The interaction of viral fusion peptides with lipid membranes.

In this paper, we studied fusogenic peptides of class I-III fusion proteins, which are relevant to membrane fusion for certain enveloped viruses, in contact with model lipid membranes. We resolved the vertical structure and examined the adsorption or penetration behavior of the fusogenic peptides at phospholipid Langmuir monolayers with different initial surface pressures with x-ray reflectometry. We show that the fusion loops of tick-borne encephalitis virus (TBEV) glycoprotein E and vesicular stomatitis virus (VSV) G-protein are not able to insert deeply into model lipid membranes, as they adsorbed mainly underneath the headgroups with only limited penetration depths into the lipid films. In contrast, we observed that the hemagglutinin 2 fusion peptide (HA2-FP) and the VSV-transmembrane domain (VSV-TMD) can penetrate deeply into the membranes. However, in the case of VSV-TMD, the penetration was suppressed already at low surface pressures, whereas HA2-FP was able to insert even into highly compressed films. Membrane fusion is accompanied by drastic changes of the membrane curvature. To investigate how the peptides affect the curvature of model lipid membranes, we examined the effect of the fusogenic peptides on the equilibration of cubic monoolein structures after a phase transition from a lamellar state induced by an abrupt hydrostatic pressure reduction. We monitored this process in presence and absence of the peptides with small-angle x-ray scattering and found that HA2-FP and VSV-TMD drastically accelerate the equilibration, while the fusion loops of TBEV and VSV stabilize the swollen state of the lipid structures. In this work, we show that the class I fusion peptide of HA2 penetrates deeply into the hydrophobic region of membranes and is able to promote and accelerate the formation of negative curvature. In contrast, we found that the class II and III fusion loops of TBEV and VSV tend to counteract negative membrane curvature.

A pressure-jump study on the interaction of osmolytes and crowders with cubic monoolein structures.

Many vital processes that take place in biological cells involve remodeling of lipid membranes. These processes take place in a milieu that is packed with various solutes, ranging from ions and small organic osmolytes to proteins and other macromolecules, occupying about 30% of the available volume. In this work, we investigated how molecular crowding, simulated with the polymer polyethylene glycol (PEG), and the osmolytes urea and trimethylamine-N-oxide (TMAO) affect the equilibration of cubic monoolein structures after a phase transition from a lamellar state induced by an abrupt pressure reduction. In absence of additives, swollen cubic crystallites form after the transition, releasing excess water over several hours. This process is reflected in a decreasing lattice constant and was monitored with small angle X-ray scattering. We found that the osmotic pressure exerted by PEG and TMAO, which are displaced from narrow inter-bilayer spaces, accelerates the equilibration. When the radius of gyration of the added PEG was smaller than the radius of the water channels of the cubic phase, the effect became more pronounced with increasing molecular weight of the polymers. As the release of hydration water from the cubic structures is accompanied by an increasing membrane curvature and a reduction of the interface between lipids and aqueous phase, urea, which has a slight affinity to reside near membrane surfaces, stabilized the swollen crystallites and slowed down the equilibration dynamics. Our results support the view that cellular solutes are important contributors to dynamic membrane processes, as they can accelerate dehydration of inter-bilayer spaces and promote or counteract membrane curvature.

The Hydrophobic Gap at High Hydrostatic Pressures.

We have gained new insight into the so-called hydrophobic gap, a molecularly thin region of decreased electron density at the interface between water and a solid hydrophobic surface, by X-ray reflectivity experiments and molecular dynamics simulations at different hydrostatic pressures. Pressure variations show that the hydrophobic gap persists up to a pressure of 5 kbar. The electron depletion in the interfacial region strongly decreases with an increase in pressure, indicating that the interfacial region is compressed more strongly than bulk water. The decrease is most significant up to 2 kbar; beyond that, the pressure response of the depletion is less pronounced.

Solid-Supported Lipid Multilayers under High Hydrostatic Pressure.

In this work, the structure of solid-supported lipid multilayers exposed to increased hydrostatic pressure was studied in situ by X-ray reflectometry at the solid-liquid interface between silicon and an aqueous buffer solution. The layers' vertical structure was analyzed up to a maximum pressure of 4500 bar. The multilayers showed phase transitions from the fluid into different gel phases. With increasing pressure, a gradual filling of the sublayers between the hydrophilic head groups with water was observed. This process was inverted when the pressure was decreased, yielding finally smaller water layers than those in the initial state. As is commonly known, water has an abrasive effect on lipid multilayers by the formation of vesicles. We show that increasing pressure can reverse this process so that a controlled switching between multi- and bilayers is possible.

Phase behavior of lysozyme solutions in the liquid-liquid phase coexistence region at high hydrostatic pressures.

We present results from small-angle X-ray scattering and turbidity measurements on the effect of high hydrostatic pressure on the phase behavior of dense lysozyme solutions in the liquid-liquid phase separation region, and characterize the underlying intermolecular protein-protein interactions as a function of temperature and pressure under charge-screening conditions (0.5 M NaCl). A reentrant liquid-liquid phase separation region is observed at elevated pressures, which may originate in the pressure dependence of the solvent-mediated protein-protein interaction. A temperature-pressure-concentration phase diagram was constructed for highly concentrated lysozyme solutions over a wide range of temperatures, pressures and protein concentrations including the critical region of the liquid-liquid miscibility gap.

X-ray reflectivity measurements of liquid/solid interfaces under high hydrostatic pressure conditions.

A high-pressure cell for in situ X-ray reflectivity measurements of liquid/solid interfaces at hydrostatic pressures up to 500 MPa (5 kbar), a pressure regime that is particularly important for the study of protein unfolding, is presented. The original set-up of this hydrostatic high-pressure cell is discussed and its unique properties are demonstrated by the investigation of pressure-induced adsorption of the protein lysozyme onto hydrophobic silicon wafers. The presented results emphasize the enormous potential of X-ray reflectivity studies under high hydrostatic pressure conditions for the in situ investigation of adsorption phenomena in biological systems.

Temperature-driven adsorption and desorption of proteins at solid-liquid interfaces.

The heat-induced desorption and adsorption of the proteins lysozyme, ribonuclease A, bovine serum albumin, and fibronectin at protein layers was investigated in two different environments: pure buffer and protein solution. Using two different environments allows us to distinguish between thermodynamic and kinetic mechanisms in the adsorption process. We observed a desorption in buffer and an adsorption in protein solution, depending upon protein properties, such as size, stability, and charge. We conclude that the desorption in buffer is mainly influenced by the mobility of the proteins at the interface, while the adsorption in protein solution is driven by conformational changes and, thereby, a gain in entropy. These results are relevant for controlling biofilm formation at solid-liquid interfaces.

Debonding energy of PDMS: A new analysis of a classic adhesion scenario.

We investigated the debonding energy between confined layers of a soft elastic solid (PDMS) and a circular steel indenter in a flat punch geometry. PDMS is extensively used in applications, but also a widespread model system for fundamental research. Varying systematically the pulling speed and the viscoelastic properties, notably the modulus, we determined scaling laws for the debonding energy. We showed that the debonding energy is independent of the sample thickness. Applying a new approach and separating the crack initiation and the propagation part of the force curves, we analyzed the thickness dependence more precisely and we demonstrated that the energy to propagate the crack at given average speed does not only depend on the modulus, but also on the sample thickness.

Cholesterol modulates the pressure response of DMPC membranes.

In this work, the effect of cholesterol on the pressure response of solid-supported phospholipid multilayers is analyzed. It is shown that DMPC multilayers become highly pressure-responsive by the incorporation of low amounts of cholesterol, resulting in a strong pressure-induced expansion of the bilayer spacing. This is accompanied by a high tendency of the multilayer system to detach from the substrate. Increasing the cholesterol concentration reduces the pressure-induced expansion and the membrane structure remains largely unchanged upon pressurization, consequently the stability of the multilayers improves. For a determination of the influence of the substrate, the pressure-dependent behavior of multilayers is compared to that of solid-supported bilayers and multi-lamellar vesicles in bulk solution. While single-supported bilayers remain largely unaffected by external pressure independent of their cholesterol content, multi-lamellar vesicles and multilayers behave similarly.

Analyzing protein-ligand and protein-interface interactions using high pressure.

All protein function is based on interactions with the environment. Proteins can bind molecules for their transport, their catalytic conversion, or for signal transduction. They can bind to each other, and they adsorb at interfaces, such as lipid membranes or material surfaces. An experimental characterization is needed to understand the underlying mechanisms, but also to make use of proteins in biotechnology or biomedicine. When protein interactions are studied under high pressure, volume changes are revealed that directly describe spatial contributions to these interactions. Moreover, the strength of protein interactions with ligands or interfaces can be tuned in a smooth way by pressure modulation, which can be utilized in the design of drugs and bio-responsive interfaces. In this short review, selected studies of protein-ligand and protein-interface interactions are presented that were carried out under high pressure. Furthermore, a perspective on bio-responsive interfaces is given where protein-ligand binding is applied to create functional interfacial structures.

Density variations of TMAO solutions in the kilobar range: Experiments, PC-SAFT predictions, and molecular dynamics simulations.

We present measurements, molecular dynamics (MD) simulations, and predictions using Perturbed-Chain Statistical Associating Fluid Theory (PC-SAFT) of the density of aqueous solutions in a pressure range from 1 bar to 5000 bar, a pressure regime that is highly relevant for both biochemical applications and the fundamental understanding of solvation. The accurate determination of density data of pressurized solutions remains challenging. We determined relative density changes from the variations in X-ray absorption through the sample and developed a new water parameter set for PC-SAFT modeling that is appropriate for high pressure conditions in the kilobar regime. As a showcase, we studied trimethylamine N-oxide (TMAO) solutions and demonstrated that their compressibility decreases with the TMAO content. This result is linked to the stabilizing effect of TMAO on the local H-bond network of water. Experiments and calculations, which represent two independent methods, are in very good agreement and are in accordance with results of force field molecular dynamics simulations of the same systems.

Structure and thermodynamics of aqueous urea solutions from ambient to kilobar pressures: From thermodynamic modeling, experiments, and first principles simulations to an accurate force field description.

Molecular simulations based on classical force fields are a powerful method for shedding light on the complex behavior of biomolecules in solution. When cosolutes are present in addition to water and biomolecules, subtle balances of weak intermolecular forces have to be accounted for. This imposes high demands on the quality of the underlying force fields, and therefore force field development for small cosolutes is still an active field. Here, we present the development of a new urea force field from studies of urea solutions at ambient and elevated hydrostatic pressures based on a combination of experimental and theoretical approaches. Experimental densities and solvation shell properties from ab initio molecular dynamics simulations at ambient conditions served as the target properties for the force field optimization. Since urea is present in many marine life forms, elevated hydrostatic pressure was rigorously addressed: densities at high pressure were measured by vibrating tube densitometry up to 500 bar and by X-ray absorption up to 5 kbar. Densities were determined by the perturbed-chain statistical associating fluid theory equation of state. Solvation properties were determined by embedded cluster integral equation theory and ab initio molecular dynamics. Our new force field is able to capture the properties of urea solutions at high pressures without further high-pressure adaption, unlike trimethylamine-N-oxide, for which a high-pressure adaption is necessary.

Pattern formation during deformation of a confined viscoelastic layer: from a viscous liquid to a soft elastic solid.

We study pattern formation during tensile deformation of confined viscoelastic layers. The use of a model system [poly(dimethylsiloxane) with different degrees of cross-linking] allows us to go continuously from a viscous liquid to an elastic solid. We observe two distinct regimes of fingering instabilities: a regime called "elastic" with interfacial crack propagation, where the fingering wavelength scales only with the film thickness, and a bulk regime called "viscoelastic," where the fingering instability shows a Saffman-Taylor-like behavior. We find good quantitative agreement with theory in both cases and present a reduced parameter describing the transition between the two regimes and allowing us to predict the observed patterns over the whole range of viscoelastic properties.