MEDUSA: A cloud-based tool for the analysis of X-ray diffuse scattering to obtain the bending modulus from oriented membrane stacks.

An important mechanical property of cells is their membrane bending modulus, κ. Here, we introduce MEDUSA (MEmbrane DiffUse Scattering Analysis), a cloud-based analysis tool to determine the bending modulus, κ, from the analysis of X-ray diffuse scattering. MEDUSA uses GPU (graphics processing unit) accelerated hardware and a parallelized algorithm to run the calculations efficiently in a few seconds. MEDUSA's graphical user interface allows the user to upload 2-dimensional data collected from different sources, perform background subtraction and distortion corrections, select regions of interest, run the fitting procedure and output the fitted parameters, the membranes' bending modulus κ, and compressional modulus B.

Alkylated EDTA potentiates antibacterial photodynamic activity of protoporphyrin.

Antibiotic resistance has garnered significant attention due to the scarcity of new antibiotics in development. Protoporphyrin IX (PpIX)-mediated photodynamic therapy shows promise as a novel antibacterial strategy, serving as an alternative to antibiotics. However, the poor solubility of PpIX and its tendency to aggregate greatly hinder its photodynamic efficacy. In this study, we demonstrate that alkylated EDTA derivatives (aEDTA), particularly C14-EDTA, can enhance the solubility of PpIX by facilitating its dispersion in aqueous solutions. The combination of C14-EDTA and PpIX exhibits potent antibacterial activity against Staphylococcus aureus (S. aureus) when exposed to LED light irradiation. Furthermore, this combination effectively eradicates S. aureus biofilms, which are known to be strongly resistant to antibiotics, and demonstrates high therapeutic efficacy in an animal model of infected ulcers. Mechanistic studies reveal that C14-EDTA can disrupt PpIX crystallization, increase bacterial membrane permeability and sequester divalent cations, thereby improving the accumulation of PpIX in bacteria. This, in turn, enhances reactive oxygen species (ROS) production and the antibacterial photodynamic activity. Overall, this effective strategy holds great promise in combating antibiotic-resistant strains.

Avidity within the N-terminal anchor drives α-synuclein membrane interaction and insertion.

In the brain, α-synuclein (aSN) partitions between free unbound cytosolic and membrane bound forms modulating both its physiological and pathological role and complicating its study due to structural heterogeneity. Here, we use an interdisciplinary, synergistic approach to characterize the properties of aSN:lipid mixtures, isolated aSN:lipid co-structures, and aSN in mammalian cells. Enabled by the isolation of the membrane-bound state, we show that within the previously described N-terminal membrane anchor, membrane interaction relies both on an N-terminal tail (NTT) head group layer insertion of 14 residues and a folded-upon-binding helix at the membrane surface. Both binding events must be present; if, for example, the NTT insertion is lost, the membrane affinity of aSN is severely compromised and formation of aSN:lipid co-structures hampered. In mammalian cells, compromised cooperativity results in lowered membrane association. Thus, avidity within the N-terminal anchor couples N-terminal insertion and helical surface binding, which is crucial for aSN membrane interaction and cellular localization, and may affect membrane fusion.

Molecular Mechanism for the Suppression of Alpha Synuclein Membrane Toxicity by an Unconventional Extracellular Chaperone.

Alpha synuclein (αS) oligomers are a key component of Lewy bodies implicated in Parkinson's disease (PD). Although primarily intracellular, extracellular αS exocytosed from neurons also contributes to PD pathogenesis through a prion-like transmission mechanism. Here, we show at progressive degrees of resolution that the most abundantly expressed extracellular protein, human serum albumin (HSA), inhibits αS oligomer (αSn) toxicity through a three-pronged mechanism. First, endogenous HSA targets αSn with sub-µM affinity via solvent-exposed hydrophobic sites, breaking the catalytic cycle that promotes αS self-association. Second, HSA remodels αS oligomers and high-MW fibrils into chimeric intermediates with reduced toxicity. Third, HSA unexpectedly suppresses membrane interactions with the N-terminal and central αS regions. Overall, our findings suggest that the extracellular proteostasis network may regulate αS cell-to-cell transmission not only by reducing the populations of membrane-binding competent αS oligomers but possibly also by shielding the membrane interface from residual toxic species.

Stabilization of Lipid Membranes through Partitioning of the Blood Bag Plasticizer Di-2-ethylhexyl phthalate (DEHP).

The safe storage of blood is of fundamental importance to health care systems all over the world. Currently, plastic bags are used for the collection and storage of donated blood and are typically made of poly(vinyl chloride) (PVC) plasticized with di-2-ethylhexyl phthalate (DEHP). DEHP is known to migrate into packed red blood cells (RBC) and has been found to extend their shelf life. It has been speculated that DEHP incorporates itself into the RBC membrane and alters membrane properties, thereby reducing susceptibility to hemolysis and morphological deterioration. Here, we used high-resolution X-ray diffraction and molecular dynamics (MD) simulations to study the interaction between DEHP and model POPC lipid membranes at high (9 mol %) and low (1 mol %) concentrations of DEHP. At both concentrations, DEHP was found to spontaneously partition into the bilayer. At high concentrations, DEHP molecules were found to aggregate in the aqueous phase before inserting as clusters into the membrane. The presence of DEHP in the bilayers resulted in subtle, yet statistically significant, alterations in several membrane properties in both the X-ray diffraction experiments and MD simulations. DEHP led to (1) an increase of membrane width and (2) an increase in the area per lipid. It was also found to (3) increase the deuterium order parameter, however, (4) decrease membrane orientation, indicating the formation of thicker, stiffer membranes with increased local curvature. The observed effects of DEHP on lipid bilayers may help to better understand its effect on RBC membranes in increasing the longevity of stored blood by improving membrane stability.

Anesthetics significantly increase the amount of intramembrane water in lipid membranes.

The potency of anesthesia was directly linked to the partitioning of the drug molecules in cell membranes by Meyer and Overton. Many molecules interact with lipid bilayers and lead to structural and functional changes. It remains an open question which change in membrane properties is responsible for a potential anesthetic effect or if anesthetics act by binding to direct targets. We studied the effect of ethanol, diethyl ether and isoflurane on the water distribution in lipid bilayers by combining all-atom molecular dynamics simulations and neutron diffraction experiments. The simulations show strong membrane-drug interactions with partitioning coefficients of 38%, 92% and 100% for ethanol, diethyl ether and isoflurane, respectively, and provide evidence for an increased water partitioning in the membrane core. The amount of intramembrane water molecules was experimentally determined by selectively deuterium labeling lipids, anesthetic drug and water molecules in neutron diffraction experiments. Four additional water molecules per lipid were observed in the presence of ethanol. Diethyl ether and isoflurane were found to significantly increase the amount of intramembrane water by 25% (8 water molecules). This increase in intramembrane water may contribute to the non-specific interactions between anesthetics and lipid membranes.

Membrane curvature allosterically regulates the phosphatidylinositol cycle, controlling its rate and acyl-chain composition of its lipid intermediates.

Signaling events at membranes are often mediated by membrane lipid composition or membrane physical properties. These membrane properties could act either by favoring the membrane binding of downstream effectors or by modulating their activity. Several proteins can sense/generate membrane physical curvature (i.e. shape). However, the modulation of the activity of enzymes by a membrane's shape has not yet been reported. Here, using a cell-free assay with purified diacylglycerol kinase ϵ (DGKϵ) and liposomes, we studied the activity and acyl-chain specificity of an enzyme of the phosphatidylinositol (PI) cycle, DGKϵ. By systematically varying the model membrane lipid composition and physical properties, we found that DGKϵ has low activity and lacks acyl-chain specificity in locally flat membranes, regardless of the lipid composition. On the other hand, these enzyme properties were greatly enhanced in membrane structures with a negative Gaussian curvature. We also found that this is not a consequence of preferential binding of the enzyme to those structures, but rather is due to a curvature-mediated allosteric regulation of DGKϵ activity and acyl-chain specificity. Moreover, in a fine-tuned interplay between the enzyme and the membrane, DGKϵ favored the formation of structures with greater Gaussian curvature. DGKϵ does not bear a regulatory domain, and these findings reveal the importance of membrane curvature in regulating DGKϵ activity and acyl-chain specificity. Hence, this study highlights that a hierarchic coupling of membrane physical property and lipid composition synergistically regulates membrane signaling events. We propose that this regulatory mechanism of membrane-associated enzyme activity is likely more common than is currently appreciated.

Modulation of DEG/ENaCs by Amphiphiles Suggests Sensitivity to Membrane Alterations.

The bile acid-sensitive ion channel is activated by amphiphilic substances such as bile acids or artificial detergents via membrane alterations; however, the mechanism of membrane sensitivity of the bile acid-sensitive ion channel is not known. It has also not been systematically investigated whether other members of the degenerin/epithelial Na+ channel (DEG/ENaC) gene family are affected by amphiphilic compounds. Here, we show that DEG/ENaCs ASIC1a, ASIC3, ENaC, and the purinergic receptor P2X2 are modulated by a large number of different, structurally unrelated amphiphilic substances, namely the detergents N-lauroylsarcosine, Triton X-100, and ß-octylglucoside; the fenamate flufenamic acid; the antipsychotic drug chlorpromazine; the natural phenol resveratrol; the chili pepper compound capsaicin; the loop diuretic furosemide; and the antiarrythmic agent verapamil. We determined the modification of membrane properties using large-angle x-ray diffraction experiments on model lipid bilayers, revealing that the amphiphilic compounds are positioned in a characteristic fashion either in the lipid tail group region or in the lipid head group region, demonstrating that they perturbed the membrane structure. Collectively, our results show that DEG/ENaCs and structurally related P2X receptors are modulated by diverse amphiphilic molecules. Furthermore, they suggest alterations of membrane properties by amphiphilic compounds as a mechanism contributing to modulation.

Dynamically Cross-Linked Self-Assembled Thermoresponsive Microgels with Homogeneous Internal Structures.

The internal morphology of temperature-responsive degradable poly(N-isopropylacrylamide) (PNIPAM) microgels formed via an aqueous self-assembly process based on hydrazide and aldehyde-functionalized PNIPAM oligomers is investigated. A combination of surface force measurements, small angle neutron scattering (SANS), and ultrasmall angle neutron scattering (USANS) was used to demonstrate that the self-assembled microgels have a homogeneously cross-linked internal structure. This result is surprising given the sequential addition process used to fabricate the microgels, which was expected to result in a densely cross-linked shell-diffuse core structure. The homogeneous internal structure identified is also significantly different than conventional microgels prepared via precipitation polymerization, which typically exhibit a diffuse shell-dense core structure. The homogeneous structure is hypothesized to result from the dynamic nature of the hydrazone cross-linking chemistry used to couple with the assembly conditions chosen that promote polymer interdiffusion. The lack of an internal cross-linking gradient within these degradable and monodisperse microgels is expected to facilitate more consistent drug release over time, improved optical properties, and other potential application benefits.

Membrane Cholesterol Reduces Polymyxin B Nephrotoxicity in Renal Membrane Analogs.

Polymyxin B (PmB) is a "last-line" antibiotic scarcely used due to its nephrotoxicity. However, the molecular basis for antibiotic nephrotoxicity is not clearly understood. We prepared kidney membrane analogs of detergent-susceptible membranes, depleted of cholesterol, and cholesterol enriched, resistant membranes. In both analogs, PmB led to membrane damage. By combining x-ray diffraction, molecular dynamics simulations, and electrochemistry, we present evidence for two populations of PmB molecules: peptides that lie flat on the membranes, and an inserted state. In cholesterol depleted membranes, PmB forms clusters on the membranes leading to an indentation of the bilayers and increase in water permeation. The inserted peptides formed aggregates in the membrane core leading to further structural instabilities and increased water intake. The presence of cholesterol in the resistant membrane analogs led to a significant decrease in membrane damage. Although cholesterol did not inhibit peptide insertion, it minimized peptide clustering and water intake through stabilization of the bilayer structure and suppression of lipid and peptide mobility.

A Cytosolic Amphiphilic α-Helix Controls the Activity of the Bile Acid-sensitive Ion Channel (BASIC).

The bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na+ channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic α-helical structure within its N-terminal domain. This α-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction.

Design of Hydrated Porphyrin-Phospholipid Bilayers with Enhanced Magnetic Resonance Contrast.

Computer simulations are used to design more hydrated bilayers, formed from amine-modified porphyrin-phospholipids (PoPs). Experiments confirm that the new constructs give rise to bilayers with greater water content. When chelated with manganese, amine-modified PoPs provide improved contrast for magnetic resonance and are safely used for imaging in vivo.

Curcumin Protects Membranes through a Carpet or Insertion Model Depending on Hydration.

Curcumin is the main ingredient in turmeric, a common Indian spice. Curcumin shows a broad spectrum of effects, including anti-Alzheimer's and antioxidant properties. An interaction between curcumin and lipid membranes has been speculated as the root cause of this activity, and the molecule is often proposed to protect the bilayer. However, the detailed molecular mechanism of this protection is disputed. There is evidence that curcumin either (a) lies flat on the bilayer and provides a "carpet" for protection by forming a steric barrier, or (b) inserts into the membrane and stiffens tails, thereby protecting against peptide insertion. We studied the interaction between curcumin and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers at different concentrations using high-resolution X-ray diffraction and molecular dynamics (MD) computer simulations. We observed curcumin molecules forming a carpet in dehydrated bilayers, whereas in hydrated membranes the curcumin molecules were found to insert into the bilayers. From calculations of the potential of mean force (PMF), we find two minima, a metastable state in the headgroup region, at |z| ≈ 22 Å, and a global minimum in the hydrophobic membrane core, at |z| ≈ 9 Å. The population of the two states depends on membrane hydration. Experiments may thus observe curcumin in a carpet or inserted position, depending on the osmotic pressure conditions created, for instance, by salts, buffer solutions, substrates, or macromolecular solutes. In the carpet model, curcumin dehydrates lipid bilayers and decreases fluidity. When inserted, curcumin leads to a further fluidification of the membranes and an increase in tail fluctuations, contrary to cholesterol's condensing effect.

Partitioning of caffeine in lipid bilayers reduces membrane fluidity and increases membrane thickness.

Caffeine is a small amphiphilic molecule, which is widely consumed as a stimulant to prevent fatigue, but is also used as a common drug adjuvant in modern medicine. Here, we show that caffeine interacts with unsaturated lipid membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). By combining X-ray diffraction and molecular dynamics simulations, we present evidence that caffeine partitions in lipid membranes and locates at the head group-tail group interface of the bilayers. By attracting water molecules from neighboring lipid molecules, it leads to the formation of "water pockets", i.e., a local increase of water density at this interface. Through this mechanism, caffeine leads to an overall decrease of the gauche defect density in the membranes and an increase of membrane thickness, indicating a loss of membrane fluidity. These non-specific membrane interactions may increase the efficacy of analgesic drugs through changes in the bioavailability and rate of metabolism of these drugs.

The organization of melatonin in lipid membranes.

Melatonin is a hormone that has been shown to have protective effects in several diseases that are associated with cholesterol dysregulation, including cardiovascular disease, Alzheimer's disease, and certain types of cancers. We studied the interaction of melatonin with model membranes made of dimyristoylphosphatidylcholine (DMPC) at melatonin concentrations ranging from 0.5mol% to 30mol%. From 2-dimensional X-ray diffraction measurements, we find that melatonin induces a re-ordering of the lipid membrane that is strongly dependent on the melatonin concentration. At low melatonin concentrations, we observe the presence of melatonin-enriched patches in the membrane, which are significantly thinner than the lipid bilayer. The melatonin molecules were found to align parallel to the lipid tails in these patches. At high melatonin concentrations of 30mol%, we observe a highly ordered melatonin structure that is uniform throughout the membrane, where the melatonin molecules align parallel to the bilayers and one melatonin molecule associates with 2 lipid molecules. Understanding the organization and interactions of melatonin in membranes, and how these are dependent on the concentration, may shed light into its anti-amyloidogenic, antioxidative and photoprotective properties and help develop a structural basis for these properties.

Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes.

Aspirin and other non-steroidal anti-inflammatory drugs have a high affinity for phospholipid membranes, altering their structure and biophysical properties. Aspirin has been shown to partition into the lipid head groups, thereby increasing membrane fluidity. Cholesterol is another well known mediator of membrane fluidity, in turn increasing membrane stiffness. As well, cholesterol is believed to distribute unevenly within lipid membranes leading to the formation of lipid rafts or plaques. In many studies, aspirin has increased positive outcomes for patients with high cholesterol. We are interested if these effects may be, at least partially, the result of a non-specific interaction between aspirin and cholesterol in lipid membranes. We have studied the effect of aspirin on the organization of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) membranes containing cholesterol. Through Langmuir-Blodgett experiments we show that aspirin increases the area per lipid and decreases compressibility at 32.5 mol% cholesterol, leading to a significant increase of fluidity of the membranes. Differential scanning calorimetry provides evidence for the formation of meta-stable structures in the presence of aspirin. The molecular organization of lipids, cholesterol and aspirin was studied using neutron diffraction. While the formation of rafts has been reported in binary DPPC/cholesterol membranes, aspirin was found to locally disrupt membrane organization and lead to the frustration of raft formation. Our results suggest that aspirin is able to directly oppose the formation of cholesterol structures through non-specific interactions with lipid membranes.

Swelling of phospholipid membranes by divalent metal ions depends on the location of the ions in the bilayers.

The Hofmeister series illustrates how salts produce a wide range of effects in biological systems, which are not exclusively explained by ion charge. In lipid membranes, charged ions have been shown to bind to lipids and either hydrate or dehydrate lipid head groups, and also to swell the water layer in multi-lamellar systems. Typically, Hofmeister phenomena are explained by the interaction of the ions with water, as well as with biological interfaces, such as proteins or membranes. We studied the effect of the divalent cations Mg(2+), Ca(2+), Fe(2+), and Zn(2+) on oriented, stacked, phospholipid bilayers made of dimyristoylphosphatidylcholine (DMPC). Using high-resolution X-ray diffraction, we observed that the cations lead to a swelling of the water layer between the bilayers, without causing significant changes to the bilayer structure. The cations swelled the bilayers in different amounts, in the order Fe(2+) > Mg(2+) > Ca(2+) > Zn(2+). By decomposing the total bilayer electron density into different molecular groups, Zn(2+) and Ca(2+) were found to interact with the glycerol groups of the lipid molecules and cause minor swelling of the bilayers. Mg(2+) and Fe(2+) were found to position near the phosphate groups and cause a strong increase in the number of hydration water molecules. Our results present a molecular mechanism-of-action for the Hofmeister series in phospholipid membranes.

Amyloid-ß(25-35) peptides aggregate into cross-ß sheets in unsaturated anionic lipid membranes at high peptide concentrations.

One of the hallmarks of Alzheimer's disease is the formation of protein plaques in the brain, which mainly consist of amyloid-ß peptides of different lengths. While the role of these plaques in the pathology of the disease is not clear, the mechanism behind peptide aggregation is a topic of intense research and discussion. Because of their simplicity, synthetic membranes are promising model systems to identify the elementary processes involved. We prepared unsaturated zwitterionic/anionic lipid membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) at concentrations of POPC/3 mol% DMPS containing 0 mol%, 3 mol%, 10 mol%, and 20 mol% amyloid-ß25-35 peptides. Membrane-embedded peptide clusters were observed at peptide concentrations of 10 and 20 mol% with a typical cluster size of ∼11 µm. Cluster density increased with peptide concentration from 59 (±3) clusters per mm(2) to 920 (±64) clusters per mm(2), respectively. While monomeric peptides take an α-helical state when embedded in lipid bilayers at low peptide concentrations, the peptides in peptide clusters were found to form cross-ß sheets and showed the characteristic pattern in X-ray experiments. The presence of the peptides was accompanied by an elastic distortion of the bilayers, which can induce a long range interaction between the peptides. The experimentally observed cluster patterns agree well with Monte Carlo simulations of long-range interacting peptides. This interaction may be the fundamental process behind cross-ß sheet formation in membranes and these sheets may serve as seeds for further growth into amyloid fibrils.

Cholesterol expels ibuprofen from the hydrophobic membrane core and stabilizes lamellar phases in lipid membranes containing ibuprofen.

There is increasing evidence that common drugs, such as aspirin and ibuprofen, interact with lipid membranes. Ibuprofen is one of the most common over the counter drugs in the world, and is used for relief of pain and fever. It interacts with the cyclooxygenase pathway leading to inhibition of prostaglandin synthesis. From X-ray diffraction of highly oriented model membranes containing between 0 and 20 mol% ibuprofen, 20 mol% cholesterol, and dimyristoylphosphatidylcholine (DMPC), we present evidence for a non-specific interaction between ibuprofen and cholesterol in lipid bilayers. At a low ibuprofen concentrations of 2 mol%, three different populations of ibuprofen molecules were found: two in the lipid head group region and one in the hydrophobic membrane core. At higher ibuprofen concentrations of 10 and 20 mol%, the lamellar bilayer structure is disrupted and a lamellar to cubic phase transition was observed. In the presence of 20 mol% cholesterol, ibuprofen (at 5 mol%) was found to be expelled from the membrane core and reside solely in the head group region of the bilayers. 20 mol% cholesterol was found to stabilize lamellar membrane structure and the formation of a cubic phase at 10 and 20 mol% ibuprofen was suppressed. The results demonstrate that ibuprofen interacts with lipid membranes and that the interaction is strongly dependent on the presence of cholesterol.

Anomalous and anisotropic nanoscale diffusion of hydration water molecules in fluid lipid membranes.

We have studied nanoscale diffusion of membrane hydration water in fluid-phase lipid bilayers made of 1,2-dimyristoyl-3-phosphocholine (DMPC) using incoherent quasi-elastic neutron scattering. Dynamics were fit directly in the energy domain using the Fourier transform of a stretched exponential. By using large, 2-dimensional detectors, lateral motions of water molecules and motions perpendicular to the membranes could be studied simultaneously, resulting in 2-dimensional maps of relaxation time, τ, and stretching exponent, ß. We present experimental evidence for anomalous (sub-diffusive) and anisotropic diffusion of membrane hydration water molecules over nanometer distances. By combining molecular dynamics and Brownian dynamics simulations, the potential microscopic origins for the anomaly and anisotropy of hydration water were investigated. Bulk water was found to show intrinsic sub-diffusive motion at time scales of several picoseconds, likely related to caging effects. In membrane hydration water, however, the anisotropy of confinement and local dynamical environments leads to an anisotropy of relaxation times and stretched exponents, indicative of anomalous dynamics.