In reference to the complexity and heterogeneity of cellular membranes, the structure and lateral ordering of lipid monolayers and bilayers composed of multi-component lipid mixtures have been investigated and compared in the present study. These complex model biomembrane systems represent valuable model systems, e. g. for studies of lipid-peptide interactions, where an integrated in situ multi-technique approach using both monolayer and bilayer techniques is required. A zwitterionic (3 components) and an anionic (5 components) heterogeneous model membrane system have been characterized that consist of saturated and unsaturated phospholipids as well as cholesterol. Lipid monolayers were analyzed by surface X-ray scattering techniques, and both the vertical structure (electron density profile) and the degree of in-plane ordering were determined as a function of surface pressure and temperature. The corresponding structure and lateral organisation of the bilayer membranes were characterized by atomic force microscopy. Both monolayers and bilayers reveal ordered domain formation, the monolayer ones being of much smaller size and different temperature stability, however. Furthermore, the charge density of the lipid monolayer has a drastic influence on the size of ordered domains as well as the intermolecular distances of the lipid molecules.

The adsorption process of proteins to surfaces is governed by the mutual interactions among proteins, the solution, and the substrate. Interactions arising from the substrate are usually attributed to the uppermost atomic layer. This actual surface defines the surface chemistry and hence steric and electrostatic interactions. For a comprehensive understanding, however, the interactions arising from the bulk material also have to be considered. Our protein adsorption experiments with globular proteins (α-amylase, bovine serum albumin, and lysozyme) clearly reveal the influence of the subsurface material via van der Waals forces. Here, a set of functionalized silicon wafers enables a distinction between the effects of surface chemistry and the subsurface composition of the substrate. Whereas the surface chemistry controls whether the individual proteins are denatured, the strength of the van der Waals forces affects the final layer density and hence the adsorbed amount of proteins. The results imply that van der Waals forces mainly influence surface processes, which govern the structure formation of the protein adsorbates, such as surface diffusion and spreading.

Osmolytes are substances that affect osmosis and are used by cells to adapt to environmental stress. Here, we present a neutron reflectivity study on the effect of various osmolytes on the structure of protein adsorbates at solid-water interfaces. Ribonuclease A (RNase A) and bovine insulin were used as model proteins adsorbing at a hydrophilic silica and a hydrophobic polystyrene planar surface. From the data, neutron scattering length densities were extracted revealing the thickness, volume fraction and roughness of the protein adsorbates in the absence and the presence of urea, trehalose, sucrose, and glycerol.

We report x-ray reflectivity and grazing incidence x-ray diffraction measurements of lipopolysaccharide (LPS) monolayers at the water-air interface. Our investigations reveal that the structure and lateral ordering of the LPS molecules is very different from phospholipid systems and can be modulated by the ionic strength of the aqueous subphase in an ion-dependent manner. Our findings also indicate differential effects of monovalent and divalent ions on the two-dimensional ordering of lipid domains. Na(+) ions interact unspecifically with LPS molecules based on their ability to efficiently screen the negative charges of the LPS molecules, whereas Ca(2+) ions interact specifically by cross-linking adjacent molecules in the monolayer. At low lateral pressures, Na(+) ions present in the subphase lead to a LPS monolayer structure ordered over large areas with high compressibility, nearly hexagonal packing of the hydrocarbon chains, and high density in the LPS headgroup region. At higher film pressures, the LPS monolayer becomes more rigid and results in a less perfect, oblique packing of the LPS hydrocarbon chains as well as a smaller lateral size of highly ordered domains on the monolayer. Furthermore, associated with the increased surface pressure, a conformational change of the sugar headgroups occurs, leading to a thickening of the entire LPS monolayer structure. The effect of Ca(2+) ions in the subphase is to increase the rigidity of the LPS monolayer, leading to an oblique packing of the hydrocarbon chains already at low film pressures, an upright orientation of the sugar moieties, and much smaller sizes of ordered domains in the plane of the monolayer. In the presence of both Na(+)- and Ca(2+) ions in the subphase, the screening effect of Na(+) is predominant at low film pressures, whereas, at higher film pressures, the structure and lateral organization of LPS molecules is governed by the influence of Ca(2+) ions. The unspecific charge-screening effect of the Na(+) ions on the conformation of the sugar moiety becomes less dominant at biologically relevant lateral pressures.

Proteinassoziation an Membranen: Eine Untersuchung der Wechselwirkungen zwischen dem Insel-Amyloid-Polypeptid (IAPP), dem β-Amyloid-Peptid (Aβ) sowie deren Mischung und anionischen Raft-Modellmembranen zeigte die dominante Wirkung von IAPP auf den Aggregationsprozess und die wasserstoffverbrückte Aggregatstruktur (siehe Bild). Die Analyse der Wechselwirkung zwischen Aβ und IAPP-GI, einem nichtamyloidogenen IAPP-Konstrukt, bestätigte die Beobachtungen.

We report X-ray reflectivity (XRR) and grazing incidence X-ray diffraction (GIXD) measurements of archaeal bipolar tetraether lipid monolayers at the air-water interface. Specifically, Langmuir films made of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at three different temperatures, i.e., 68, 76, and 81 °C, were examined. The dependence of the structure and packing properties of PLFE monolayers on surface pressure were analyzed in a temperature range between 10 and 50 °C at different pH values. Additionally, the interaction of PLFE monolayers (using lipids derived from cells grown at 76 °C) with the ion channel peptide gramicidin was investigated as a function of surface pressure. A total monolayer thickness of approximately 30 Å was found for all monolayers, hinting at a U-shaped conformation of the molecules with both head groups in contact with the interface. The monolayer thickness increased with rising film pressure and decreased with increasing temperature. At 10 and 20 °C, large, highly crystalline domains were observed by GIXD, whereas at higher temperatures no distinct crystallinity could be observed. For lipids derived from cells grown at higher temperatures, a slightly more rigid structure in the lipid dibiphytanyl chains was observed. A change in the pH of the subphase had an influence only on the structure of the lipid head groups. The addition of gramicidin to an PLFE monolayer led to a more disordered state as observed by XRR. In GIXD measurements, no major changes in lateral organization could be observed, except for a decrease of the size of crystalline domains, indicating that gramicidin resides mainly in the disordered areas of the monolayer and causes local membrane perturbation, only.

Osmolytes are substances that affect osmosis and are used by cells to adapt to environmental stress. Here, we report a neutron reflectivity study on the influence of some osmolytes on protein adsorption at solid liquid interfaces. Bovine ribonuclease A (RNase) and bovine insulin were used as model proteins adsorbing at a hydrophilic silica and at a hydrophobic polystyrene surface. From the neutron reflectivity data, the adsorbed protein layers were characterized in terms of layer thickness, protein packing density, and adsorbed protein mass in the absence and presence of urea, trehalose, sucrose, and glycerol. All data point to the clear effect of these nonionic cosolvents on the degree of protein adsorption. For example, 1 M sucrose leads to a reduction of the adsorbed amount of RNase by 39% on a silica surface and by 71% on a polystyrene surface. Trehalose was found to exhibit activity similar to that of sucrose. The changes in adsorbed protein mass can be attributed to a decreased packing density of the proteins in the adsorbed layers. Moreover, we investigated insulin adsorption at a hydrophobic surface in the absence and presence of glycerol. The degree of insulin adsorption is decreased by even 80% in the presence of 4 M of glycerol. The results of this study demonstrate that nonionic cosolvents can be used to tune and control nonspecific protein adsorption at aqueous solid interfaces, which might be relevant for biomedical applications.

In a biological cell, proteins perform their functions in a highly complex environment comprising crowding and confinement effects as well as interactions with interfaces, cosolvents, and other biomolecules. Cosolvents can stabilize or destabilize the native folded structure of proteins in solution. Here, we present recent studies on how ionic and non-ionic cosolvents affect the interfacial affinity and structure of proteins at hydrophilic and hydrophobic surfaces [1,2]. We studied the adsorption of bovine ribonuclease A (RNase A) at the silica-water, the polystyrene-water, and the air-water interface as model systems that were analyzed applying optical reflectometry as well as neutron and X-ray scattering techniques. The degree of protein adsorption and the density profile of the adsorbed protein films were determined in the absence and the presence of cosolvents. It has been found that both the protein stabilizing glycerol and the destabilizing urea cause a distinct reduction in the interfacial affinity of RNase A, which may represent a rather unexpected result. At the hydrophobic polystyrene surface, it has been found that both the kosmotropic salts, (NH4)2SO4 and Na2SO4, and the chaotropic salts, NaSCN and Ca(SCN)2, significantly reduce the amount of adsorbed protein, while maximum adsorption is observed in the presence of NaCl.

This study reports on the influence of nonionic cosolvents on the interfacial structure of ribonuclease A (RNase) adsorbed at the air-water interface. We applied X-ray reflectometry to obtain detailed volume fraction profiles of the adsorbed layers and to follow the effect of glycerol and urea on the adsorbate structure as a function of cosolvent concentration. Under all conditions studied, the adsorbed RNase layer maintains its compact shape, and the adsorbed RNase molecules adopt a flat-on orientation at the interface. Both kosmotropic glycerol and chaotropic urea exert profound effects on the adsorbate: The surface excess decreases linearly with glycerol content and is also reduced at low urea concentration. However, at high urea concentration, parts of the adsorbed layer are dehydrated and become exposed to air. The electron density and volume fraction profiles of the adsorbed protein provide clear evidence that these effects are ruled by different mechanisms.

The effects of various salts on the adsorption of ribonuclease A (RNase) at a hydrophobic poly(styrene) film was analyzed in this study applying neutron reflectometry. It has been found that both the kosmotropic salts, (NH4)2SO4 and Na2SO4, and the chaotropic salts, NaSCN and Ca(SCN)2, significantly reduce the amount of adsorbed protein. Maximum adsorption is observed in the presence of NaCl. Apparently, there is no single Hofmeister effect on the degree of protein adsorption at an aqueous-solid interface which ranges from kosmotropic to chaotropic ions. The observed variations in the adsorbed amount can be attributed to variations in the packing density of the adsorbed protein molecules. The results suggest that kosmotropic salts reduce the degree of protein adsorption by disfavoring a conformational adaptation and a dehydration of the protein molecules at a hydrophobic poly(styrene) film. On the other hand, chaotropic salts shield hydrophobic interactions between the protein molecules and the substrate by saturating hydrophobic patches on the protein surface. Remarkably, the results of this study corroborate earlier findings on the effects of non-ionic cosolvents, glycerol and urea, on protein adsorption.

A planar poly(acrylic acid) (PAA) brush provides an unusual substrate for the unspecific immobilization of proteins on material surfaces. At neutral pH-values, proteins adsorb at a PAA brush when the ionic strength of the protein solution is low. In contrast, raising the ionic strength to a few 100 mM transforms a PAA brush into a rather protein-resistant surface coating. Moreover, a PAA brush represents a mild environment for adsorbed proteins which preserves their secondary structure and biological activity. In this study, we focus on the biocompatibility of a PAA brush with an insulin solution. Insulin can form amyloid fibrils, which may also be triggered by interfaces. Using neutron reflectometry and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, the effects of pD value, ionic strength, and glycerol concentration on the density profile and the secondary structure of adsorbed insulin molecules at a PAA brush have been studied. At pD 7, insulin adsorbs at a PAA brush despite its negative net charge. As has been found for other proteins in earlier studies, increasing the ionic strength of the insulin solution to 500 mM decreases the amount of adsorbed insulin drastically. In contrast, at pD 2, addition of salt to the insulin solution induces a thick insulin adsorbate at a PAA brush suggesting both protein-brush and protein-protein interactions, i.e., insulin adsorption and aggregation to be effective. However, in the presence of 2 M glycerol, insulin adsorption is largely suppressed. Furthermore, no major alterations of the secondary structure of adsorbed insulin can be detected by ATR-FTIR spectroscopy under all conditions studied. Hence, the performed experiments demonstrate that a PAA brush does not promote the formation of insulin amyloid structures, which represents a fundamentally new aspect of the biocompatibility of this material surface coating.

In a biological cell, proteins face a highly complex environment comprising crowding and confinement effects as well as interactions with interfaces, cosolvents, and other biomolecules. In this study, the X-ray reflectivity technique has been used for the in-situ characterization of adsorbed protein layers at solid-liquid interfaces. The adsorption of bovine serum albumin at the hydrophobic polystyrene-water interface has been investigated in the presence and in the absence of salts. The data indicate that enhanced adsorption occurs at high salt concentrations.

The islet amyloid polypeptide (IAPP) or amylin is a pancreatic hormone and crucially involved in the pathogenesis of type-II diabetes mellitus (T2DM). Aggregation and amyloid formation of IAPP is considered as the primary culprit for pancreatic beta-cell loss in T2DM patients. In this study, first X-ray reflectivity (XRR) measurements on IAPP at lipid interfaces have been carried out, providing a molecular level characterization of the first steps of the lipid-induced fibrillation process of IAPP, which is initiated by lipid-induced nucleation, oligomerization, followed by detachment of larger IAPP aggregate structures from the lipid membrane, and terminated by the formation of mature fibrils in the bulk solution. The adsorption process of IAPP at lipid interfaces in the absence and presence of negatively charged lipid has also been studied by complementary ATR-FTIR spectroscopic measurements. The morphological properties were followed by atomic force microscopy (AFM). Moreover, we show that the polyphenolic red wine compound resveratrol is able to inhibit IAPP aggregation also in the presence of aggregation-fostering negatively charged lipid interfaces, revealing its potential as a drug candidate for T2DM.

The effects of various salts on the adsorption of ribonuclease A (RNase) at a hydrophobic poly(styrene) film was analyzed in this study applying neutron reflectometry. It has been found that both the kosmotropic salts, (NH4)2SO4 and Na2SO4, and the chaotropic salts, NaSCN and Ca(SCN)2, significantly reduce the amount of adsorbed protein. Maximum adsorption is observed in the presence of NaCl. Apparently, there is no single Hofmeister effect on the degree of protein adsorption at an aqueous-solid interface that ranges from kosmotropic to chaotropic ions. The observed variations in the adsorbed amount can be attributed to variations in the packing density of the adsorbed protein molecules. The results suggest that kosmotropic salts reduce the degree of protein adsorption by disfavoring a conformational adaptation and a dehydration of the protein molecules at a hydrophobic poly(styrene) film. On the other hand, chaotropic salts shield hydrophobic interactions between the protein molecules and the substrate by saturating hydrophobic patches on the protein surface. Remarkably, the results of this study corroborate earlier findings on the effects of nonionic cosolvents on protein adsorption (Koo, J.; Gutberlet, T.; Czeslik, C. J. Phys. Chem. B 2008, 112, 6292-6295).

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.

An X-ray reflectivity set-up to study buried interfaces at beamline BL9 of the electron storage ring DELTA is presented. The structure of solid-gas and solid-liquid interfaces can be investigated using X-rays with incident energies of about 27 keV. A detailed description of the set-up is given and its performance is demonstrated by a discussion of selected applications, i.e. protein adsorption at the solid-liquid interface and gas adsorption at the solid-gas interface at elevated pressures.

Using a novel layer-by-layer approach we have deposited metal-organic open frameworks (MOFs) based on benzenetricarboxylic acid ligands and Cu(II)-ions on a COOH-terminated organic surface. The deposited layers were characterized using a number of surface analysis techniques. XRD measurements show that the MOFs deposited using this method have the same bulk structure of HKUST-1.

Two-photon photoemission (2PPE) from silver nanoparticles on a thin Al2O3 film on NiAl(110) has been investigated using femtosecond lasers. The 2PPE spectra show a feature similar to the surface state of Ag(111). The total 2PPE yield possesses a maximum at about 3.6eV which corresponds to the surface plasmon resonance of the silver particles. For particle sizes of about 10nm an enhancement by a factor 450 compared to Ag(111) is found. Up to this diameter the photoemission yield increases but levels off for larger sizes due to interactions between the particles. It is inferred that the 2PPE process is a direct transition via a virtual state, and that the enhanced 2PPE yield results from the enhancement of the electromagnetic field at the surface of nanoparticles.