Claus Czeslik

Temperature- and pressure-dependent phase behavior of monoacylglycerides monoolein and monoelaidin.

We used x-ray and neutron diffraction to study the temperature- and pressure-dependent structure and phase behavior of the monoacylglycerides 1-monoelaidin (ME) and 1-monoolein (MO) in excess water. The monoacylglycerides were chosen for investigation of their phase behavior because they exhibit mesomorphic phases with one-, two-, and three-dimensional periodicity, such as lamellar, an inverted hexagonal and bicontinuous cubic phases, in a rather easily accessible temperature and pressure range. We studied the structure, stability, and transformations of the different phases over a wide temperature and pressure range, explored the epitaxial relations that exist between different phases, and established a relationship between the chemical structure of the lipid molecules and their phase behavior. For both systems, a temperature-pressure phase diagram has been determined in the temperature range from 0 to 100 degrees C at pressures from ambient up to 1400 bar, and drastic differences in phase behavior are found for the two systems. In MO-water dispersions, the cubic phase Pn3m extends over a large phase field in the T,p-plane. At temperatures above 95 degrees C, the inverted hexagonal phase is found. In the lower temperature region, a crystalline lamellar phase is induced at higher pressures. The phases found in ME-water include the lamellar crystalline Lc phase, the L beta gel phase, the L alpha liquid-crystalline phase, and two cubic phases belonging to the crystallographic space groups Im3m and Pn3m. In addition, the existence of metastable phases has been exploited. Between coexisting metastable cubic structures, a metric relationship has been found that is predicted theoretically on the basis of the curvature elastic energy approximation only.

Effect of temperature on the conformation of lysozyme adsorbed to silica particles

Steady-state and lifetime fluorescence spectroscopy was used to study the conformation of hen egg white lysozyme adsorbed to differently charged colloidal silica particles as a function of temperature. While electrostatic interactions appear to be the driving force for adsorption, a decreased charge density of the substrate was found to enhance attractive protein–silica interactions. In the adsorbed state the temperature of unfolding is lowered by about 12–20°C, reflecting a decreased thermal protein stability. Applying a two-state thermodynamic model significantly smaller enthalpy and entropy changes have been found for the temperature-induced unfolding of lysozyme when it is adsorbed to the silica particles. From intrinsic fluorescence lifetime measurements a characteristic change of the lifetime distribution of lysozyme due to adsorption has been observed over a wide temperature range. These results were found to be consistent with an adsorption-induced modification of the lysozyme structure and a spreading of lysozyme on the silica particles in the process of unfolding.

Pressure effects on the structure of lyotropic lipid mesophases and model biomembrane systems

Lipid systems, which provide valuable model systems for biological membranes, display a variety of polymorphic phases, depending on their molecular structure and environmental conditions. By use of X-ray and neutron diffraction the temperature- and pressure-dependent structure and phase behavior of lipid systems, differing in chain configuration and headgroup structure, have been studied. Besides lamellar phases also nonlamellar phases have been investigated. Hydrostatic pressure has been used as a physical parameter for studying the stability and energetics of lyotropic lipid mesophases, but also because high pressure is an important feature of certain natural membrane environments (e.g., marine biotopes) and because the high pressure phase behavior of biomolecules is of biotechnological interest (e.g., high pressure food processing). We demonstrate that temperature and pressure have noncongruent effects on the structural and phase behavior. By using the pressure-jump relaxation technique in combination with time-resolved synchrotron X-ray diffraction, the kinetics of different lipid phase transformations was also investigated. The time constants for completion of the transitions depend on the direction of the transition, the symmetry and topology of the structures involved, and also on the pressure-jump amplitude. In addition, the effect of incorporating ions, steroids and polypeptides into bilayers on the temperature- and pressure-dependent phase behavior of the lipid systems is discussed.

Salt-induced protein resistance of polyelectrolyte brushes studied using fluorescence correlation spectroscopy and neutron reflectometry

We used two-photon excitation fluorescence correlation spectroscopy (FCS) and neutron reflectometry to study in situ the effect of salt concentration on the degree of protein binding to polyelectrolyte brushes. The binding of bovine serum albumin (BSA) to poly(acrylic acid) (PAA) brushes was characterized at neutral pH values where both the protein and the brushes carry a negative charge. Spherical PAA brush particles were used in the FCS experiments, whereas a planar PAA brush served as protein substrate in the neutron reflectometry experiments. It has been found that BSA binds strongly to both the spherical and the planar PAA brushes under electrostatic repulsion at low ionic strength. The BSA volume fraction profile, as determined from the neutron reflectivities, indicates a deep penetration of the BSA molecules into the PAA brush. However, the analysis of the FCS data reveals that the protein affinity of the spherical PAA brush particles decreases drastically when increasing the concentration of sodium chloride to a few 100 mM. This observation is in line with the measured neutron reflectivities of the planar PAA brush. The reflectivity curve obtained in the absence of protein is virtually overlapping with that measured when the PAA brush is in contact with a BSA solution but containing 500 mM sodium chloride which suggests protein resistance of the planar PAA brush at this elevated salt concentration. The results of this study provide evidence for a new kind of protein-resistant interfaces. Whereas protein binding to the PAA brush is likely to be dominated by the release of counterions, this driving force vanishes as the ionic strength of the solution is raised and protein molecules are repelled from the interface by steric interactions. In a general view, the “switching” of the protein affinity of a PAA brush by varying the ionic strength of the protein solution over a relatively small range may appear to be useful for biotechnological applications.

Effect of high pressure on the structure of dipalmitoylphosphatidylcholine bilayer membranes: a synchrotron-X-ray diffraction and FT-IR spectroscopy study using the diamond anvil technique

Abstract The effect of high pressure, up to 20 kbar, on multilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) was examined by using FT-IR spectroscopy and Synchrotron-X-ray diffraction in the small- and wide-angle scattering region. The temperature and pressure range studied permitted us to explore the phase behaviour of DPPC from the liquid–crystalline phase through various gel phases up to the pressure region where bulk water freezes and freeze-induced dehydration of the membrane occurs. To our knowledge this is the first study where the structure and phase behaviour of an amphiphilic lipid system has been studied up to these high pressures. The use of pressure to study the lipid gel state has revealed several new phases which may not exist at very low temperatures at ambient pressure.

Reorientational Dynamics of Enzymes Adsorbed on Quartz: A Temperature-Dependent Time-Resolved TIRF Anisotropy Study

The preservation of enzyme activity and protein binding capacity upon protein adsorption at solid interfaces is important for biotechnological and medical applications. Because these properties are partly related to the protein flexibility and mobility, we have studied the internal dynamics and the whole-body reorientational rates of two enzymes, staphylococcal nuclease (SNase) and hen egg white lysozyme, over the temperature range of 20-80 degrees C when the proteins are adsorbed at the silica/water interface and, for comparison, when they are dissolved in buffer. The data were obtained using a combination of two experimental techniques, total internal reflection fluorescence spectroscopy and time-resolved fluorescence anisotropy measurements in the frequency domain, with the protein Trp residues as intrinsic fluorescence probes. It has been found that the internal dynamics and the whole-body rotation of SNase and lysozyme are markedly reduced upon adsorption over large temperature ranges. At elevated temperatures, both protein molecules appear completely immobilized and the fractional amplitudes for the whole-body rotation, which are related to the order parameter for the local rotational freedom of the Trp residues, remain constant and do not approach zero. This behavior indicates that the angular range of the Trp reorientation within the adsorbed proteins is largely restricted even at high temperatures, in contrast to that of the dissolved proteins. The results of this study thus provide a deeper understanding of protein activity at solid surfaces.

Exploring the interfacial structure of protein adsorbates and the kinetics of protein adsorption: an in situ high-energy X-ray reflectivity study.

The high energy X-ray reflectivity technique has been applied to study the interfacial structure of protein adsorbates and protein adsorption kinetics in situ. For this purpose, the adsorption of lysozyme at the hydrophilic silica-water interface has been chosen as a model system. The structure of adsorbed lysozyme layers was probed for various aqueous solution conditions. The effect of solution pH and lysozyme concentration on the interfacial structure was measured. Monolayer formation was observed for all cases except for the highest concentration. The adsorbed protein layers consist of adsorbed lysozyme molecules with side-on or end-on orientation. By means of time-dependent X-ray reflectivity scans, the time-evolution of adsorbed proteins was monitored as well. The results of this study demonstrate the capabilities of in situ X-ray reflectivity experiments on protein adsorbates. The great advantages of this method are the broad wave vector range available and the high time resolution.

Effect of pressure on the stability, phase behaviour and transformation kinetics between structures of lyotropic lipid mesophases and model membrane systems

Lipids, which provide valuable model systems for membranes, display a variety of polymorphic phases, depending on their molecular structure and environmental conditions. By use of x-ray and neutron diffraction, infrared spectroscopy and calorimetry, the temperature and pressure dependent structure and phase behaviour of several lipid systems, differing in chain configuration and headgroup structure, have been studied. Besides lamellar phases also non-lamellar phases, such as the inverted hexagonal phase and bicontinuous cubic phases, have been investigated. Hydrostatic pressure has been used as a physical parameter for studying the stability and energetics of lyotropic mesophases, but also because high pressure is an important feature of certain natural membrane environments (e.g., marine biotopes) and because the high-pressure phase behaviour of biomolecules is of biotechnological interest. Neutron scattering in combination with the H/D contrast variation technique has been used to the study of lateral organization of phase-separated binary lipid mixtures with distinct mixing properties. Within their two-phase coexistence regions large-scale concentration fluctuations appear, and the morphology of these fluctuations can be characterized as a complex heterogeneous system of coexisting clusters having fractal-like properties. By using the pressure-jump relaxation technique in combination with time-resolved synchrotron x-ray diffraction, the kinetics of different lipid phase transformations were also investigated. The time constants for completion of the transitions are dependent on the direction of the transition, the symmetry and topology of the structures involved, and also on the pressure-jump amplitude. In several cases also intermediate structures can be detected under non-equilibrium conditions.

Influence of pressure and crowding on the sub-nanosecond dynamics of globular proteins.

The influence of high hydrostatic pressure on the internal sub-nanosecond dynamics of highly concentrated lysozyme in aqueous solutions was studied by elastic incoherent neutron scattering (EINS) up to pressures of 4 kbar. We have found, with increasing pressure, a reduction in the dynamics of H atoms of folded lysozyme, suggesting a loss in protein mobility that follows a change in the local energy landscape upon the increase in packing density. Moreover, the amplitude of the protein fluctuations depends drastically on the protein concentration, and protein structural and interaction parameters as well as the dynamical properties are affected by pressure in a nonlinear way. A significant reduction of the mean squared displacement of H atoms occurs already at rather low pressures of a few hundred bars for lysozyme in bulk water solution. This trend is lifted at ∼2 kbar, which is probably due to a solvent-mediated effect. Conversely, for high protein concentrations (e.g., 160 mg mL(-1)), that is, under strong self-crowding conditions, as they are also encountered in the biological cell, strong restriction of the dynamics of protein motions takes place, reducing the mean squared displacement of H atoms by 60% and rendering its pressure dependence almost negligible. These results are also important for understanding the pressure stability of highly concentrated protein solutions in organisms thriving under hydrostatic pressure conditions such as in the deep sea, where pressures up to the kbar level are reached.

Interaction of IAPP and Insulin with Model Interfaces Studied Using Neutron Reflectometry

The islet amyloid polypeptide (IAPP) and insulin are coproduced by the beta-cells of the pancreatic islets of Langerhans. Both peptides can interact with negatively charged lipid membranes. The positively charged islet amyloid polypeptide partially inserts into these membranes and subsequently forms amyloid fibrils. The amyloid fibril formation of insulin is also accelerated by the presence of negatively charged lipids, although insulin has a negative net charge at neutral pH-values. We used water-polymer model interfaces to differentiate between the hydrophobic and electrostatic interactions that can drive these peptides to adsorb at an interface. By applying neutron reflectometry, the scattering-length density profiles of IAPP and insulin, as adsorbed at three different water-polymer interfaces, were determined. The islet amyloid polypeptide most strongly adsorbed at a hydrophobic poly-(styrene) surface, whereas at a hydrophilic, negatively charged poly-(styrene sulfonate) interface, the degree of adsorption was reduced by 50%. Almost no IAPP adsorption was evident at this negatively charged interface when we added 100 mM NaCl. On the other hand, negatively charged insulin was most strongly attracted to a hydrophilic, negatively charged interface. Our results suggest that IAPP is strongly attracted to a hydrophobic surface, whereas the few positive charges of IAPP cannot warrant a permanent immobilization of IAPP at a hydrophilic, negatively charged surface at an ionic strength of 100 mM. Furthermore, the interfacial accumulation of insulin at a hydrophilic, negatively charged surface may represent a favorable precondition for nucleus formation and fibril formation.