High-pressure study of magnetic nanoparticles with a polyelectrolyte brush as carrier particles for enzymes.

The recovery of enzymes from a reaction medium can be achieved in a convenient way by using magnetic nanoparticles (MNP) as carriers. Here, we present MNP with a polyelectrolyte brush composed of poly(ethylene imine) (PEI) to provide a benign environment for the immobilized enzyme molecules. Yeast alcohol dehydrogenase (ADH) has been tested for enzymatic activity when it is free in solution or adsorbed on the PEI brush-MNP. Furthermore, the effect of pressure on the enzymatic activity has been studied to reveal activation volumes, which are a sensitive probe of the transition state geometry. The results of this study indicate that the secondary structure of ADH is pressure-stable up to 9 kbar. The enzymatic activity of ADH can be analyzed using Michaelis-Menten kinetics free in solution and adsorbed on the PEI brush-MNP. Remarkably, no significant changes of the Michaelis constant and the activation volume are observed upon adsorption. Thus, it can be assumed that the turnover number of ADH is also the same in the free and adsorbed state. However, the maximum enzymatic rate is reduced when ADH is adsorbed, which must be explained by a lower effective enzyme concentration due to steric hindrance of the enzyme inside the PEI brush of the MNP. In this way, the pressure experiments carried out in this study enable a distinction between steric and kinetic effects on the enzymatic rate of adsorbed ADH.

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.

Interaction of calmodulin with poly(acrylic acid) brushes: Effects of high pressure, pH-value and ligand binding.

Poly(acrylic acid) (PAA) brushes are well-known to interact with proteins in an ionic strength-dependent way. Moreover, they provide a native-like environment that largely maintains the secondary structure and biological activity of adsorbed proteins. Recently, it has been shown that the application of high pressure can lead to a reduced protein adsorption at a PAA brush in the case of a positively charged protein. Here, we analyze the effect of pressure on the interactions between a protein and a PAA brush in more detail. We use calmodulin as model protein that has a negative net charge at neutral pH-value and determine the degree of adsorption at a planar PAA brush applying total internal reflection fluorescence (TIRF) spectroscopy. Remarkably, the degree of calmodulin adsorption at a PAA brush is increasing with increasing pressure, when the protein is negatively charged. However, at low pH-value, where calmodulin is positively charged, high pressure leads to a partial desorption of the protein. Moreover, in the presence of trifluoperazine, which binds to calmodulin as a ligand, the pressure effect is diminished. The results of this study indicate that protein adsorption at a PAA brush at the "wrong" side of the isoelectric point, i.e. under net electrostatic repulsion, can involve a volume reduction that is favored under high pressure. It is suggested that this volume reduction is related to a hydration of counterions that are released from the PAA chains and the protein surface. In contrast, at pH-values close to the isoelectric point, the obtained data are consistent with a charge regulation mechanism that involves a volume increase. Thus, the application of high pressure in combination with pH-variation, as carried out in this study, provides the volume changes of adsorption that need to be consistent with any proposed mechanism of protein interaction with a PAA brush.

Inhibitor and peptide binding to calmodulin characterized by high pressure Fourier transform infrared spectroscopy and Förster resonance energy transfer.

We compare the binding of an inhibitor with that of a natural peptide to Ca2+ saturated calmodulin (holo-CaM). As inhibitor we have chosen trifluoperazine (TFP) that is inducing a huge conformational change of holo-CaM from the open dumbbell-shaped to the closed globular conformation upon binding. On the other hand, melittin is used as model peptide, which is a well-known natural binding partner of holo-CaM. The experiments are carried out as a function of pressure to reveal the contribution of volume or packing effects to the stability of the calmodulin-ligand complexes. From high-pressure Fourier transform infrared (FTIR) spectroscopy, we find that the holo-CaM/TFP complex has a much higher pressure stability than the holo-CaM/melittin complex. Although the analysis of the secondary structure of holo-CaM (without and with ligand) indicates no major changes up to several kbar, pressure-induced exposure of α-helices to water is most pronounced for holo-CaM without ligand, followed by holo-CaM/melittin and then holo-CaM/TFP. Moreover, structural pressure resistance of the holo-CaM/TFP complex in comparison with the holo-CaM/melittin complex is also clearly visible by higher Ca2+ affinity. Förster resonance energy transfer (FRET) from the Tyr residues of holo-CaM to the Trp residue of melittin even suggests some partial dissociation of the complex under pressure which points to void volumes at the protein-ligand interface and to electrostatic binding. Thus, all results of this study show that the inhibitor TFP binds to holo-CaM with higher packing density than the peptide melittin enabling a favorable volume contribution to the inhibitor efficiency.

A high pressure study of calmodulin-ligand interactions using small-angle X-ray and elastic incoherent neutron scattering.

Calmodulin (CaM) is a Ca2+ sensor and mediates Ca2+ signaling through binding of numerous target ligands. The binding of ligands by Ca2+-saturated CaM (holo-CaM) is governed by attractive hydrophobic and electrostatic interactions that are weakened under high pressure in aqueous solutions. Moreover, the potential formation of void volumes upon ligand binding creates a further source of pressure sensitivity. Hence, high pressure is a suitable thermodynamic variable to probe protein-ligand interactions. In this study, we compare the binding of two different ligands to holo-CaM as a function of pressure by using X-ray and neutron scattering techniques. The two ligands are the farnesylated hypervariable region (HVR) of the K-Ras4B protein, which is a natural binding partner of holo-CaM, and the antagonist trifluoperazine (TFP), which is known to inhibit holo-CaM activity. From small-angle X-ray scattering experiments performed up to 3000 bar, we observe a pressure-induced partial unfolding of the free holo-CaM in the absence of ligands, where the two lobes of the dumbbell-shaped protein are slightly swelled. In contrast, upon binding TFP, holo-CaM forms a closed globular conformation, which is pressure stable at least up to 3000 bar. The HVR of K-Ras4B shows a different binding behavior, and the data suggest the dissociation of the holo-CaM/HVR complex under high pressure, probably due to a less dense protein contact of the HVR as compared to TFP. The elastic incoherent neutron scattering experiments corroborate these findings. Below 2000 bar, pressure induces enhanced atomic fluctuations in both holo-CaM/ligand complexes, but those of the holo-CaM/HVR complex seem to be larger. Thus, the inhibition of holo-CaM by TFP is supported by a low-volume ligand binding, albeit this is not associated with a rigidification of the complex structure on the sub-ns Å-scale.

Lipid Phase Control and Secondary Structure of Viral Fusion Peptides Anchored in Monoolein Membranes.

The fusion of lipid membranes involves major changes of the membrane curvatures and is mediated by fusion proteins that bind to the lipid membranes. For a better understanding of the way fusion proteins steer this process, we have studied the interaction of two different viral fusion peptides, HA2-FP and TBEV-FP, with monoolein mesophases as a function of temperature and pressure at limited hydration. The fusion peptides are derived from the influenza virus hemagglutinin fusion protein (HA2-FP) and from the tick-borne encephalitis virus envelope glycoprotein E (TBEV-FP). By using synchrotron X-ray diffraction, the changes of the monoolein phase behavior upon binding the peptides have been determined and the concomitant secondary structures of the peptides have been analyzed by FTIR spectroscopy. As main results we have found that the fusion peptides interact differently with monoolein and change the pressure and temperature dependent lipid phase behavior to different extents. However, they both destabilize the fluid lamellar phase and favor phases with negative curvature, i.e. inverse bicontinuous cubic and inverse hexagonal phases. These peptide-induced phase changes can partially be reversed by the application of high pressure, demonstrating that the promotion of negative curvature is achieved by a less dense packing of the monoolein membranes by the fusion peptides. Pressure jumps across the cubic-lamellar phase transition reveal that HA2-FP has a negligible effect on the rates of the cubic and the lamellar phase formation. Interestingly, the secondary structures of the fusion peptides appear unaffected by monoolein fluid-fluid phase transitions, suggesting that the fusion peptides are the structure dominant species in the fusion process of lipid membranes.

Bioresponsive interfaces composed of calmodulin and poly(ethylene glycol): Toggling the interfacial film thickness by protein-ligand binding.

Responsive interfaces are often realized by polymer films that change their structure and properties upon changing the pH-value, ionic strength or temperature. Here, we present a bioresponsive interfacial structure that is based on a protein, calmodulin (CaM), which undergoes a huge conformational change upon ligand binding. At first, we characterize the conformational functionality of a double Cys mutant of CaM by small-angle X-ray scattering (SAXS) and Fourier transform infrared (FTIR) spectroscopy. The CaM mutant is then used to cross-link poly(ethylene glycol) (PEG) chains, which are bound covalently to a supporting planar Si surface. These films are characterized by X-ray reflectometry (XR) in a humidity chamber providing full hydration. It is well known that Ca2+-saturated holo-CaM binds trifluoperazine (TFP) and changes its conformation from an open, dumbbell-shaped to a closed, globular one in solution. At the interface, we observe an increase of the PEG-CaM film thickness, when TFP is binding and inducing the closed conformation, whereas the removal of Ca2+-ions and a concomitant release of TFP is associated with a decrease of the film thickness. This toggling of the film thickness is largely reversible. In this way, a structural change of the interface is achieved via protein functionality which has the advantage of being selective for ligand molecules without changing the environmental conditions in a harsh way via physico-chemical parameters.

Building Polyelectrolyte Multilayers with Calmodulin: A Neutron and X-ray Reflectivity Study.

We have studied the formation and functional properties of polyelectrolyte multilayers where calmodulin (CaM) is used as a polyanion. CaM is known to populate distinct conformational states upon binding Ca2+ and small ligand molecules. Therefore, we have also probed the effects of Ca2+ ions and trifluoperazine (TFP) as ligand molecule on the interfacial structures. Multilayers with the maximum sequence PEI-(PSS-PAH)x-CaM-PAH-CaM-PAH have been deposited on silicon wafers and characterized by X-ray and neutron reflectometry. From the analysis of all data, several remarkable conclusions can be drawn. When CaM is deposited for the second time, a much thicker sublayer is produced than in the first CaM deposition step. However, upon rinsing with PAH, very thin CaM-PAH sublayers remain. There are no indications that ligand TFP can be involved in the multilayer buildup due to strong CaM-PAH interactions. However, there is a significant increase in the multilayer thickness upon removal of Ca2+ ions from holo-CaM and an equivalent decrease in the multilayer thickness upon subsequent saturation of apo-CaM with Ca2+ ions. Presumably, CaM can still be toggled between an apo and a holo state, when it is embedded in polyelectrolyte multilayers, providing an approach to design bioresponsive interfaces.

Improved activity of α-chymotrypsin on silica particles - A high-pressure stopped-flow study.

Pressure is well known to affect the catalytic rate of enzymes dissolved in solution. To better understand enzyme kinetics at aqueous-solid interfaces, we have carried out a high-pressure stopped-flow activity study of α-chymotrypsin (α-CT) that is adsorbed on silica particles and, for comparison, dissolved in solution. The enzyme reaction was modulated using pressures up to 2000bar and recorded using the high-pressure stopped-flow technique. The results indicate an 8-fold enhancement of the turnover number upon α-CT adsorption and a further increase of the catalytic rate in the pressure range up to 1000bar. From the pressure dependence of the catalytic rate, apparent activation volumes have been determined. In the adsorbed state of α-CT, a pronounced change of the activation volume is found with increasing pressure. Furthermore, owing to suppression of its autolysis, a significantly longer storage time of α-CT can be achieved when the enzyme is adsorbed on silica particles. The results obtained are discussed in terms of a surface-induced selection of conformational substates of the enzyme-substrate complex.

Volume profile of α-chymotrypsin during adsorption and enzymatic reaction on a poly(acrylic acid) brush.

Poly(acrylic acid) (PAA) brushes are known to provide a native-like environment for proteins. In this study, we explore this biocompatibility under high pressure conditions. Using α-chymotrypsin (α-CT) as a model enzyme, we report on the pressure dependencies of the enzymatic activity and the neutron scattering length density profile, when this enzyme is adsorbed on a PAA brush. From high pressure total internal reflection fluorescence spectroscopy, an increasing enzymatic activity has been observed up to 1000 bar, but a rather pressure independent enzymatic activity at higher pressures up to 2000 bar. This finding suggests a non-constant activation volume of α-CT on the PAA brush that is negative below 1000 bar. Thus, the compact nature of the transition state of α-CT is largely preserved upon adsorption. We have also performed high pressure neutron reflectivity experiments to determine the spatial distribution of α-CT inside the PAA brush. Apparently, the enzyme is strongly binding to the PAA chains with 2.3 mg m(-2) of adsorbed enzyme that is reduced to about 1.7 mg m(-2) at 1000-2000 bar. This change of adsorbed mass is consistent with a positive volume change of adsorption, which is probably reflecting electrostriction upon protein-PAA interaction. Thus, the performed high pressure experiments provide new insights into the volume profile of α-CT during adsorption and enzymatic activity on the PAA brush. They also demonstrate that the biocompatible properties of a PAA brush can even be enhanced by pressure.

Effect of interfacial properties on the activation volume of adsorbed enzymes.

We have studied the enzymatic activities of α-chymotrypsin (α-CT) and horseradish peroxidase (HRP) that are adsorbed on various chemically modified planar surfaces under aqueous solution. The enzymes were adsorbed on bare quartz, hydrophobic poly(styrene) (PS), positively charged poly(allylamine hydrochloride) (PAH), and negatively charged poly(styrene sulfonate) (PSS). Activation volumes of the enzymes at the aqueous-solid interfaces were determined by using high-pressure total internal reflection fluorescence (TIRF) spectroscopy. Apparently, the pressure response of the adsorbed enzymes strongly depends on the interfacial properties. α-CT can be activated by pressure (increasing enzymatic rate) on negatively charged surfaces like quartz and PSS, whereas HRP is activated by pressure on hydrophobic PS. Corresponding negative activation volumes of -29 mL mol(-1) for α-CT on quartz, -23 mL mol(-1) for α-CT on PSS, and -35 mL mol(-1) for HRP on PS are found. In addition, the absolute activities of α-CT and HRP on quartz, PS, PAH and PSS were determined by UV absorption at ambient pressure. Remarkably, large activities are found on those surfaces that are associated with negative activation volumes. However, Fourier transform infrared (FTIR) spectra collected in attenuated total reflection (ATR) mode do not indicate major adsorption induced conformational changes of the enzymes at any interface studied. Overall, the results of this study show that the activity of immobilized enzymes can largely be enhanced by the right combination of adsorbent material and applied pressure.

Secondary structure and folding stability of proteins adsorbed on silica particles - Pressure versus temperature denaturation.

We present a systematic study of the pressure and temperature dependent unfolding behavior of proteins that are adsorbed on silica particles. Hen egg white lysozyme and bovine ribonuclease A (RNase) were used as model proteins, and their secondary structures were resolved by Fourier transform infrared (FTIR) spectroscopy in the temperature range of 10-90°C and the pressure range of 1-16,000bar. Apparently, the secondary structures of both proteins do not change significantly when they are adsorbing on the silica particles. Remarkably, the changes of the secondary structure elements upon protein unfolding are very similar in the adsorbed and the free states. This similarity could be observed for both lysozyme and RNase using both high pressures and high temperatures as denaturing conditions. However, the pressures and temperatures of unfolding of lysozyme and RNase are drastically lowered upon adsorption indicating lower folding stabilities of the proteins on the silica particles. Moreover, the temperature ranges, where changes in secondary structure occur, are broadened due to adsorption, which is related to smaller enthalpy changes of unfolding. For both proteins, free or adsorbed, pressure-induced unfolding always leads to less pronounced changes in secondary structure than temperature-induced unfolding. In the case of lysozyme, high pressure also favors a different unfolded conformation than high temperature. Overall, the results of this study reveal that adsorption of proteins on silica particles decreases the folding stability against high pressures and temperatures, whereas the unfolding pathways are mainly preserved in the adsorbed state.

Activation volumes of enzymes adsorbed on silica particles.

The immobilization of enzymes on carrier particles is useful in many biotechnological processes. In this way, enzymes can be separated from the reaction solution by filtering and can be reused in several cycles. On the other hand, there is a series of examples of free enzymes in solution that can be activated by the application of pressure. Thus, a potential loss of enzymatic activity upon immobilization on carrier particles might be compensated by pressure. In this study, we have determined the activation volumes of two enzymes, α-chymotrypsin (α-CT) and horseradish peroxidase (HRP), when they are adsorbed on silica particles and free in solution. The experiments have been carried out using fluorescence assays under pressures up to 2000 bar. In all cases, activation volumes were found to depend on the applied pressure, suggesting different compressions of the enzyme-substrate complex and the transition state. The volume profiles of free and adsorbed HRP are similar. For α-CT, larger activation volumes are found in the adsorbed state. However, up to about 500 bar, the enzymatic reaction of α-CT, which is adsorbed on silica particles, is characterized by a negative activation volume. This observation suggests that application of pressure might indeed be useful to enhance the activity of enzymes on carrier particles.

Solvent effects on the dynamics of amyloidogenic insulin revealed by neutron spin echo spectroscopy.

Insulin is well known to self-associate under specific solvent conditions. At low pH values, in the presence of sodium chloride (NaCl) and at elevated temperatures, insulin readily aggregates and forms amyloid fibrils. Without NaCl, but in the presence of ethanol, the lag time of this temperature-induced aggregation is increased drastically. In this study, we have analyzed the dynamical properties of bovine insulin under these two solvent conditions by using neutron spin echo (NSE) spectroscopy. In addition, small-angle X-ray scattering (SAXS) and thioflavin T (ThT) fluorescence experiments were carried out to track the concomitant structural changes of insulin. Measurements have mainly been performed at 318 K, where amyloid fibrils are formed over 25 h, when the insulin solution contains 100 mmol L(-1) of NaCl at pD = 2.4. In contrast, no amyloid fibrils are formed during 25 h at 318 K, when the insulin solution contains ethanol with a volume fraction of 20% at pD = 2.4. Remarkably, the NSE data reveal distinct dynamic signatures of insulin depending on the chosen solvent conditions. Collective diffusion of insulin molecules can be inferred from an increased diffusion coefficient at low wave vector transfers in the nonfibrillating sample, whereas self-diffusion is observed in the other case. The SAXS data confirm these dynamic behaviors because a pronounced correlation peak is only observed under conditions of collective diffusion. The dynamic responses of insulin, as revealed here by NSE spectroscopy, are in agreement with intermolecular interaction potentials derived recently from measurements of the static structure factors of insulin and lysozyme.

Pressure-induced protein adsorption at aqueous-solid interfaces.

There seems to be a general relation between the standard Gibbs energy change of unfolding, ΔG°unf, of a protein and its affinity to aqueous-solid interfaces. So-called "hard" proteins (ΔG°unf is large) are found to adsorb less strongly to such interfaces than "soft" proteins (ΔG°unf is small). Here, we provide direct support for this rule by using high pressure to modulate the folding stability of a protein. We have performed high-pressure total internal reflection fluorescence (HP-TIRF) spectroscopy and high-pressure neutron reflectometry (HP-NR) to measure the degree of adsorption and the structure of lysozyme on planar solid surfaces as a function of pressure for the first time. By carrying out these experiments at hydrophilic and hydrophobic surfaces with varying concentrations of glycerol, we have found strong evidence that ΔG°unf has indeed a direct influence. At high pressures, there is a larger degree of lysozyme adsorption, probably because lysozyme becomes a "soft" protein under these conditions. The results of this study demonstrate that high pressure is a very useful tool to explore thermodynamics of protein-interface interactions.

High pressure sample cell for total internal reflection fluorescence spectroscopy at pressures up to 2500 bar.

Total internal reflection fluorescence (TIRF) spectroscopy is a surface sensitive technique that is widely used to characterize the structure and dynamics of molecules at planar liquid-solid interfaces. In particular, biomolecular systems, such as protein adsorbates and lipid membranes can easily be studied by TIRF spectroscopy. Applying pressure to molecular systems offers access to all kinds of volume changes occurring during assembly of molecules, phase transitions, and chemical reactions. So far, most of these volume changes have been characterized in bulk solution, only. Here, we describe the design and performance of a high pressure sample cell that allows for TIRF spectroscopy under high pressures up to 2500 bar (2.5 × 10(8) Pa), in order to expand the understanding of volume effects from the bulk phase to liquid-solid interfaces. The new sample cell is based on a cylindrical body made of Nimonic 90 alloy and incorporates a pressure transmitting sample cuvette. This cuvette is composed of a fused silica prism and a flexible rubber gasket. It contains the sample solution and ensures a complete separation of the sample from the liquid pressure medium. The sample solution is in contact with the inner wall of the prism forming the interface under study, where fluorescent molecules are immobilized. In this way, the new high pressure TIRF sample cell is very useful for studying any biomolecular layer that can be deposited at a planar water-silica interface. As examples, high pressure TIRF data of adsorbed lysozyme and two phospholipid membranes are presented.

Physical properties of archaeal tetraether lipid membranes as revealed by differential scanning and pressure perturbation calorimetry, molecular acoustics, and neutron reflectometry: effects of pressure and cell growth temperature.

The polar lipid fraction E (PLFE) is a major tetraether lipid component in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Using differential scanning and pressure perturbation calorimetry as well as ultrasound velocity and density measurements, we have determined the compressibilities and volume fluctuations of PLFE liposomes derived from different cell growth temperatures (T(g) = 68, 76, and 81 °C). The compressibility and volume fluctuation values of PLFE liposomes, which are substantially less than those detected from diester lipid membranes (e.g., DPPC), exhibit small but significant differences with T(g). Among the three T(g)s employed, 76 °C leads to the least compressible and most tightly packed PLFE membranes. This temperature is within the range for optimal cell growth (75-80 °C). It is known that a decrease in T(g) decreases the number of cyclopentane rings in archael tetraether lipids. Thus, our data enable us to present the new view that membrane packing in PLFE liposomes varies with the number of cyclopentane rings in a nonlinear manner, reaching maximal tightness when the tetraether lipids are derived from cells grown at optimal T(g)s. In addition, we have studied the effects of pressure on total layer thickness, d, and neutron scattering length density, ρ(n), of a silicon-D(2)O interface that is covered with a PLFE membrane using neutron reflectometry (NR). At 55 °C, d and ρ(n) are found to be rather insensitive to pressure up to 1800 bar, suggesting minor changes of the thickness of the membrane's hydrophobic core and headgroup orientation upon compression only.

Probing aggregation and fibril formation of insulin in polyelectrolyte multilayers.

Ultrathin films are useful for coating materials and controlling drug delivery processes. Here, we explore the use of polyelectrolyte multilayers as templates for the formation of two-dimensional protein networks, which represent biocompatible and biodegradable ultrathin films. In a first step, we have studied the lateral aggregation and amyloid fibril formation of bovine insulin that is adsorbed at and confined within planar polyelectrolyte multilayers, assembled with poly(diallyldimethylammonium chloride) (PDDA), poly(styrenesulfonic acid) (PSS), and hyaluronic acid (HA). Si-PDDA-PSS-(insulin-PSS)(x) and Si-PDDA-PSS-(insulin-HA)(x) multilayers (x=1-4) have been prepared and characterized in the fully hydrated state by using X-ray reflectometry, attenuated total reflection-Fourier transform infrared spectroscopy and confocal fluorescence microscopy. The obtained data demonstrate a successful build-up of the insulin-polyelectrolyte multilayers on silicon wafers that grow strongly in thickness upon insulin adsorption on PSS and HA layers. The secondary structure analysis of insulin, based on the vibrational amide I'-band, indicates an enhanced intermolecular ß-sheet formation within the multilayers at 70°C and pD=2, i.e. at conditions that promote insulin amyloid fibrils rich in ß-sheet contents. However, insulin that is confined between two polyelectrolyte layers rather forms amorphous aggregates as can be inferred from confocal fluorescence images. Remarkably, when insulin is deposited as the top-layer, a partial conversion into a two-dimensional fibrillar network can be induced by adding amyloid seeds to the solution. Thus, the results of this study illustrate the capability of polyelectrolyte multilayers as templates for the growth of protein networks.

Reduced protein adsorption by osmolytes.

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.

High pressure cell for neutron reflectivity measurements up to 2500 bar.

The design of a high pressure (HP) cell for neutron reflectivity experiments is described. The cell can be used to study solid-liquid interfaces under pressures up to 2500 bar (250 MPa). The sample interface is based on a thick silicon block with an area of about 14 cm(2). This area is in contact with the sample solution which has a volume of only 6 cm(3). The sample solution is separated from the pressure transmitting medium, water, by a thin flexible polymer membrane. In addition, the HP cell can be temperature-controlled by a water bath in the range 5-75°C. By using an aluminum alloy as window material, the assembled HP cell provides a neutron transmission as high as 41%. The maximum angle of incidence that can be used in reflectivity experiments is 7.5°. The large accessible pressure range and the low required volume of the sample solution make this HP cell highly suitable for studying pressure-induced structural changes of interfacial proteins, supported lipid membranes, and, in general, biomolecular systems that are available in small quantities, only. To illustrate the performance of the HP cell, we present neutron reflectivity data of a protein adsorbate under high pressure and a lipid film which undergoes several phase transitions upon pressurization.