of GATE-16 and GABARAP proteins promote extensive membrane tethering and fusion, whereas lipidated LC3 does so to a much lesser extent. Moreover, we characterize the GATE-16/GABARAP-mediated membrane fusion as a phenomenon of full membrane fusion, independently demonstrating vesicle aggregation, intervesicular lipid mixing and intervesicular mixing of aqueous content, in the absence of vesicular content leakage. Multiple fusion events give rise to large vesicles, as seen by cryo-EM observations. We also show that both vesicle diameter and selected curvature-inducing lipids (cardiolipin, diacylglycerol and lyso-phosphatidylcholine) can modulate the fusion process, smaller vesicle diameters and negative intrinsic curvature lipids (cardiolipin, diacylglycerol) facilitating fusion. These results strongly support the hypothesis of a highly bent structural fusion intermediate (“stalk”) during AP biogenesis and add to the growing body of evidence that identifies lipids as important regulators of autophagy.

Lipid Geometry and Bilayer Curvature Modulate LC3/GABARAP-Mediated Model Autophagosomal Elongation

Autophagy, an important catabolic pathway involved in a broad spectrum of human diseases, implies the formation of double-membrane-bound structures called autophagosomes (AP), which engulf material to be degraded in lytic compartments. How APs form, especially how the membrane expands and eventually closes upon itself, is an area of intense research. Ubiquitin-like ATG8 has been related to both membrane expansion and membrane fusion, but the underlying molecular mechanisms are poorly understood. Here, we used two minimal reconstituted systems (enzymatic and chemical conjugation) to compare the ability of human ATG8 homologs (LC3, GABARAP, and GATE-16) to mediate membrane fusion. We found that both enzymatically and chemically lipidated forms of GATE-16 and GABARAP proteins promote extensive membrane tethering and fusion, whereas lipidated LC3 does so to a much lesser extent. Moreover, we characterize the GATE-16/GABARAP-mediated membrane fusion as a phenomenon of full membrane fusion, independently demonstrating vesicle aggregation, intervesicular lipid mixing, and intervesicular mixing of aqueous content, in the absence of vesicular content leakage. Multiple fusion events give rise to large vesicles, as seen by cryo-electron microscopy observations. We also show that both vesicle diameter and selected curvature-inducing lipids (cardiolipin, diacylglycerol, and lyso-phosphatidylcholine) can modulate the fusion process, smaller vesicle diameters and negative intrinsic curvature lipids (cardiolipin, diacylglycerol) facilitating fusion. These results strongly support the hypothesis of a highly bent structural fusion intermediate (stalk) during AP biogenesis and add to the growing body of evidence that identifies lipids as important regulators of autophagy.

High Cholesterol Obviates a Prolonged Hemifusion Intermediate in Fast SNARE-Mediated Membrane Fusion

Cholesterol is essential for exocytosis in secretory cells, but the exact molecular mechanism by which it facilitates exocytosis is largely unknown. Distinguishing contributions from the lateral organization and dynamics of membrane proteins to vesicle docking and fusion and the promotion of fusion pores by negative intrinsic spontaneous curvature and other mechanical effects of cholesterol have been elusive. To shed more light on this process, we examined the effect of cholesterol on SNARE-mediated membrane fusion in a single-vesicle assay that is capable of resolving docking and elementary steps of fusion with millisecond time resolution. The effect of cholesterol on fusion pore formation between synaptobrevin-2 (VAMP-2)-containing proteoliposomes and acceptor t-SNARE complex-containing planar supported bilayers was examined using both membrane and content fluorescent markers. This approach revealed that increasing cholesterol in either the t-SNARE or the v-SNARE membrane favors a mechanism of direct fusion pore opening, whereas low cholesterol favors a mechanism leading to a long-lived (>5 s) hemifusion state. The amount of cholesterol in the target membrane had no significant effect on docking of synaptobrevin vesicles. Comparative studies with α-tocopherol (vitamin E) show that the negative intrinsic spontaneous curvature of cholesterol and its presumed promotion of a very short-lived (<50 ms) lipid stalk intermediate is the main factor that favors rapid fusion pore opening at high cholesterol. This study also shows that this single-vesicle fusion assay can distinguish between hemifusion and full fusion with only a single lipid dye, thereby freeing up a fluorescence channel for the simultaneous measurement of another parameter in fast time-resolved fusion assays.

Small-Anion Selective Transmembrane “Holes” Induced by an Antimicrobial Peptide Too Short to Span Membranes

Whereas many membrane-destabilization modes have been suggested for membrane-spanning antimicrobial peptides (AMPs), few are available for those too short to span membrane thickness. Here we show that ORB-1, a 15-residue disulfide-bridged AMP that is only ∼20 Å long even when fully stretched like a hairpin, may act by inducing small anion-selective transmembrane "holes" of negative mean curvature. In model membranes of Gram-negative bacteria, ORB-1 induces chloride transmembrane transport and formation of transmembrane channels of negative mean curvature, whereas the inactive analogue, ORB-N, does not, suggesting a correlation between antibacterial activity and ability to induce transmembrane channels. Given that ORB-N is the C-terminus amidated form of ORB-1, our results further suggest that formation of membrane-spanning dimers may be required to initiate the observed channel induction. Moreover, ORB-1 renders model bacterial membranes permeable to anions with effective hydration diameters of <1 nm (e.g., Cl(-) and NO3(-)), but not cations of similar sizes (e.g., H3O(+)), indicative of anion-selective transmembrane channels with an effective inner diameter of ≤1 nm. In addition, negative-intrinsic-curvature (NIC) lipids such as phosphoethanolamine (PE) may facilitate the membrane-destabilization process of ORB-1. Our findings may expand current understandings on how AMPs destabilize membranes and facilitate the pharmaceutical development of ORB-1.

PH Alters PEG-Mediated Fusion of Phosphatidylethanolamine-Containing Vesicles

Here, we examine the different mechanisms of poly(ethylene glycol)-mediated fusion of small unilamellar vesicles composed of dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamine (DOPE)/sphingomyelin/cholesterol in a molar ratio of 35:30:15:20 at pH 7.4 versus pH 5. In doing so, we test the hypothesis that fusion of this lipid mixture should be influenced by differences in hydration of DOPE at these two pH values. An examination of the literature reveals that DOPE should be less hydrated at pH 5 (where influenza virus particles fuse with endosome membranes) than at pH 7.4 (where synaptic vesicles or HIV virus particles fuse with plasma membrane). Ensemble kinetic experiments revealed substantial differences in fusion of this plasma membrane mimetic system at these two pH values. The most dramatic difference was the observation of two intermediates at pH 5 but loss of one of these fusion intermediates at pH 7.4. Analysis of data collected at several temperatures also revealed that formation of the initial fusion intermediate (stalk) was favored at pH 7.4 due to increased activation entropy. Our observations support the hypothesis that the different negative intrinsic curvature of DOPE can account for different fusion paths and activation thermodynamics in steps of the fusion process at these two pH values. Finally, the effects of 2 mol % hexadecane on fusion at both pH values seemed to have similar origins for step 1 (promotion of acyl chain or hydrocarbon excursion into interbilayer space) and step 3 (reduction of interstice energy leading to expansion to a critical stalk radius). Different hexadecane effects on activation thermodynamics at these two pH values can also be related to altered DOPE hydration. The results support our kinetic model for fusion and offer insight into the critical role of phosphatidylethanolamine in fusion.

Lipids as Regulators of Effective Membrane Rigidity

The rigidity of lipid bilayer, the structural core of all cellular membranes, is one of the key force factor in membrane remodeling. For most of “biomimetic” lipid compositions the rigidity is thought to be only a weak function of the composition. However, for multicomponent membranes the composition is coupled to geometry, resulting in lateral redistribution of components in curvature gradients. Such redistribution can substantially facilitate local membrane deformations. We detected that such a decrease of apparent membrane rigidity in pure lipid bilayers containing physiological amounts of dioleoylphosphotidylethanolamin (DOPE), the lipid characterized by highly negative intrinsic curvature. Analyzing fast shape transformations of membrane nanotubes containing different amounts of DOPE we found that the membrane softening followed concentration-dependent redistribution of DOPE towards negative membrane curvature. The apparent bending rigidity of DOPE-containing membranes decreased almost twofold at 30mol% of DOPE, indicating that similar amount of DOPE in cellular membranes can substantially facilitate deformations.

Lagrangian theory of structure formation in relativistic cosmology. II. Average properties of a generic evolution model

Kinematical and dynamical properties of a generic inhomogeneous cosmological model, spatially averaged with respect to free-falling (generalized fundamental) observers, are investigated for the matter model irrotational dust. Paraphrasing a previous Newtonian investigation, we present a relativistic generalization of a backreaction model based on volume-averaging the Relativistic Zeldovich Approximation. In this model we investigate the effect of kinematical backreaction on the evolution of cosmological parameters as they are defined in an averaged inhomogeneous cosmology, and we show that the backreaction model interpolates between orthogonal symmetry properties by covering subcases of the plane-symmetric solution, the Lemaitre-Tolman-Bondi solution and the Szekeres solution. We so obtain a powerful model that lays the foundations for quantitatively addressing curvature inhomogeneities as they would be interpreted as Dark Energy or Dark Matter in a quasi-Newtonian cosmology. The present model, having a limited architecture due to an assumed FLRW background, is nevertheless capable of replacing 1/4 of the needed amount for Dark Energy on domains of 200 Mpc in diameter for typical (one-sigma) fluctuations in a CDM initial power spectrum. However, the model is far from explaining Dark Energy on larger scales (spatially), where a 6% effect on 400 Mpc domains is identified that can be traced back to an on average negative intrinsic curvature today. One drawback of the quantitative results presented is the fact that the epoch when backreaction is effective on large scales and leads to volume acceleration lies in the future. We discuss this issue in relation to the initial spectrum, the Dark Matter problem, the coincidence problem, and the fact that large-scale Dark Energy is an effect on the past light cone (not spatial), and we pinpoint key elements of future research.

The High-Resolution Structures of Two Piscidin Isoforms in Two Microbial Lipid Membrane Mimics Using Solid-State NMR

Piscidins 1 and 3 are twenty-two-residue-long amphipathic, cationic, antimicrobial peptides that have been isolated from hybrid striped bass. Since pathogens are becoming increasingly resistant to traditional antibiotics, antimicrobial peptides such as piscidin may prove to be useful in the future due to their broad-spectrum of activity against microbes and low induction of bacterial resistance. While membrane-active and fast-acting piscidins 1 and 3 have been shown to bind and disrupt bacterial lipid membrane mimics, their mode of action against microbes such as bacteria and yeasts is not known.In earlier studies, we used solid-state NMR to determine the three-dimensional atomic-resolution structures of piscidin 1 and piscidin 3 in aligned bilayers consisting of 3:1 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG). Here, the specific aim has been to solve the structures of these peptides in 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) so that we could compare and contrast these structures with those in 3:1 DMPC/DMPG, a lipid system that has lower anionic content and does not exhibit the negative intrinsic membrane curvature of bacterial membranes. We have collected two-dimensional solid-state NMR spectra on 15N-backbone labeled piscidins in the presence of aligned 1:1 POPE/POPG bilayers. 15N chemical shifts and 15N-1H dipolar couplings have been combined to obtain the topological orientation of the peptides and calculate their atomic level backbone structures in the membrane-bound state. The data indicate that the carboxyl- and amino-ends of the peptides are affected differently by the changes in lipid composition. We will discuss the implications in terms of structural features and peptide-lipid interactions that may be important for piscidin's mode of action. Overall, these studies performed using native-like conditions provide us with a better understanding of structure-function relationships in piscidins and related antimicrobial peptides.

Interaction between Lipids and Antimicrobial Oligomers Studied by Solid-State NMR

Antimicrobial peptides and their synthetic analogues are well known to interact with the cell membrane, which has complex distributions of lipids. The phase behavior of DOPE/DOPG mixed lipids and the interaction between the lipids and several synthetic amphiphilic antimicrobial oligomers (AMOs) were studied by solid-state nuclear magnetic resonance (NMR). A phase diagram of the lipids over a broad window of water content was constructed. There are large areas in the phase diagram where multiple phases coexist, and the fraction of each phase at a given state is dependent on the sample's preparation and thermal history. The comparable stability of the different phases implies that even slight changes in the lipid condition could result in substantial changes to the phase structure, which may be utilized by living organisms to achieve many membrane functions. Nuclear Overhauser spectroscopy (NOESY) and several other NMR experiments indicated that the AMO primarily resides in the head group region of the lipids and that DOPE, the negative intrinsic curvature lipid, does not selectively enrich in the inverted hexagonal phase.