Bilayer Setup Research Articles

Hydrophobin Bilayer as Water Impermeable Protein Membrane.

One of the most important properties of membranes is their permeability to water and other small molecules. A targeted change in permeability allows the passage of molecules to be controlled. Vesicles made of membranes with low water permeability are preferable for drug delivery, for example, because they are more stable and maintain the drug concentration inside. This study reports on the very low water permeability of pure protein membranes composed of a bilayer of the amphiphilic protein hydrophobin HFBI. Using a droplet interface bilayer setup, we demonstrate that HFBI bilayers are essentially impermeable to water. HFBI bilayers withstand far larger osmotic pressures than lipid membranes. Only by disturbing the packing of the proteins in the HFBI bilayer is a measurable water permeability induced. To investigate possible molecular mechanisms causing the near-zero permeability, we used all-atom molecular dynamics simulations of various HFBI bilayer models. The simulations suggest that the experimental HFBI bilayer permeability is compatible neither with a lateral honeycomb structure, as found for HFBI monolayers, nor with a residual oil layer within the bilayer or with a disordered lateral packing similar to the packing in lipid bilayers. These results suggest that the low permeabilities of HFBI and lipid bilayers rely on different mechanisms. With their extremely low but adaptable permeability and high stability, HFBI membranes could be used as an osmotic pressure-insensitive barrier in situations where lipid membranes fail such as desalination membranes.

Sulfone-Modified Perylene Acceptors with Improved Permittivity for Bilayer Organic Solar Cells Processed from Non-halogenated Solvents

Organic photovoltaics show high promise as a technology for sustainable energy conversion. A prominent strategy to reduce the substantial energy loss of organic solar cells is to synthesize high-permittivity (high-ε) active layer materials. However, despite the increase in permittivity, many of the high-ε materials achieved only inferior efficiencies, which is generally explained with a worse bulk heterojunction morphology. In this work, we tackled this issue by preparing high-ε acceptors and incorporating them in a bilayer setup, which we optimized using the systematic Design of Experiment (DoE) approach. The prepared acceptors are based on a perylene-linker-perylene scaffold, to which we attached polar sulfone-containing side chains. The relative permittivity of these acceptors increased by over 50% compared to their alkylated analogues. Simultaneously, some of the acceptors have greatly improved solubilities in non-halogenated “green” solvents. Both improvements enabled us to build bilayer organic solar cells from o-xylene and THF with PTQ10 as the donor, while simultaneously increasing the efficiency to 5.51% with a high open-circuit voltage of 1.3 V. Our results show that using a bilayer setup can successfully prevent morphology-related efficiency losses when employing high-ε materials. Combining this approach with a systematic optimization method (DoE) can unlock the theoretical potential of permittivity modification in organic solar cell research.

Munc13 Recruits SNAP25 to Facilitate SNARE Complex Assembly

Synaptic vesicle exocytosis in neurons requires SNARE complex formation during which VAMP2 from vesicles zippers with t-SNAREs (SNAP25 and Syntaxin1a) on the plasma membrane. Munc13-1 acts as a chaperone for this process by both tethering vesicles to PIP2-microdomains on the plasma membrane containing Syntaxin1a and by activating Syntaxin1a. How membrane-anchored SNAP25 enters this reaction is unknown. Here we report a novel function of Munc13 in the recruitment of SNAP25 to initiate the SNARE priming process. Using biochemical analyses, including quantitative binding studies, we show the MUN domain of Munc13 directly binds the SNAP25 loop region (connecting the two helices). Indeed, replacing the SNAP25 loop with the loop region of closely-related SNAP23 or with a non-specific GGGGS repeat sequence abrogated the Munc13 interaction. Interestingly, the Munc13 mutant NF->AA (1128/1131) that has been shown to be incapable of activating Syntaxin1a, still bound SNAP25 suggesting the binding sites on Munc13 for Syntaxin1a and SNAP25 are different. Furthermore, total internal reflection fluorescence (TIRF) microscopy analysis revealed that Munc13 co-clusters with SNAP25 on a supported lipid bilayer setup and these clusters typically contain 5-6 molecules each of Munc13 and SNAP25. This co-clustering was absent in the GGGGS mutant. Overall, our data suggest Munc13 recruits SNAP25 to the site of vesicle docking via direct MUN-SNAP25 loop interaction and by virtue of increasing its local concentration, it facilitates the initiation of the SNARE complex formation.

Voltage-tunable Majorana bound states in time-reversal symmetric bilayer quantum spin Hall hybrid systems

We investigate hybrid structures based on a bilayer quantum spin Hall system in proximity to an s-wave superconductor as a platform to mimic time-reversal symmetric topological superconductors. In this bilayer setup, the induced pairing can be of intra- or inter-layer type, and domain walls of those different types of pairing potentials host Kramers partners (time-reversal conjugate pairs) of Majorana bound states. Interestingly, we discover that such topological interfaces providing Majorana bound states can also be achieved in an otherwise homogeneous system by a spatially dependent inter-layer gate voltage. This gate voltage causes the relative electron densities of the two layers to vary accordingly which suppresses the inter-layer pairing in regions with strong gate voltage. We identify particular transport signatures (zero-bias anomalies) in a five-terminal setup that are uniquely related to the presence of Kramers pairs of Majorana bound states.

Rashba sandwiches with topological superconducting phases

We introduce a versatile heterostructure harboring various topological superconducting phases characterized by the presence of helical, chiral, or unidirectional edge states. Changing parameters, such as an effective Zeeman field or chemical potential, one can tune between these three topological phases in the same setup. Our model relies only on conventional non-topological ingredients. The bilayer setup consists of an s-wave superconductor sandwiched between two two-dimensional electron gas layers with strong Rashba spin-orbit interaction. The interplay between two different pairing mechanisms, proximity induced direct and crossed Andreev superconducting pairings, gives rise to multiple topological phases. In particular, helical edge states occur if crossed Andreev superconducting pairing is dominant. In addition, an in-plane Zeeman field leads to a 2D gapless topological phase with unidirectional edge states, which were previously predicted to exist only in non-centrosymmetric superconductors. If the Zeeman field is tilted out of the plane, the system is in a topological phase hosting chiral edge states.

Computational Investigation of the Serotonin Transporter Conformation and Reset Mechanism

The aid of monoamine transporter (MAT) proteins in terminating the effect of neurotransmitters in the synaptic cleft is crucial for the maintenance of proper neurotransmitter concentrations. Termination of effect is achieved via the process of reuptake, where the proteins bind neurotransmitters to a central substrate binding site and transport the substrate to the other side of the membrane through a permeation pathway. The MAT family of proteins includes the serotonin transporter (SERT), which is responsible for the reuptake of serotonin from the synaptic cleft. SERT, as well as the other MAT proteins are implicated in several psychological disorders, including depression, anxiety, and addiction. Treatments for these disorders are focused on the inhibition of the MAT system, especially SERT. These drugs are known as selective serotonin reuptake inhibitors, which act by binding to the substrate site of SERT, preventing the reuptake of serotonin. Recent work in our lab has focused on identifying the complete transport mechanism of SERT computationally, employing both a single bilayer and dual bilayer system to investigate this phenomenon. The work presented here represents the data collected from our most recent dual bilayer system. Herein we utilized the newly crystallized human SERT protein (PDB 5I6X), embedded in POPE lipid membranes. The dual bilayer setup allows the system to maintain ion concentrations on either side of the membrane when periodic boundary conditions are implemented. Ions were added to either side of the membrane at physiological concentrations, keeping the membrane potential at around −70mV, representative of resting potential. Understanding more fully the complete transport process, with specific attention paid to the reset mechanism, can aid in drug discovery and design for treatments that better alleviate symptoms of depression and related disorders. Presented here are the molecular dynamics and conformational data from our ongoing simulations of the dual bilayer SERT system.

Increased Binding of Calcium Ions at Positively Curved Phospholipid Membranes.

Calcium ion is the ubiquitous messenger in cells and plays a key role in neuronal signaling and fusion of synaptic vesicles. These vesicles are typically ∼20-50 nm in diameter, and thus their interaction with calcium ions cannot be modeled faithfully with a conventional flat membrane bilayer setup. Within our newly developed molecular dynamics simulations setup, we characterize here interactions of the calcium ion with curved membrane interfaces with atomistic detail. The present molecular dynamics simulations together with time-dependent fluorescence shift experiments suggest that the mode and strength of interaction of calcium ion with a phospholipid bilayer depends on its curvature. Potential of mean force calculations demonstrate that the binding of calcium ion to the positively curved side of the bilayer is significantly stronger compared with that to a flat membrane.

Pseudo painting/air bubble technique for planar lipid bilayers.

A functional reconstitution of channel proteins in planar lipid bilayers is still very versatile to study structure/function correlates under well-defined conditions at the single protein level. In this study we present an improved planar lipid bilayer technique in which an air bubble is used for stabilizing unstable/leaky bilayers or for removing excess lipids. The bubble can also be used as a tool for reducing the number of channels in the bilayer with the goal of having only one active channel in the membrane. Stable planar lipid bilayers are formed within seconds to minutes. In the case of multiple channel insertion the air bubble can be used to reduce the number of channels within minutes. The simple improvement of the classical folding technique guarantees a very fast creation of stable bilayers even with difficult phospholipids in a conventional vertical bilayer set-up; it requires no modifications of the existing set-up. This technique is very easy to handle and guarantees successful single channel recordings for any kind of planar lipid bilayer experiment.

Polar molecules in bilayers with high population imbalance

We investigate a dilute Fermi gas of polar molecules confined into a bilayer setup with dipole moments polarized perpendicular to the layers. In particular, we consider the extreme case of population imbalance, where we have only one particle in one layer and many particles in the other one. The single molecule is attracted by the dilute Fermi-gas through the inter-layer dipole-dipole force presenting an interesting impurity problem with longrange anistropic interaction. We calulate the chemical potential of the impurity, in second order perturbation theory and in ladder approximation with a Brueckner-Hartree-Fock approach. Moreover, we determine the momentum relaxation rate of the impurity, which is related to the "dipolar" drag effect. For a confined system we relate the results for the chemical potential with the measurement of the collective modes of the impurity. The momentum relaxation rate provide instead an estimate on how quickly the oscillations are damped.

Water Channel Formation and Ion Transport in Linear and Branched Lipid Bilayers

The composition of natural lipid membranes varies greatly depending on the type of organism, and it has been observed that small variations in lipid composition affect dramatically the membrane properties, such as structural stability and solute permeability. One of the variations is the methyl-branched lipids commonly found in archaea and bacteria. Using molecular dynamics simulations, we studied the influence of methyl branching on the electric-field induced formation of water channels in lipid bilayers and ion transports through them. We employed a double lipid bilayer setup to create within a periodic box two water compartments separated by those two bilayers. One of the compartments contains an excess of cations, while the other an equal excess of anions. This setup creates an initial transmembrane potential controlled by the ion concentration in each water compartment. We compared the response of diphytanoylphosphatidylcholine (DPhPC) lipid bilayers that have multiple methyl-branches with that of the straight-chain dipalmitoylphosphatidylcholine (DPPC) lipids. We found that compared to the straight-chain DPPC lipids, branched DPhPC lipids require a higher critical transmembrane potential and a longer time for the membrane to break down, followed by water channel formation, and transport of anions and cations through the channel. We demonstrated that while adding methyl branches reduces the lateral diffusion of the lipids, different transport properties of branched lipids are mainly due to the bulkiness of the branched lipid tails resulting in different water channel morphologies. The transmembrane potential creates toroidal pores in the straight-chain lipid bilayers, but barrel stave pores in the branched-lipid bilayers the formation of which requires a higher transmembrane potential. Our results provided a deeper understanding of the ion transport process through lipid bilayer membranes and shed light on the transport of various molecules across the lipid membranes.

Nanopore Force Spectroscopy on Nucleic Acid Structures & their Target Complexes using Biological and Synthetic Ion Channels

Nanopore force spectroscopy (NFS) is a versatile tool for the investigation of single molecule interactions with high throughput and sensitivity. We parallelize this method using a 16-channel chip-based bilayer setup (Nanion-Technologies), making it suitable for screening assays.In our experiments, we study the binding of the ATP aptamer to its target in terms of complex stability as well as binding affinity. Aptamer-target complexes show a stability that is considerably higher compared to unbound aptamer structures. This allows us to determine the binding affinity of the ATP aptamer to its target molecules by comparing populations in the stability distributions for varying target concentrations. Due to the high force resolution of NFS we observe that the stability of bound aptamer structures splits up into several sub-populations that reveal more detailed information about the binding process.We extend the use of NFS from DNA to RNA-based molecular structures, which play important roles in biological processes e.g. in the form of aptamers or in gene regulation. In particular RNA hairpin structures are investigated, which are found to display an increased stability compared to their DNA analogues, as their predicted thermodynamic stability suggested. Furthermore, first experiments on RNA aptamers have been performed.Previous NFS studies were based on naturally occurring membrane channels like α-hemolysin or solid-state nanopores. Tailoring these systems to specific applications remains a challenging task, when altering the channels' geometry or chemical modification becomes necessary. We report on the use of a novel synthetic membrane channel constructed entirely from DNA to perform nanopore-sensing experiments. Scaffolded DNA origami was used to create the channel, allowing for easy modifications in terms of geometry and chemical features. We demonstrate its functionality by studying DNA hairpin unzipping and G-quadruplex unfolding.

Dimers, Effective Interactions, and Pauli Blocking Effects in a Bilayer of Cold Fermionic Polar Molecules

We consider a bilayer setup with two parallel planes of cold fermionic polar molecules when the dipole moments are oriented perpendicular to the planes. The binding energy of two-body states with one polar molecule in each layer is determined and compared to various analytic approximation schemes in both coordinate- and momentum-space. The effective interaction of two bound dimers is obtained by integrating out the internal dimer bound state wave function and its robustness under analytical approximations is studied. Furthermore, we consider the effect of the background of other fermions on the dimer state through Pauli blocking, and discuss implications for the zero-temperature many-body phase diagram of this experimentally realizable system.

Pore formation by human stefin B in its native and oligomeric states and the consequent amyloid induced toxicity

It is well documented that amyloid forming peptides and proteins interact with membranes and that this correlates with cytotoxicity. To introduce the theme we give a brief description of some amyloidogenic proteins and note their similarities with pore forming toxins (PFTs) and cell penetrating peptides. Human stefin B, a member of the family of cystatins, is an amyloidogenic protein in vitro. This review describes our studies of the interaction of stefin B oligomers and prefibrillar aggregates with model membranes leading to pore formation. We have studied the interaction between human stefin B and artificial membranes of various compositions. We also have prepared distinct sizes and morphologies of stefin B prefibrillar states and assessed their toxicity. Furthermore, we have measured electrical currents through pores formed by stefin B prefibrillar oligomers in a planar lipid bilayer setup. We finally discuss the possible functional and pathological significance of such pores formed by human stefin B.

Voltage-controlled insertion of single α-hemolysin and Mycobacterium smegmatis nanopores into lipid bilayer membranes

One of the prerequisites for single molecule nanopore translocation experiments is the availability of a single nanopore embedded into a lipid bilayer membrane. Using two alternative experimental setups, microdroplets, and a classical planar lipid bilayer setup, we here show that at near-neutral pH and high salt concentration the incorporation rates of the pore-forming proteins α-hemolysin and porin A from Mycobacterium smegmatis are exponentially enhanced at elevated voltages. This fact can be utilized to establish an experimental procedure by which a voltage-controlled insertion of single pores into lipid membranes can be achieved.

Theoretical analysis of drag resistance in amorphous thin films exhibiting superconductor-insulator transitions

The magnetical field tuned superconductor-insulator transition in amorphous thin films, e.g., Ta and InO, exhibits a range of yet unexplained curious phenomena, such as a putative low-resistance metallic phase intervening the superconducting and the insulating phase, and a huge peak in the magnetoresistance at large magnetic field. Qualitatively, the phenomena can be explained equally well within several significantly different pictures, particularly the condensation of quantum vortex liquid, and the percolation of superconducting islands embedded in normal region. Recently, we proposed and analyzed a new measurement that should be able to decisively point to the correct picture: a drag resistance measurement in an amorphous thin-film bilayer setup. Neglecting interlayer tunneling, we found that the drag resistance within the vortex paradigm has opposite sign and is orders of magnitude larger than that in competing paradigms. For example, two identical films as in Sambandamurthy et al. 2004 with 25 nm layer separation at 0.07 K would produce a drag resistance $\sim10^{-4}\Omega$ according the vortex theory, but only $\sim10^{-12}\Omega$ for the percolation theory. We provide details of our theoretical analysis of the drag resistance within both paradigms, and report some new results as well.

A semi-synthetic ion channel platform for detection of phosphatase and protease activity.

Sensitive methods to probe the activity of enzymes are important for clinical assays and for elucidating the role of these proteins in complex biochemical networks. This paper describes a semi-synthetic ion channel platform for detecting the activity of two different classes of enzymes with high sensitivity. In the first case, this method uses single ion channel conductance measurements to follow the enzyme-catalyzed hydrolysis of a phosphate group attached to the C-terminus of gramicidin A (gA, an ion channel-forming peptide) in the presence of alkaline phosphatase (AP). Enzymatic hydrolysis of this phosphate group removes negative charges from the entrance of the gA pore, resulting in a product with measurably reduced single ion channel conductance compared to the original gA-phosphate substrate. This technique employs a standard, commercial bilayer setup and takes advantage of the catalytic turnover of enzymes and the amplification characteristics of ion flux through individual gA pores to detect picomolar concentrations of active AP in solution. Furthermore, this technique makes it possible to study the kinetics of an enzyme and provides an estimate for the observed rate constant (k(cat)) and the Michaelis constant (K(M)) by following the conversion of the gA-phosphate substrate to product over time in the presence of different concentrations of AP. In the second case, modification of gA with a substrate for proteolytic cleavage by anthrax lethal factor (LF) afforded a sensitive method for detection of LF activity, illustrating the utility of ion channel-based sensing for detection of a potential biowarfare agent. This ion channel-based platform represents a powerful, novel approach to monitor the activity of femtomoles to picomoles of two different classes of enzymes in solution. Furthermore, this platform has the potential for realizing miniaturized, cost-effective bioanalytical assays that complement currently established assays.

Determining the Local Shear Viscosity of a Lipid Bilayer System by Reverse Non‐Equilibrium Molecular Dynamics Simulations

The parallel shear viscosity of a dipalmitoylphosphatidylcholine (DPPC) bilayer system is studied by reverse non-equilibrium molecular dynamics simulations (RNEMD) with two different united-atom force fields. The results are related to diffusion coefficients and structural distributions obtained by equilibrium molecular simulations. We investigate technical issues of the algorithm in the bilayer setup, namely, the dependence of the velocity profiles on the imposed flux and the influence of the thermostat on the calculated shear viscosity. We introduce the concept of local shear viscosity and investigate its dependence on the slip velocity of the monolayers and the particle density at the headgroup-water interface and the tail-tail interface. With this we demonstrate that the lipid bilayer is more viscous than the surrounding water phase, and that slip takes place near the headgroup region and in the centre of the bilayer where the alkyl tails meet. We also quantify the apparent increase in viscosity of the water molecules entangled at the water-headgroup interface.

Folding Peptides Into Lipid Bilayer Membranes

The folding and integration of peptides into lipid bilayers remains one of the most intriguing processes in biophysics, as it cannot be directly observed at sufficient temporal and spatial resolutions. From a physical chemistry perspective transfer of solvated peptides into a hydrocarbon phase should follow a two-stage pathway, where helical segments fold at the phase boundary before inserting, due to the large energetic penalty associated with de-solvating exposed peptide bonds.The adsorption, folding and membrane insertion of a model peptide (WALP) was studied via microsecond-timescale molecular simulations at atomic resolution. Both an implicit model and an explicit lipid bilayer setup were used. The implicit simulations generally follow the theoretically predicted two-stage pathway. The vastly increased sampling yields fully converged thermodynamic properties such as the free-energy of folding and membrane insertion. In contrast, the explicit bilayer simulations show that after spontaneous adsorption the peptide immediately crosses the polar interface to locate at the hydrophobic membrane core. Remarkably, there is no interfacial state and the dominant configurations are deeply inserted unfolded and beta-hairpin conformers. The native trans-membrane helix formed for several hundred nanoseconds is not stable. At present the reasons for this unexpected behavior remain unclear.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Folding Peptides into Lipid Bilayer Membranes.

The adsorption, insertion, and folding of a synthetic 16-residue WALP peptide was studied at physiological time scales (>μs) by atomic detail molecular dynamics simulation using a fully explicit DPPC/DMPC lipid bilayer setup. The temperature was elevated to 80 °C/44 °C respectively to increase sampling. After spontaneous adsorption the peptide crosses the polar interfaces to locate at the hydrophobic bilayer core. Remarkably, insertion occurs before folding, and the dominant configurations are inserted beta-hairpins. For the DPPC simulation a trans-membrane helix formed but was not stable. Unfolded membrane insertion of WALP was first observed by Nymeyer and co-workers using a replica exchange method. However, both results are in stark contrast to current theory and simulations with implicit membrane models, which rule out unfolded insertion into the hydrophobic core. At present the exact reasons for this unexpected behavior cannot be unambiguously determined, due to the lack suitable experimental and simulation data to compare to. Nevertheless, the results demonstrate that simulation studies can now in principle provide atomic detail insights into complex biophysical phenomena at physiologically relevant time scales. Future effort must now concentrate on suitable ways to verify current force fields and methodologies for such simulations.

Properties of iron-induced alpha-synuclein oligomers determined on a single particle level

Formation of fibrillar protein aggregates is a key event in several neurodegenerative diseases including Parkinson's disease, which is characterized by Lewy bodies and degeneration of dopaminergic neurons. Several lines of evidence implicate involvement of alpha-synuclein oligomers as the principal toxic aggregate species as well as iron ions in the disease progression in Parkinson's disease. Using three independent single particle-based methods we could recently analyze aggregation pathways and oligomer formation of alpha-synuclein. Biophysical and structural characterization was performed by confocal single molecule fluorescence techniques and atomic force microscopy. Functional characterization included single pore electrophysiology in a lipid bilayer set-up. We characterized two different oligomer species. Organic solvents were used to trigger aggregation, which resulted in small oligomers („intermediate I“). Under these conditions, additional Fe3+- ions at low micromolar concentrations dramatically increased aggregation and induced formation of larger oligomers („intermediate II“). Both oligomer species were on-pathway to amyloid fibrils and could seed amyloid formation [1]. Notably, only Fe3+-induced oligomers were SDS-resistant. In contrast to monomers and intermediate I, only Fe3+-induced intermediate II species were able to bind to unilamelar vesicles composed of POPC (palmitoyl-oleoyl-phosphatidylcholine). Deferoxamine and Vitamin C were able to dissolute the aggregates formed in presence of Fe3+. Interestingly, Deferoxamine shows positive effects in an animal model of PD. In regard to toxicity, we could show that the intermediate II species were able to form ion-permeable pores in a planar lipid bilayer.