Julia Nase

Exploring the Piezophilic Behavior of Natural Cosolvent Mixtures

Proteins are only marginally stable and are hence very sensitive to environmental conditions, such as high and low temperatures or high hydrostatic pressures. In nature, living organisms are able to compensate for extreme environmental conditions and hence rescue proteins from denaturation by using osmolytes. Organic osmolytes are accumulated under anhydrobiotic, thermal, and pressure stresses. Among those osmolytes are amino acids, polyols and sugars (e.g., glycerol and trehalose), methylamines such as trimethylamine-Noxide (TMAO), and urea. TMAO has been found to enhance protein folding and ligand binding most efficiently. On the other hand, urea, a highly concentrated waste product in mammalian kidneys, is a perturbant. It is also a major organic osmolyte in marine elasmobranch fishes. Interestingly, TMAO has been found to counteract perturbations imposed by urea and hydrostatic pressure in deep-sea animals, most effectively at a 2:1 urea:TMAO ratio. In the deep sea, hydrostatic pressures up to the 1 kbar (100 MPa) range prevail, and living organisms have to cope with such extreme environmental conditions. High hydrostatic pressure generally destabilizes the protein structure, inhibits polymerization of proteins and ligand binding. Interestingly, TMAO has been shown to largely offset these pressure effects. In fact, it was found that the amount of TMAO in the cells of a series of marine organisms increases linearly with the depth of the ocean. For that reason, TMAO is thought to serve as pressure counteractant. The term “piezolyte” has been coined for such kind of cosolute. About the underlying mechanism of stabilization by TMAO at ambient pressure conditions several experimental and theoretical (molecular dynamics simulations) articles have been published in recent years. TMAO is largely excluded from the protein surface and enhances the water structure causing greater organization through more and stronger hydrogen bonding among water molecules. However, the mechanism of this “chemical chaperon” at high hydrostatic pressure (HHP) conditions is still unclear. To yield a deeper understanding of this phenomenon, we determined the intermolecular interaction of dense protein solutions in the absence and presence of cosolvent mixtures of TMAO and urea also under HHP conditions. Small-angle Xray scattering (SAXS) experiments on dense lysozyme solutions have been carried out in the pressure range from 1 bar up to 4 kbar. The SAXS technique accurately monitors structural alterations of the protein solution and yields quantitative information on the state-dependent protein– protein interaction potential. As lysozyme is a highly stable protein, pressure-induced effects will only be attributed to changes in the protein–protein interaction of the native protein and how this is influenced by osmolytes. No pressureinduced unfolding of the protein occurs in the pressure range covered. Complementary thermodynamic data, that is, the temperature of unfolding and the volume change upon unfolding of the protein, were obtained by differential scanning (DSC) and pressure perturbation calorimetry (PPC), respectively. To verify that the protein is folded at all solution conditions studied, SAXS measurements on diluted lysozyme solutions (cP= 10 mgmL ) were carried out in the whole pressure range covered. For diluted protein solutions, the scattering intensity I(q) is proportional to the form factor P(q) (q= (4p/l)sin(V/2) is the wave vector transfer, l the wavelength of the X-rays, and V the scattering angle), which depends on the structure and size of the protein. For dilute lysozyme solutions, the radius of gyration of the particle, Rg, could be determined. We found a constant Rg value of (15.1 0.4) up to 4 kbar, indicating the absence of unfolding even at the highest pressure applied. In the case of concentrated protein solutions, the interaction between the particles gives rise to an additional scattering contribution. This SAXS signal can be described as the product of the form factor and an effective structure factor, which is related to the intermolecular structure factor S(q). To relate the structure factor to the protein–protein interaction potential, statistical mechanical model approaches have to be employed. Here, the mean-spherical approximation (MSA) in combination with the DLVO (Derjaguin– Landau–Verwey–Overbeek) potential V(r) has been used. The pair potential V(r) is given as the sum of a hard sphere potential VHS(r), a repulsive screened Coulomb-like potential VSC(r) and an attractive Yukawian potential VY(r), which is frequently used to describe protein–protein interactions (for details, see the Supporting Information). [*] Y. Zhai, Prof. Dr. R. Winter Fakult t Chemie, TU Dortmund Physikalische Chemie—Biophysikalische Chemie Otto-Hahn Str. 6, 44227 Dortmund (Germany) E-mail: roland.winter@tu-dortmund.de

Measurement of the receding contact angle at the interface between a viscoelastic material and a rigid surface

We present the first quantitative measurement of the receding contact angle between a soft viscoelastic material and a solid surface during the debonding of the material. We developed a new method of observation, based on the technique pioneered by Yamaguchi et al. (T. Yamaguchi, K. Koike, and M. Doi, Euro. Phys. Lett., 2007, 77, 64002). In a probe-tack geometry, we obtained a 3D-like view of the debonding pattern as seen through the adhesive/probe interface, providing thus an excellent view of the details of the contact geometry near the interface. Combining visual and force-displacement information in a probe tack test, we identified three different categories of mechanisms and contact geometries as a function of the material parameters. For very weakly crosslinked viscoelastic materials around the gel point, air fingers form in the bulk of the layer. For higher degrees of crosslinking, the air fingers form and propagate at the interface, with a large deformation of the bulk of the layer. A well defined and reproducible receding contact angle φ < 90° depending on the viscoelastic properties of the layer was observed at the leading edge of the moving finger. Finally for well crosslinked samples the failure occurs by interfacial crack propagation with little deformation of the layer; the contact angle at the leading edge then was close to 90° as expected from elastic fracture mechanics.

Dynamic evolution of fingering patterns in a lifted Hele–Shaw cell

We present a study on pattern formation in a Newtonian liquid during lifting of a circular Hele–Shaw cell. When a confined layer of oil is subject to such a stretch flow, air penetrates into the liquid from the sides and a fingering instability, a variant of the classical Saffman–Taylor instability, evolves. This setting has the particularity that the finger growth takes place in a conserved volume of liquid and that the dimensionless surface tension, the control parameter which governs the Saffman–Taylor instability, is changing with time. This leads to a constantly evolving pattern, which we investigate with regard to number of fingers and finger amplitude. We distinguish in the pattern at each instant growing fingers and stagnant fingers. Systematically varying the properties of the viscous oil and the geometry of the Hele–Shaw cell, we show that the number of growing fingers is at each moment well described by a simple approach based on linear stability analysis and depends only on the dimensionless s...

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.Graphical abstract

Polaron-induced lattice distortion of (In,Ga)As/GaAs quantum dots by optically excited carriers

We report on a high resolution x-ray diffraction study unveiling the effect of carriers optically injected into (In,Ga)As quantum dots on the surrounding GaAs crystal matrix. We find a tetragonal lattice expansion with enhanced elongation along the [001] crystal axis that is superimposed on an isotropic lattice extension. The isotropic contribution arises from excitation induced lattice heating as confirmed by temperature dependent reference studies. The tetragonal expansion on the femtometer scale is tentatively attributed to polaron formation by carriers trapped in the quantum dots.

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.

The adsorption behavior of octafluoropropane at the water/gas interface

We studied the adsorption behavior of the gas octafluoropropane at the water/gas interface as a function of different pressures. In a custom-made measurement cell, the gas pressure was varied in a range between 1 bar and close to the condensation pressure of octafluoropropane. The electron density profiles of the adsorption layers show that the layer thickness increases with pressure. The evolution of the layer electron density indicates that the bulk electron density is reached if a layer consisting of more than one monolayer of octafluoropropane is adsorbed on the water surface.

Study of time and pressure dependent phenomena at the hard x-ray beamline BL9 of DELTA

The beamline BL9 of DELTA (Dortmund ELecTron Accelerator) is a multi-purpose beamline operating in an energy range between 4 and 27 keV. A short overview of the beamline and the experimental endstation is given. Exemplarily three typical applications, namely x-ray diffraction from interfaces, small angle x-ray scattering under high hydrostatic pressure and fast x-ray reflectivity measurements, are discussed in some detail in order to demonstrate the capabilities of the beamline.