Bilayer Charge Asymmetry and Oil Residues Destabilize Membranes upon Poration.

Transmembrane asymmetry is ubiquitous in cells, particularly with respect to lipids, where charged lipids are mainly restricted to one monolayer. We investigate the influence of anionic lipid asymmetry on the stability of giant unilamellar vesicles (GUVs), minimal plasma membrane models. To quantify asymmetry, we apply the fluorescence quenching assay, which is often difficult to reproduce, and caution in handling the quencher is generally underestimated. We first optimize this assay and then apply it to GUVs prepared with the inverted emulsion transfer protocol by using increasing fractions of anionic lipids restricted to one leaflet. This protocol is found to produce highly asymmetric bilayers but with ∼20% interleaflet mixing. To probe the stability of asymmetric versus symmetric membranes, we expose the GUVs to porating electric pulses and monitor the fraction of destabilized vesicles. The pulses open macropores, and the GUVs either completely recover or exhibit leakage or bursting/collapse. Residual oil destabilizes porated membranes, and destabilization is even more pronounced in asymmetrically charged membranes. This is corroborated by the measured pore edge tension, which is also found to decrease with increasing charge asymmetry. Using GUVs with imposed transmembrane pH asymmetry, we confirm that poration-triggered destabilization does not depend on the approach used to generate membrane asymmetry.

Elucidating the Morphology of the Endoplasmic Reticulum: Puzzles and Perspectives.

Artificial or synthetic organelles are a key challenge for bottom-up synthetic biology. So far, synthetic organelles have typically been based on spherical membrane compartments, used to spatially confine selected chemical reactions. In vivo, these compartments are often far from being spherical and can exhibit rather complex architectures. A particularly fascinating example is provided by the endoplasmic reticulum (ER), which extends throughout the whole cell by forming a continuous network of membrane nanotubes connected by three-way junctions. The nanotubes have a typical diameter of between 50 and 100 nm. In spite of much experimental progress, several fundamental aspects of the ER morphology remain elusive. A long-standing puzzle is the straight appearance of the tubules in the light microscope, which form irregular polygons with contact angles close to 120°. Another puzzling aspect is the nanoscopic shapes of the tubules and junctions, for which very different images have been obtained by electron microcopy and structured illumination microscopy. Furthermore, both the formation and maintenance of the reticular networks require GTP and GTP-hydrolyzing membrane proteins. In fact, the networks are destroyed by the fragmentation of nanotubes when the supply of GTP is interrupted. Here, it is argued that all of these puzzling observations are intimately related to each other and to the dimerization of two membrane proteins anchored to the same membrane. So far, the functional significance of this dimerization process remained elusive and, thus, seemed to waste a lot of GTP. However, this process can generate an effective membrane tension that stabilizes the irregular polygonal geometry of the reticular networks and prevents the fragmentation of their tubules, thereby maintaining the integrity of the ER. By incorporating the GTP-hydrolyzing membrane proteins into giant unilamellar vesicles, the effective membrane tension will become accessible to systematic experimental studies.

Biomimetic asymmetric bacterial membranes incorporating lipopolysaccharides.

Gram-negative bacteria are equipped with a cell wall that contains a complex matrix of lipids, proteins, and glycans, which form a rigid layer protecting bacteria from the environment. Major components of this outer membrane are the high-molecular weight and amphiphilic lipopolysaccharides (LPSs). They form the extracellular part of a heterobilayer with phospholipids. Understanding LPS properties within the outer membrane is therefore important to develop new antimicrobial strategies. Model systems, such as giant unilamellar vesicles (GUVs), provide a suitable platform for exploring membrane properties and interactions. However, LPS molecules contain large polysaccharide parts that confer high water solubility, which makes LPS incorporation in artificial membranes difficult; this hindrance is exacerbated for LPS with long polysaccharide chains, i.e., the smooth LPS. Here, a novel emulsification step of the inverted emulsion method is introduced to incorporate LPS in the outer or the inner leaflet of GUVs, exclusively. We developed an approach to determine the LPS content on individual GUVs and quantify membrane asymmetry. The asymmetric membranes with outer leaflet LPS show incorporations of 1-16 mol % smooth LPS (corresponding to 16-79 wt %), while vesicles with inner leaflet LPS reach coverages of 2-7 mol % smooth LPS (28-60 wt %). Diffusion coefficient measurements in the obtained GUVs showed that increasing LPS concentrations in the membranes resulted in decreased diffusivity.

Binding of His-tagged fluorophores to lipid bilayers of giant vesicles.

His-tagged molecules can be attached to lipid bilayers via certain anchor lipids, a method that has been widely used for the biofunctionalization of membranes and vesicles. To observe the membrane-bound molecules, it is useful to consider His-tagged molecules that are fluorescent as well. Here, we study two such molecules, green fluorescence protein (GFP) and green-fluorescent fluorescein isothiocyanate (FITC), both of which are tagged with a chain of six histidines (6H) that bind to the anchor lipids within the bilayers. The His-tag 6H is much smaller than the GFP molecule but somewhat larger than the FITC dye. The lipid bilayers form giant unilamellar vesicles (GUVs), the behavior of which can be directly observed in the optical microscope. We apply and compare three well-established preparation methods for GUVs: electroformation on platinum wire, polyvinyl alcohol (PVA) hydrogel swelling, and electroformation on indium tin oxide (ITO) glass. Microfluidics is used to expose the GUVs to a constant fluorophore concentration in the exterior solution. The brightness of membrane-bound 6H-GFP exceeds the brightness of membrane-bound 6H-FITC, in contrast to the quantum yields of the two fluorophores in solution. In fact, 6H-FITC is observed to be strongly quenched by the anchor lipids which bind the fluorophores via Ni2+ ions. For both 6H-GFP and 6H-FITC, the membrane fluorescence is measured as a function of the fluorophores' molar concentration. The theoretical analysis of these data leads to the equilibrium dissociation constants Kd = 37.5 nM for 6H-GFP and Kd = 18.5 nM for 6H-FITC. We also observe a strong pH-dependence of the membrane fluorescence.

Cell Permeable Imidazole-Desferrioxamine Conjugates: Synthesis and In Vitro Evaluation.

Desferrioxamine (DFO), a clinically approved iron chelator used for iron overload, is unable to chelate labile plasma iron (LPI) because of its limited cell permeability. Herein, alkyl chain modified imidazolium cations with varied hydrophobicities have been conjugated with DFO. The iron binding abilities and the antioxidant properties of the conjugates were found to be similar to DFO. The degree of cellular internalization was much higher in the octyl-imidazolium-DFO conjugate (IV) compared with DFO, and IV was able to chelate LPI in vitro. This opens up a new avenue in using N-alkyl imidazolium salts as a delivery vector for hydrophilic cell-impermeable drugs.

Spontaneous Fluctuations Can Guide Drug Design Strategies for Structurally Disordered Proteins.

Structure-based "rational" drug design strategies fail for diseases associated with intrinsically disordered proteins (IDPs). However, structural disorder allows large-amplitude spontaneous intramolecular dynamics in a protein. We demonstrate a method that exploits this dynamics to provide quantitative information about the degree of interaction of an IDP with other molecules. A candidate ligand molecule may not bind strongly, but even momentary interactions can be expected to perturb the fluctuations. We measure the amplitude and frequency of the equilibrium fluctuations of fluorescently labeled small oligomers of hIAPP (an IDP associated with type II diabetes) in a physiological solution, using nanosecond fluorescence cross-correlation spectroscopy. We show that the interterminal distance fluctuates at a characteristic time scale of 134 ± 10 ns, and 6.4 ± 0.2% of the population is in the "closed" (quenched) state at equilibrium. These fluctuations are affected in a dose-dependent manner by a series of small molecules known to reduce the toxicity of various amyloid peptides. The degree of interaction increases in the following order: resveratrol < epicatechin ∼ quercetin < Congo red < epigallocatechin 3-gallate. Such ordering can provide a direction for exploring the chemical space for finding stronger-binding ligands. We test the biological relevance of these measurements by measuring the effect of these molecules on the affinity of hIAPP for lipid vesicles and cell membranes. We find that the ability of a molecule to modulate intramolecular fluctuations correlates well with its ability to lower membrane affinity. We conclude that structural disorder may provide new avenues for rational drug design for IDPs.

Exposure of Aggregation-Prone Segments is the Requirement for Amyloid Fibril Formation.

Arranging into well-organized fibrillar aggregate, commonly known as amyloid fibril is an inherent property of any polypeptide chain. Amyloid fibrils are associated with a number of severe human pathologies like the Alzheimer's disease, Parkinson's disease, type2 diabetes and many more. Recent studies suggest that most of the fibrils are inert and extremely stable, thus could be used for the bionanotechnological applications. As the native state is protected by evolution from aggregation under physiological condition, understanding the structure of aggregation precursor state (APS) will be of extreme importance to decode mechanism of its formation and prevention. This review article includes the recent studies of identification and characterization of possible conformations of proteins which can act as APS. The literature regarding the research in this field revealed that any conformation ranging from native-like state to completely unfolded state could be an APS. The structural characteristics of the APS depend on the protein and on its surrounding environment. From this review of literatures, we conclude that exposure of aggregation-prone segments is the requirement for amyloid fibril formation and the amyloid state seems to be the most stable known physical state of the proteins. This means all conformations of proteins with exposed aggregation-prone segments can promote intermolecular interactions and channel to amyloid fibril pathway to acquire their minimum energy state.