Insertion of tear proteins into a meibomian lipids film

The eyelid meibomian gland secretions form the outer layer of the tear film. That layer functions as a lubricant during a blink, and as a barrier against intrusion of foreign bodies. The lipid film is also exposed to proteins present in the aqueous phase that may adsorb there, and thus form an integral part of the surface of the tear film, or possibly, cause disruption to the outermost layer. Therefore, the adsorption of tear proteins to the meibomian lipid layer was object of the present investigation. A model tear was set up coating a pendant drop of saline with a film of meibomian lipids and measuring variations of the interfacial pressure after the injection of tear proteins into the aqueous subphase at their physiological concentration. All tear proteins adsorbed at the interface causing the initial surface pressure to increase. For each protein, a limiting surface pressure at which a given protein was no longer able to insert into the lipid layer was found. Among the proteins tested, lipocalin was the most surface active one and inserted into the lipid layer in the whole range of surface pressure exerted by the meibomian lipid mixture. Lactoferrin, lysozyme and IgA also interacted with the lipids whereas albumin interacted more weakly. The timescale of the protein insertion into the lipid layer was of the order of 10(2) s. It was hypothesized that protein adsorption at the interface could be associated with structural changes. Indeed, the enzymatic activity of lysozyme was maintained in the presence of an outermost meibomian lipid layer that prevented its denaturation while exposure at the air/aqueous interface induced significant lysozime degradation. meibomian lipid composition is therefore functional to maintain tear proteins activity.     

Diffusion of glycosylphosphatidylinositol (GPI)-anchored bovine prion protein (PrP) in supported lipid membranes studied by single-molecule and complementary ensemble methods

In this work cellular bovine prion protein (PrP^c) was incorporated in supported lipid membranes and its lateral diffusion was studied by single-dye tracking (SDT) and a complementary ensemble method, fluorescence recovery after photobleaching (FRAP). PrP^c was purified from calf brain with its native glycosylphosphatidylinositol (GPI) anchor and reconstituted into DMPC lipid vesicles. Homogeneous spreading on solid supports over macroscopic areas was confirmed with fluorescence microscopy. FRAP results demonstrated very high mobile fractions of up to 94%, confirming that most of the GPI-anchored PrP^c are freely diffusive in the fluid supported membrane matrix. Moreover, the lateral diffusivity of PrP^c significantly depends on the pH of the buffer, suggesting that the conformation of PrP^c and thus the frictional drag exerted to the protein molecule (and thus the effective hydrodynamic radius) is influenced by the effective net charge. To complement the ensemble results obtained by FRAP, the statistical variation of lateral diffusion coefficients of individual PrP^c molecules in the supported membranes were measured with SDT. Simulation-based statistical analysis indicated that in addition to the expected statistical scatter there is a significant spread of diffusion coefficients, while the average of the diffusion coefficients of individual proteins obtained by SDT is in excellent agreement with those measured by ensemble FRAP. In further experiments, PrP^c was laterally concentrated in the membrane by the application of tangential electric fields (membrane electrophoresis). However, the equilibrium concentration profile reached after 20 min was different from an exponential gradient. This finding suggests that PrP^c purified from bovine brain possesses non-uniform net charges. As the lateral diffusion coefficient of proteins in two-dimensional lipid membranes sensitively depends upon the frictional drag, the combination of SDT, ensemble FRAP, and membrane electrophoresis can be used as a powerful tool to gain insights into protein–protein binding and oligomer formation that would play a crucial role in infectious protein transmitted diseases such as BSE.     

Insight into the role of hydration on protein dynamics

The potential energy surface of a protein is rough. This intrinsic energetic roughness affects diffusion, and hence the kinetics. The dynamics of a system undergoing Brownian motion on this surface in an implicit continuum solvent simulation can be tuned via the frictional drag or collision frequency to be comparable to that of experiments or explicit solvent simulations. We show that the kinetic rate constant for a local rotational isomerization in stochastic simulations with continuum solvent and a collision frequency of 2 ps(-1) is about 10(4) times faster than that in explicit water and experiments. A further increase in the collision frequency to 60 ps(-1) slows down the dynamics, but does not fully compensate for the lack of explicit water. We also show that the addition of explicit water does not only slow down the dynamics by increasing the frictional drag, but also increases the local energetic roughness of the energy landscape by as much as 1.0 kcal/mol.     

Displacement of fibrinogen from the air/aqueous interface by dilauroylphosphatidylcholine lipid

Fibrinogen (FB) and other serum proteins leak into the aqueous alveolar lining layer due to lung injuries. The adsorption of these serum proteins at the air/aqueous interface can produce higher surface tensions than the pulmonary lipids, and acute respiratory distress syndrome (ARDS) can ensue. By having a molecular adsorption mechanism, as compared to a particulate adsorption mechanism of other longer chain lipids, dilauroylphosphatidylcholine (DLPC) lipid can expel FB from the air/aqueous interface at 25 degrees C, in water or in phosphate-buffered saline, as proven by tensiometry (also at 37 degrees C), ellipsometry, and infrared reflection-absorption spectroscopy. Moreover, before FB is displaced by DLPC at the interface, there is a substantial initial enhancement in the FB adsorption, consistent with some interaction or binding of DLPC with FB to produce a more hydrophobic protein surface. After the FB molecules have been displaced by DLPC, or when DLPC has already adsorbed at the interface, FB molecules are less favored to adsorb near the DLPC monolayer with the lecithin headgroups facing toward them. The results have implications for possible uses of DLPC lipid in potential lung surfactant formulations in treating patients with ARDS.     

Nanoscale electrowetting effects observed by using friction force microscopy

We report the study of electrowetting (EW) effects under strong electric field on poly(methyl methacrylate) (PMMA) surface by using friction force microscopy (FFM). The friction force dependence on the electric field at nanometer scale can be closely related to electrowetting process based on the fact that at this scale frictional behavior is highly affected by capillary phenomena. By measuring the frictional signal between a conductive atomic force microscopy (AFM) tip and the PMMA surface, the ideal EW region (Young-Lippmann equation) and the EW saturation were identified. The change in the interfacial contact between the tip and the PMMA surface with the electric field strength is closely associated with the transition from the ideal EW region to the EW saturation. In addition, a reduction of the friction coefficient was observed when increasing the applied electric field in the ideal EW region.     

Aligning nanodiscs at the air-water interface, a neutron reflectivity study

Nanodiscs are self-assembled nanostructures composed of a belt protein and a small patch of lipid bilayer, which can solubilize membrane proteins in a lipid bilayer environment. We present a method for the alignment of a well-defined two-dimensional layer of nanodiscs at the air-water interface by careful design of an insoluble surfactant monolayer at the surface. We used neutron reflectivity to demonstrate the feasibility of this approach and to elucidate the structure of the nanodisc layer. The proof of concept is hereby presented with the use of nanodiscs composed of a mixture of two different lipid (DMPC and DMPG) types to obtain a net overall negative charge of the nanodiscs. We find that the nanodisc layer has a thickness or 40.9 ± 2.6 ? with a surface coverage of 66 ± 4%. This layer is located about 15 ? below a cationic surfactant layer at the air-water interface. The high level of organization within the nanodiscs layer is reflected by a low interfacial roughness (~4.5 ?) found. The use of the nanodisc as a biomimetic model of the cell membrane allows for studies of single membrane proteins isolated in a confined lipid environment. The 2D alignment of nanodiscs could therefore enable studies of high-density layers containing membrane proteins that, in contrast to membrane proteins reconstituted in a continuous lipid bilayer, remain isolated from influences of neighboring membrane proteins within the layer.     

Dynamics of protein and mixed protein/surfactant adsorption layers at the water/fluid interface

The adsorption behaviour of proteins and systems mixed with surfactants of different nature is described. In the absence of surfactants the proteins mainly adsorb in a diffusion controlled manner. Due to lack of quantitative models the experimental results are discussed partly qualitatively. There are different types of interaction between proteins and surfactant molecules. These interactions lead to protein/surfactant complexes the surface activity and conformation of which are different from those of the pure protein. Complexes formed with ionic surfactants via electrostatic interaction have usually a higher surface activity, which becomes evident from the more than additive surface pressure increase. The presence of only small amounts of ionic surfactants can significantly modify the structure of adsorbed proteins. With increasing amounts of ionic surfactants, however, an opposite effect is reached as due to hydrophobic interaction and the complexes become less surface active and can be displaced from the interface due to competitive adsorption. In the presence of non-ionic surfactants the adsorption layer is mainly formed by competitive adsorption between the compounds and the only interaction is of hydrophobic nature. Such complexes are typically less surface active than the pure protein. From a certain surfactant concentration of the interface is covered almost exclusively by the non-ionic surfactant. Mixed layers of proteins and lipids formed by penetration at the water/air or by competitive adsorption at the water/chloroform interface are formed such that at a certain pressure the components start to separate. Using Brewster angle microscopy in penetration experiments of proteins into lipid monolayers this interfacial separation can be visualised. A brief comparison of the protein adsorption at the water/air and water/n-tetradecane shows that the adsorbed amount at the water/oil interface is much stronger and the change in interfacial tension much larger than at the water/air interface. Also some experimental data on the dilational elasticity of proteins at both interfaces measured by a transient relaxation technique are discussed on the basis of the derived thermodynamic model. As a fast developing field of application the use of surface tensiometry and rheometry of mixed protein/surfactant mixed layers is demonstrated as a new tool in the diagnostics of various diseases and for monitoring the progress of therapies.     

DNA translocation through polyelectrolyte‐modified nanopores: An analytical approximation

An analytical model for the electrophoretic speed of DNA translocating through nanopore functionalized with polyelectrolyte (PE) brush is developed for the first time. The electrophoretic speed depends on DNA surface potential, applied electric field, viscosity, and permittivity of solution along with velocity and electrostatic potential at liquid–polyelectrolyte layer (PEL) interface where the interface seemed to behave similar to that of a solid‐state nanopore wall. Under the limit of Debye–Hückel linearization, the electrostatic potential at liquid–PEL interface and at DNA surface have been calculated. Velocity at liquid–PEL interface has been estimated by assuming a linear variation of hydrodynamic frictional force within the PEL. It is observed that velocity and electrostatic potential at liquid–PEL interface strongly depend on PE charge density and softness parameter. Present analytical results show excellent agreement with exact numerical results (i.e., without any approximation) at a higher salt concentration where Debye–Hückel linearization is applicable.     

Protein-lipid interactions at the oil-water interface

The distribution of proteins and lipids in food emulsions and foams is determined by competitive and cooperative adsorption between the two types of emulsifiers at the fluid-fluid interfaces, and by the nature of protein-lipid interactions, both at the interface and in the bulk phase. The existence of protein-lipid interactions can have a pronounced impact on the surface rheological properties of these systems. Therefore, these results are of practical importance for food emulsion formulation, texture, and stability. In this study, the existence of protein-lipid interactions at the interface was determined by surface dynamic properties (interfacial tension and surface dilational modulus). Systematic experimental data on surface dynamic properties, as a function of time and at long-term adsorption, for protein (whey protein isolate (WPI)), lipids (monoglycerides), and protein-lipid mixed films at the oil-water interface were measured in an automated drop tensiometer. The dynamic behaviour of protein+lipid mixed films depends on the adsorption time, the lipid and the protein/lipid ratio in a rather complicated manner. The protein determined the interfacial characteristics of the mixed film as the protein at WPI>/=10(-2)% wt/wt saturated the film, no matter what the concentration of the lipid. However, there exists a competitive or cooperative adsorption of the emulsifier (WPI and monoglycerides), as the concentration of protein in the bulk phase is far lower than that for interfacial saturation.     

Construction of supported lipid membrane modified piezoelectric biosensor for sensitive assay of cholera toxin based on surface-agglutination of ganglioside-bearing liposomes

A novel piezoelelctric biosensor has been developed for cholera toxin (CT) detection based on the analyte-mediated surface-agglutination of ganglioside (GM1)-functionalized liposomes. To achieve a CT-specific agglutination at the surface, the gold electrode is modified by a GM1-functionalized supported lipid membrane via spontaneous spread of the liposomes on a self-assembled monolayer of a long-chain alkanethiol. In the presence of CT, the GM1-incorporated liposomes in assay medium will rapidly specifically agglutinate at the electrode surface through the binding of CT to GM1 on the electrode surface and the liposome interface. This results in an enormous mass loading on the piezoelelctric crystal as well as a significant increase of density and viscosity at the interface, thereby generating a decrease in frequency of the piezoelelctric crystal. The combination of mass loading with interfacial change in the surface-agglutination reaction allows the developed piezoelelctric biosensor to show substantial signal amplification in response to the analyte CT. The detection limit can be achieved as low as 25 ng mL⁻¹ CT. This is the first demonstration on CT detection based on specific surface-agglutination of GM1-modified liposomes. The supported lipid layer based sensing interface can be prepared readily and renewably, making the developed technique especially useful for simple, reusable and sensitive determination of proteins.     

Boojum and stripe textures in long-range orientationally ordered monolayers on solid substrates

Long-range organization of molecular tilt azimuth is a striking feature in monolayers at the air-water interface. We show that the boojum and stripe textures of pentadecanoic acid (PDA) with the continuous variations of molecular tilt azimuth formed at the air-water interface at temperatures lower than room temperature can be preserved after being transferred to glass substrates at low dipping speeds. The long-range tilt order in the transferred boojums and stripes is resolved by frictional force microscopy at room temperature, suggesting that the tilt order is "frozen" through the interaction of PDA molecules with the glass surface. The transferred stripe structure can be used as a unique alignment layer to induce a continuously azimuthal orientation of nematic liquid crystals.     

Density dependent friction of lipid monolayers

We measure frictional properties of liquid-expanded and liquid-condensed phases of lipid Langmuir-Blodgett monolayers by interfacial force microscopy. We find that over a reasonably broad surface-density range, the friction shear strength of the lipid monolayer film is proportional to the surface area (42-74 A2/molecule) occupied by each molecule. The increase in frictional force (i.e., friction shear strength with molecular area can be attributed to the increased conformational freedom and the resulting increase in the number of available modes for energy dissipation.     

Electrokinetic potentials at the gas-aqueous interface by spinning cylinder electrophoresis

The natural buoyancy of gas bubbles has hampered conventional electrokinetic methods of evaluating charge at the gas-liquid interface. In this study, bubbles are held by centripetal force at the centre of a horizontal, rotating cylinder filled completely with liquid. Migration of the bubble along the axis of the cylinder can be achieved by applying a gravity force or an electrophoretic force. An expression for the drag coefficient has been developed and used to calculate surface charge densities and electrokinetic potentials. In the presence of potassium chloride, anionic and non-ionic surfactants, the electrokinetic potentials were negative (～2–5 mV) at the air-aqueous interface. A cationic surfactant rendered the zeta potential positive. A knowledge of the air-aqueous interfacial rheology is desirable if the drag coefficient, and consequently the zeta potential, at all types of gas-liquid interfaces is to be evaluated.     

Protein adsorption at the electrified air-water interface: implications on foam stability

The surface chemistry of ions, water molecules, and proteins as well as their ability to form stable networks in foams can influence and control macroscopic properties such as taste and texture of dairy products considerably. Despite the significant relevance of protein adsorption at liquid interfaces, a molecular level understanding on the arrangement of proteins at interfaces and their interactions has been elusive. Therefore, we have addressed the adsorption of the model protein bovine serum albumin (BSA) at the air-water interface with vibrational sum-frequency generation (SFG) and ellipsometry. SFG provides specific information on the composition and average orientation of molecules at interfaces, while complementary information on the thickness of the adsorbed layer can be obtained with ellipsometry. Adsorption of charged BSA proteins at the water surface leads to an electrified interface, pH dependent charging, and electric field-induced polar ordering of interfacial H(2)O and BSA. Varying the bulk pH of protein solutions changes the intensities of the protein related vibrational bands substantially, while dramatic changes in vibrational bands of interfacial H(2)O are simultaneously observed. These observations have allowed us to determine the isoelectric point of BSA directly at the electrolyte-air interface for the first time. BSA covered air-water interfaces with a pH near the isoelectric point form an amorphous network of possibly agglomerated BSA proteins. Finally, we provide a direct correlation of the molecular structure of BSA interfaces with foam stability and new information on the link between microscopic properties of BSA at water surfaces and macroscopic properties such as the stability of protein foams.     

Membrane surface dynamics of DNA-threaded nanopores revealed by simultaneous single-molecule optical and ensemble electrical recording

We describe a method for simultaneous single-molecule optical and electrical characterization of membrane-based sensors that contain ion-channel nanopores. The technique is used to study the specific and nonspecific interactions of streptavidin-capped DNA polymers with lipid bilayers composed of diphytanoyl phosphatidylcholine and diphytanoyl phosphatidylglycerol. Biotinylated DNA that is bound to fluorescently labeled streptavidin is electrophoretically driven into, or away from, the lumen of alpha hemolysin (alphaHL) ion channels by an external electric field. Confocal microscopy simultaneously captures single-molecule fluorescence dynamics from the membrane interface at different applied potentials. Fluorescence correlation analysis is used to determine the surface number density and diffusion constant of membrane-associated complexes. The dual optical and electrical approach can detect membrane-associated species at a surface coverage below 10(-5) monolayers of streptavidin, a sensitivity that surpasses most other in vitro surface analysis techniques. By comparing the change in transmembrane current to the number of fluorescent molecules leaving the bilayer when the electrical potential is reversed, we demonstrate the general utility of the approach within the context of nanopore-based sensing and discuss a mechanism by which DNA-streptavidin complexes can be nonspecifically retained at the membrane interface.     

Reciprocal influence between the protein and lipid components of a lipid-protein membrane model

The crystallization of bacterial surface layer proteins (S-layer proteins) at phosphoethanolamine monolayers on aqueous (buffer) surfaces has been investigated with dual label fluorescence microscopy, FTIR spectroscopy, and electron microscopy. The phase state of the lipid exerts a marked influence on protein crystallization: when the surface monolayer is in the phase-separated state between the isotropic and anisotropic fluid phases, the S-layer protein is preferentially adsorbed at the isotropic phase. Protein crystals nucleate at the boundary lines between the coexisting lipid phases and crystallization proceeds underneath the anisotropic fluid. Crystal growth is much slower under the fluid lipid and the entire interface is overgrown only after prolonged protein incubation. In turn, as indicated by characteristic frequency shifts of the methylene stretch vibrations on the lipids, protein crystallization affects the order of the alkane chains and drives the fluid lipid into a state of higher order. Most probably, the protein does not interpenetrate the lipid surface monolayer and the coupling between protein and lipid occurs via the lipid head groups.     

Modeling solvent effects in molecular dynamics simulations of proteins

A series of computer simulations has been carried out on bovine pancreatic trypsin inhibitor using various models to mimic the effects of explicit bulk solvent on the structure of the protein. The solvent properties included are the polarization of the solute by the polar bulk solvent and the restraining effect on the motional freedom of the solute due to frictional drag at the solvent–protein surface interface. The former has been included by using a distance–dependent dielectric permittivity to screen the electrostatic interactions, whereas the latter is simulated by adding a limited number of solvent molecules near the protein surface. To achieve the proper mobility of the water molecules, their motion was restrained by adding a harmonic restraining force. It was found that a very small force constant was sufficient to model the static and dynamical behavior of the fully solvated solute, but that it was necessary to include enough explicit waters to occupy the first solvation shell. © 1992 John Wiley & Sons, Inc.     

Subnanometer actuation of a tethered lipid bilayer monitored with fluorescence resonance energy transfer

Lipid membrane nanotechnology can play a key role in preserving the function of transmembrane proteins on biofunctional substrates. We show here that rational nanoscopic actuation of a polymer-tethered lipid bilayer can be achieved by modulating the dielectric environment at the membrane-substrate interface. This provides a hydrated platform with increased lipid mobility compared to bilayers supported directly onto silica. We suggest that this construct may be used for promoting the functional reconstitution of transmembrane proteins on planar surfaces for bioanalytical devices.     

Effect of temperature on the frictional forces between polystyrene brushes

Normal and shear forces between opposing polystyrene (PS) brushes made from preferentially assembled PS–polyvinylpyridine diblock copolymers were measured in toluene and in near‐theta cyclohexane at 32, 40, and 50 °C, using a modified surface forces apparatus. In cyclohexane, over the temperature window probed, the normal forces of interaction are repulsive and the range of those force profiles changes only slightly; however, for both of the PS brushes studied, the onset of shear forces in near‐theta cyclohexane is strongly influenced by changes in temperature: As the temperature is increased, the onset of the frictional interactions between the brushes in cyclohexane shifts to smaller distances, approaching the distances where frictional forces are observed for brushes in the good solvent toluene. The pattern of behavior seen in the frictional response between the limits of good and theta condition is attributed to composition fluctuations, which increase near the theta condition because of the decrease in excluded volume interactions. These fluctuations may give rise to increased drag during shearing motion because of interfacial roughness or interchain coupling across the brush–brush interface. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 649–655, 2006     

Designing a Useful Lipid Raft Model Membrane for Electrochemical and Surface Analytical Studies

A model biomimetic system for the study of protein reconstitution or drug interactions should include lipid rafts in the mixed lipid monolayer, since they are usually the domains embedding membrane proteins and peptides. Four model lipid films composed of three components: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Chol) and sphingomyelin (SM) mixed in different molar ratios were proposed and investigated using surface pressure measurements and thermodynamic analysis of the monolayers at the air–water interface and imaged by Brewster angle microscopy. The ternary monolayers were transferred from the air–water onto the gold electrodes to form bilayer films and were studied for the first time by electrochemical methods: alternative current voltammetry and electrochemical impedance spectroscopy and imaged by atomic force microscopy. In excess of DOPC, the ternary systems remained too liquid for the raft region to be stable, while in the excess of cholesterol the layers were too solid. The layers with SM in excess lead to the formation of Chol:SM complexes but the amount of the fluid matrix was very low. The equimolar content of the three components lead to the formation of a stable and well-organized assembly with well-developed raft microdomains of larger thickness, surrounded by the more fluid part of the bilayer. The latter is proposed as a convenient raft model membrane for further physicochemical studies of interactions with drugs or pollutants or incorporation of membrane proteins.