The spontaneous adsorption of proteins at aqueous-solid interfaces plays an important role in nature, medicine, and biotechnology. A sound understanding of the molecular mechanisms and interactions upon protein adsorption is required for the development of new materials coatings and for the choice of solvent conditions. So far, protein adsorption studies have been carried out mainly as a function of protein solution concentration, pH-value, and surface chemistry. In addition, several temperature-dependent studies have shed some light on the thermodynamics underlying protein adsorption. In contrast, effects of pressure on this process are widely unknown. Applying pressure to molecular systems generally offers access to all kinds of volume changes occurring during assembly of molecules, phase transitions, and chemical reactions. In the case of protein adsorption, using pressure as a thermodynamic variable allows for the determination of volume changes of adsorption, volume changes of unfolding in the adsorbed state, and changes of protein-interface interactions that determine the degree of protein adsorption. We have designed new high pressure cells for total internal reflection fluorescence (TIRF) spectroscopy and neutron reflectometry (NR) for pressures up to 2500 bar in order to access these quantities. The results obtained so far indicate a pressure-induced increase of the degree of protein adsorption at both the water-silica and water-poly(styrene) interfaces. Moreover, a drastic decrease of the volume change of unfolding has been found when proteins are adsorbed at the water-silica interface. Apparently, pressure exerts a distinct influence on the process of protein adsorption and provides a new complemental view on the underlying mechanism.

There seems to be a general relation between the standard Gibbs energy change of unfolding, Delta G(unf)degrees, of a protein and its affinity to aqueous solid interfaces. So-called "hard" proteins (Delta G(unf)degrees is large) are found to adsorb less strongly to such interfaces than "soft" proteins (Delta G(unf)degrees 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 Delta G(unf)degrees 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.

Proteins often stay at interfaces, where their conformation and biological activity can be altered. In this study, we investigate the underlying changes in the folding stability and molecular volume of proteins using staphylococcal nuclease (SNase) as the model protein and colloidal silica particles as the model adsorbent. The folding stability and molecular volume have been determined by high-pressure fluorescence experiments in the range of 1-2500 bar utilizing the intrinsic Trp fluorescence of SNase. At pH = 7.0 and 25 degrees C, SNase, dissolved in bulk solution, is characterized by a volume change of unfolding of -73 mL mol(-1). This value is drastically reduced, when SNase is adsorbed on silica particles. Here, volume changes in the range of 41 to 32 mL mol(-1) can be measured at silica particles differing in their surface charge density. In addition, the standard Gibbs energy change and the pressure of unfolding are strongly reduced in the adsorbed state of SNase indicating a surface-induced destabilization of the protein native structure. The effect of the pH-value has been studied as well. Whereas a pH-change in the range of 7-10 has no significant effect on the pressure-driven unfolding of dissolved SNase, strong pH-dependence is observed for the adsorbed SNase. As the pH is approaching the isoelectric point of SNase, the conformational stability of the adsorbed protein is lowered drastically. The results of this study reveal a novel view on conformational changes upon protein adsorption. It is suggested that the molecular volume of SNase is lowered by a partial filling of the protein void volume corresponding to about two water molecules.

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 β-sheet formation within the multilayers at 70°C and pD=2, i.e. at conditions that promote insulin amyloid fibrils rich in β-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.

Protein adsorption often plays the central role in a wide variety of processes occurring in medicine, biochemistry and biotechnology. In order to develop novel material coatings, a detailed insight into the underlying adsorption mechanisms at the molecular level and the knowledge of thermodynamic parameters is of great importance. So far, protein adsorption has been investigated in terms of concentration, pH-value and temperature of the protein solution. However, volume effects on protein adsorption are still unknown in spite of their fundamental contribution to protein-interface interactions. in order to investigate volume effects and protein conformational transitions, two model proteins, SNase and lysozyme, were adsorbed onto silica nanoparticles and subjected to high hydrostatic pressures. At pressures up to 2500 bar, we have observed much smaller volumes of protein unfolding in the adsorbed state as compared to the solution behavior. These changes are directly linked to volume changes upon protein adsorption at the aqueous-solid model interface.

Ultrathin films are useful for coating materials and controlling drug delivery processes. Here, we explore the use of two-dimensional amyloidal networks as biodegradable ultrathin films. In a first step, we have studied the lateral aggregation and fibril formation of insulin that is adsorbed at and confined within planar polyelectrolyte multilayers containing poly(diallyldimethylammonium chloride) (PDDA), poly(styrenesulfonic acid) (PSS), and hyaluronic acid (Hyal). Si-PDDA-PSS-(Ins-PSS)x and Si-PDDA-PSS-(Ins-Hyal)x multilayers have been prepared and characterized in the hydrated state by using X-ray reflectometry, ATR-FTIR spectroscopy and confocal fluorescence microscopy. The obtained data demonstrate a successful build-up of insulin-polyelectrolyte multilayers on silicon wafers that grow strongly in thickness upon insulin adsorption on PSS and Hyal layers. The secondary structure analysis of insulin, based on the insulin infrared amide I' band, indicates intermolecular β-sheet formation within the multilayers at 70 °C and pH = 2, i.e. at conditions that promote amyloid fibrils rich in β-sheet contents. However, insulin that is incorporated in polyelectrolyte multilayers rather forms amorphous aggregates as can be inferred from confocal fluorescence images. Only when insulin is the top-layer, formation of a fibrillar network can be observed after adding seeds to the buffer solution.

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.

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.

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. In this study, we show that nonionic cosolvents also affect the interfacial affinity of proteins. We use bovine ribonuclease A and a planar silica-water interface as model system and apply neutron and optical reflectometry to analyze this system. The degree of protein adsorption and-the density profile of adsorbed protein molecules were determined in the absence and the presence of cosolvents. It has been found that both the protein stabilizing glycerol and the protein destabilizing urea cause a distinct reduction in protein interfacial affinity, which may represent a rather unexpected result. However, it is suggested that different mechanisms are underlying the similar effects of glycerol and urea.