Characterization of secondary and tertiary conformational changes of beta-lactoglobulin adsorbed on silica nanoparticle surfaces

Nanoparticles possess unique properties as a result of their large surface area per unit volume and therefore can be functionalized by the immobilization of enzymes for a variety of biosensing applications. Changes in the tertiary conformation of beta-lactoglobulin adsorbed on 90 nm silica nanoparticles with time were inferred using tryptophan fluorescence and Fourier transform infrared spectroscopy (FTIR) for different surface concentrations, temperature, pH, ionic strength, and 2,2,2-trifluoroethanol (TFE) and dithiothreitol (DTT) concentrations. Rapid initial unfolding followed by a much slower rate at longer times was observed, with the extent of unfolding being higher at lower surface concentrations, higher ionic strengths, higher temperature, higher TFE and DTT concentrations, and pI. The effect of temperature on the unfolding of adsorbed protein on the nanoparticle surface was similar to that in the bulk even though the extent of unfolding was higher for adsorbed protein molecules. The results of the extent of change in tertiary conformation using FTIR as indicated by the change in the ratio of amide II'/amide I were consistent with those obtained by tryptophan fluorescence whereas the rates of conformational changes given by FTIR were found to be much faster. Circular dichroism (CD) spectra showed that altering the surface concentration by itself did not change the secondary structure of beta-lactoglobulin on the surface. TFE was found to increase the alpha helix content at the expense of the fraction of the beta sheet, whereas the beta sheet was converted to an unordered conformation in the presence of DTT.     

Adsorption-induced conformational changes of proteins onto ceramic particles: differential scanning calorimetry and FTIR analysis

Three model proteins, bovine serum albumin, hen's egg lysozyme and bovine serum fibrinogen, were adsorbed from aqueous solution onto finely dispersed ceramic particles, namely different kinds of alumina and hydroxyapatite particles. The influence of adsorption on protein secondary structure was investigated. The FTIR spectroscopic findings were compared with the results of DSC measurements. In almost all cases it was found that adsorption results in destabilisation and structural loss of the bound protein. A decrease in transition enthalpy is correlated with a loss in alpha-helical structure, which seems to be the most sensitive structure on adsorption-induced rearrangements. A total collapse of structure in the adsorbed proteins was not determined on any ceramic surface. Some residual structure is always retained. Structural changes in the D- or E-domains of fibrinogen could be independently observed by two different calorimetric signals. The two techniques applied in the present study -- micro-DSC and FTIR spectroscopy -- can be concluded to provide complementary information on adsorption-induced structural changes on both the molecular (thermal stability, overall structure) and the sub-molecular level (secondary structure).     

Formation of intermolecular beta-sheet structures: a phenomenon relevant to protein film structure at oil-water interfaces of emulsions

Oil-in-water emulsions stabilized with beta-lactoglobulin (beta-lg) were made using a homogenizer or a high-speed blender. The protein was studied by Fourier transform infrared (FTIR) spectroscopy in the raw emulsion, in the bulk phase, and at the interface, as a function of pH, oil content, and homogenizing pressure. Results show that the amount of adsorbed protein varies with the available interfacial area. The protein that remains in the aqueous phase exhibit no spectral change, which suggests that homogenization causes no conformational modification or reversible ones. Strong and irreversible changes were observed in the adsorbed protein. Our findings reveal the formation of intermolecular antiparallel beta-sheets upon adsorption due to the protein self-aggregation. As deduced from transmission electronic microscopy, this surface aggregation leads to the formation of continuous and homogeneous membranes coating the globules. The structure of the adsorbed proteins is unaffected by the homogenizing pressures used in our study and slightly modified by the pH. FTIR spectroscopy allows to characterize the type of aggregates formed at the interface. An analysis of the spectra of beta-lg heat-induced gels shows that the aggregates at the interface are very close at a molecular scale to those that constitute particulate gels near the protein's isoelectric point. Since the type of aggregates is similar when the emulsion water phase is pure D(2)O and D(2)O at pD 4.4, the interface not only seems to induce aggregation, but seems to determine the type of aggregation as well. The mechanism that drives the formation of particulate aggregates (rather than fine-stranded ones) may reside in strong protein-protein interactions that are promoted by adverse oil-protein interactions.     

Effect of thermal treatment on interfacial properties of beta-lactoglobulin

The changes in the secondary conformation and surface hydrophobicity of beta-lactoglobulin subjected to different thermal treatments were characterized at pH values of 7, 5.5 and 4 using circular dichroism (CD) and hydrophobic dye binding. Heating resulted in a decrease in alpha-helix content with a corresponding increase in random coil at all pH values, this change being more pronounced for small heating times. Heating also resulted in an increase in surface hydrophobicity as a result of partial denaturation, this increase being more pronounced at pH 4. Thermal treatment resulted in a shift of the spread monolayer isotherm at air-water interface to smaller area per molecule due to increased flexibility and more loop formation. Thermal treatment led to an increase in interfacial shear elasticity and viscosity of adsorbed beta-lactoglobulin layer at pH 5.5 and 7. Interfacial shear elasticity, shear viscosity, stability of beta-lactoglobulin stabilized emulsion and average coalescence time of a single droplet at a planar oil-water interface with adsorbed protein layer exhibited a maximum for protein subjected to 15 min heat treatment at pH 7. At pH 5.5, the interfacial shear rheological properties and average single drop coalescence time were maximum for 15 min heat treatment whereas emulsion stability was maximum for 5 min heat treatment. At pH 7, thermal treatment was found to enhance foam stability. Analysis of thin film drainage indicated that interfacial shear rheological properties do not influence thin film drainage.     

A differential microcalorimetric study of whey proteins and their behaviour in oil-in-water emulsions

Oil-in-water emulsions (20% soya oil, 1% protein) have been prepared containing lysozyme or isolates of -lactalbumin and β-lactoglobulin from whey protein. The structural characteristics of these proteins adsorbed at an oil/water interface were determined by following their thermal transitions using differential scanning microcalorimetry. Thermograms of the proteins in the adsorbed state differed markedly from the corresponding transitions seen for the proteins in solution. This suggests that the proteins underwent substantial changes in secondary and tertiary structure upon adsorption. In general, for all the proteins studied, a net decrease in the total energy absorbed during denaturation was found when the proteins were in an adsorbed state. Both lysozyme and -lactalbumin were in part “surface denatured”, and they showed a certain degree of reversibility between solution and the adsorbed state. β-Lactoglobulin showed the highest degree of denaturation upon adsorption and the conformational change was irreversible.     

Monoglycerides and beta-lactoglobulin adsorbed films at the air-water interface. structure, microscopic imaging, and shear characteristics

In this work we have analyzed the structural, topographical, and shear characteristics of mixed monolayers formed by adsorbed beta-lactoglobulin (beta-lg) and spread monoglyceride (monopalmitin or monoolein) on a previously adsorbed protein film. Measurements of the surface pressure (pi)-area (A) isotherm, Brewster angle microscopy (BAM), and surface shear characteristics were obtained at 20 degrees C and at pH 7 in a modified Wilhelmy-type film balance. The pi-A isotherm and BAM images deduced for adsorbed beta-lactoglobulin-monoglyceride mixed films at pi lower than the equilibrium surface pressure of beta-lactoglobulin (pi(e)(beta-lg)) indicate that beta-lactoglobulin and monoglyceride coexist at the interface. However, the interactions between protein and monoglyceride are somewhat weak. At higher surface pressures (at pi > or = pi(e)(beta-lg)) a protein displacement by the monoglyceride from the interface takes place. The surface shear viscosity (eta(s)) of mixed films is very sensitive to protein-monoglyceride interactions and displacement as a function of monolayer composition (protein/monoglyceride fraction) and surface pressure. Shear can induce change in the morphology of monoglyceride and beta-lactoglobulin domains, on the one hand, and segregation between domains of the film-forming components on the other hand. In addition, the displacement of beta-lactoglobulin by the monoglycerides is facilitated under shear conditions.     

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.     

Conformational changes of protein adsorbed on tailored flat substrates with different chemistries

Changes in the bioactivity of a protein after being adsorbed on a material surface may result from conformational changes of the protein. Unfortunately, however, direct evidence of such conformational changes of proteins adsorbed on a flat material surface is sparse so far. This is because probing the conformation of an adsorbed protein on material surfaces, especially flat ones, remains a challenge due to considerable experimental difficulties. In this study, the surface-enhanced Raman scattering (SERS) technique is used to characterize the conformational changes of a protein (lysozyme) adsorbed on tailored flat gold substrates with different chemistries. Two such substrates are formed by self-assembly of octadecanethiol and thiolated PEG on gold chips (Au-C18 and Au-PEG). Preliminary results reveal that, compared to the hydrophobic Au-C18 surface, the hydrophilic Au-PEG surface has much smaller effect on the conformation of lysozyme in aqueous solution, which thereby keeps its high bioactivity. The conformational changes of lysozyme adsorbed on material surfaces with different chemistries are well correlated with changes in its bioactivity.     

Interfacial dilatational elasticity and viscosity of beta-lactoglobulin at air-water interface using pulsating bubble tensiometry

The ability of proteins to provide stability in foams is greatly influenced by their interfacial dilatational rheological properties. Surface tension response of a pulsatingbubble with an adsorbed layer of beta-lactoglobulin was measured for different frequencies and protein concentrations using a pulsating bubble tensiometer. A methodology, accounting for adsorption/desorption as well as variation of surface concentration due to expansion/contraction, was developed for the evaluation of surface dilatational elasticity and viscosity at different frequencies from these measurements. The adsorption rate constants were inferred from the surface pressure dynamics of protein adsorption using a Langmuir minitrough. The desorption rates were shown to be negligible for beta-lactoglobulin from the surface pressure response of a spread monolayer when subjected to compression in a Langmuir minitrough. The proposed model was employed to infer the interfacial dilatational viscosity and elasticity of an adsorbed beta-lactoglobulin layer at the air-water interface from experimental pulsating bubble data for protein concentrations in the range of 0.01-0.5 wt % at pH 7. As expected, the interfacial dilatational rheological properties were found to be higher at higher protein concentrations, this effect being less pronounced for dilatational elasticity. Heating at 80 degrees C for 30 min was found to result in higher interfacial dilatational viscosity and lower interfacial dilatational elasticity though this difference was within experimental error. The traditional approach for the inference of interfacial dilatational rheological properties is found to overpredict the interfacial dilatational elasticity whereas the viscosity values do not differ significantly from those obtained using the current analysis.     

Pressure-induced structural and hydration changes of proteins in aqueous solutions

The effects of elevated hydrostatic pressure on four representative proteins, lysozyme, human serum albumin, ubiquitin and RNase A, were investigated by using Fourier transform infrared (FTIR) spectroscopy, by principal component analysis (PCA) and by moving-window two-dimensional (MW2D) correlation analysis. In addition, we revealed the pressure-induced changes of secondary structure elements using curve fitting. With pressure increase, the amide I band shifted to lower wavenumbers, with a transition at 200 MPa, which was indicative of hydration enhancement. Moreover, the pressure-induced behavior of pure water was studied, similar transition pressure was observed with protein in aqueous solution, suggesting that structure change of water around 200 MPa caused a hydration enhancement of protein. Under pressure higher than 200 MPa, the structural changes of the four proteins were obviously different except for the common features shifting to lower wavenumbers with pressure, basically due to the distinct structural differences among them.     

Characterizing protein activities on the lysozyme and nanodiamond complex prepared for bio applications

Recently, nanodiamond particles have attracted increasing attention as a promising nanomaterial for its biocompatibility, easy functionalization and conjugation with biomolecules, and its superb physical/chemical properties. Nanodiamonds are mainly used as markers for cell imaging, using its fluorescence or Raman signals for detection, and as carriers for drug delivery. For the success of these applications, the biomolecule associated with the nanodiamond has to retain its functionality. In this work, the protein activities of egg white lysozyme adsorbed on nanodiamond particles of different sizes is investigated. The lysozyme nanodiamond complex is used here as a protein model for analyzing its structural conformation changes and, correspondingly, its enzymatic activity after the adsorption. Fourier-transform infrared spectroscopy (FTIR) is used for the analysis of the sensitive protein secondary structure. To access the activities of the adsorbed lysozyme, a fluorescence-based assay is used. The process of adsorption is also analyzed using UV-visible spectroscopic measurements in combination with analysis of nanodiamond properties with FTIR, Raman spectroscopy, and ζ-potential measurements. It is found that the activity of lysozyme upon adsorption depends on the nanodiamond's size and surface properties, and that the nanodiamond particles can be selected and treated, which do not alter the lysozyme functional properties. Such nanodiamonds can be considered convenient nanoparticles for various bioapplications.     

Polysaccharide charge density regulating protein adsorption to air/water interfaces by protein/polysaccharide complex formation

Because the formation of protein/polysaccharide complexes is dominated by electrostatic interaction, polysaccharide charge density is expected to play a major role in the adsorption behavior of the complexes. In this study, pullulan (a non-charged polysaccharide) carboxylated to four different charge densities (fraction of carboxylated subunits: 0.1, 0.26, 0.51, and 0.56) was used to investigate the effect of charge density on the properties of mixed protein/polysaccharide adsorbed layers at air/water interfaces. With all pullulan samples, soluble complexes with beta-lactoglobulin could be formed at low ionic strength, pH 4.5. It was shown that the higher was the pullulan charge density, the more the increase of surface pressure in time was retarded as compared to that for pure beta-lactoglobulin. The retardation was even more pronounced for the development of the dilatational modulus. The lower dilatational modulus can be explained by the ability of the polysaccharides to prevent the formation of a compact protein layer at the air/water interface due to electrostatic repulsion. This ability of the polysaccharides to prevent "layer compactness" increases with the net negative charge of the complexes. If charge density is sufficient (> or = 0.26), polysaccharides may enhance the cohesion between complexes within the adsorbed layer. The charge density of polysaccharides is shown to be a dominant regulator of both the adsorption kinetics as well as the resulting surface rheological behavior of the mixed layers formed. These findings have significant value for the application of complex protein-polysaccharide systems.     

Effects of surface curvature and surface chemistry on the structure and activity of proteins adsorbed in nanopores

The interactions of proteins with the surface of cylindrical nanopores are systematically investigated to elucidate how surface curvature and surface chemistry affect the conformation and activity of confined proteins in an aqueous, buffered environment. Two globular proteins, lysozyme and myoglobin, with different catalytic functions, were used as model proteins to analyze structural changes in proteins after adsorption on ordered mesoporous silica SBA-15 and propyl-functionalized SBA-15 (C(3)SBA-15) with carefully controlled pore size. Liquid phase ATR-FTIR spectroscopy was used to study the amide I and II bands of the adsorbed proteins. The amide I bands showed that the secondary structures of free and adsorbed protein molecules differ, and that the secondary structure of the adsorbed protein is influenced by the local geometry as well as by the surface chemistry of the nanopores. The conformation of the adsorbed proteins inside the nanopores of SBA-15 and C(3)SBA-15 is strongly correlated with the local geometry and the surface properties of the nanoporous materials, which results in different catalytic activities. Adsorption by electrostatic interaction of proteins in nanopores of an optimal size provides a favorably confining and protecting environment, which may lead to considerably enhanced structural stability and catalytic activity.     

Modulating surface rheology by electrostatic protein/polysaccharide interactions

There is a large interest in mixed protein/polysaccharide layers at air-water and oil-water interfaces because of their ability to stabilize foams and emulsions. Mixed protein/polysaccharide adsorbed layers at air-water interfaces can be prepared either by adsorption of soluble protein/polysaccharide complexes or by sequential adsorption of complexes or polysaccharides to a previously formed protein layer. Even though the final protein and polysaccharide bulk concentrations are the same, the behavior of the adsorbed layers can be very different, depending on the method of preparation. The surface shear modulus of a sequentially formed beta-lactoglobulin/pectin layer can be up to a factor of 6 higher than that of a layer made by simultaneous adsorption. Furthermore, the surface dilatational modulus and surface shear modulus strongly (up to factors of 2 and 7, respectively) depend on the bulk -lactoglobulin/pectin mixing ratio. On the basis of the surface rheological behavior, a mechanistic understanding of how the structure of the adsorbed layers depends on the protein/polysaccharide interaction in bulk solution, mixing ratio, ionic strength, and order of adsorption to the interface (simultaneous or sequential) is derived. Insight into the effect of protein/polysaccharide interactions on the properties of adsorbed layers provides a solid basis to modulate surface rheological behavior.     

Investigation of the mechanism of protein adsorption on ordered mesoporous silica using flow microcalorimetry

The adsorption of bovine serum albumin (BSA) and lysozyme (LYS) on siliceous SBA-15 with 24 nm pores was studied using flow microcalorimetry; this is the first attempt to understand the thermodynamics of protein adsorption on SBA-15 using flow microcalorimetry. The adsorption mechanism is a strong function of protein structure. Exothermic events were observed when protein–surface interactions were attractive. Entropy-driven endothermic events were also observed in some cases, resulting from lateral protein–protein interactions and conformational changes in the adsorbed protein. The magnitudes of the enthalpies of adsorption for primary protein–surface interactions decrease with increased surface coverage, indicating the possibility of increased repulsion between adsorbed protein molecules. Secondary exothermic events were observed for BSA adsorption, presumably due to secondary adsorption made possible by conformational changes in the soft BSA protein. These secondary adsorption events were not observed for lysozyme, which is structurally robust. The results of this study emphasize the influence of solution conditions and protein structure on conformational changes of the adsorbed protein and the value of calorimetry in understanding protein–surface interactions.     

Proteins at liquid interfaces: II. Adsorption isotherms

Adsorption isotherms for the three proteins β-casein, bovine serum albumin, and lysozyme at the air-water and oil-water interfaces have been determined independently using ellipsometry and surface radioactivity methods; the surface pressure and surface potential were also monitored. Saturated monolayer coverage occurs via irreversible adsorption of 2–3 mg M^?2 of protein; the resultant films generate surface pressures of about 20 mN m^?1 and are 50–60 Å thick. Molecules adsorbed in the first layer dominate the film pressures so that further adsorption causes no change in the pressure although the film thickness can increase to more than 100 Å. The molecules which give rise to this increase in film thickness are reversibly adsorbed with respect to aqueous substrate exchange. The experimental isotherm data and the Langmuir adsorption isotherm are in close agreement at low protein concentrations. However, comparison with the Gibbs adsorption equation is not valid, although reasonable agreement can be achieved if some account is taken of the fact that the protein molecules in the first layer are irreversibly adsorbed.     

Effect of the air-water interface on the structure of lysozyme in the presence of guanidinium chloride

We report observations of the changes in the surface structure of lysozyme adsorbed at the air-water interface produced by the chemical denaturant guanidinium chloride. A primary result is the durability of the adsorbed surface layer to denaturation, as compared to the molecule in the bulk solution. Data on the surface film were obtained from X-ray and neutron reflectivity measurements and modeled simultaneously. The behavior of lysozyme in G.HCl solutions was determined by small-angle X-ray scattering. For the air-water interface, determination of the adsorbed protein layer dimensions shows that at low to moderate denaturant concentrations (up to 2 mol L(-1)), there is no significant distortion of the protein's tertiary structure at the interface, as changes in the orientation of the protein are sufficient to model data. At higher denaturant concentrations, time-dependent multilayer formation occurred, indicating molecular aggregation at the surface. Methodologies to predict the protein orientation at the interface, based on amino acid residues' surface affinities and charge, were critiqued and validated against our experimental data.     

Reduction of protein adsorption to a solid surface by a coating composed of polymeric micelles with a glass-like core

Adsorption studies by optical reflectometry show that complex coacervate core micelles (C3Ms) composed of poly([4-(2-amino-ethylthio)-butylene] hydrochloride)(49)-block-poly(ethylene oxide)(212) and poly([4-(2-carboxy-ethylthio)-butylene] sodium salt)(47)-block-poly(ethylene oxide)(212) adsorb in equal amounts to both silica and cross-linked 1,2-polybutadiene (PB). The C3Ms have an almost glass-like core and atomic force microscopy of a dried layer of adsorbed C3Ms shows densely packed flattened spheres on silica, which very probably are adsorbed C3Ms. Experiments were performed with different types of surfaces, solvents, and proteins; bare silica and cross-linked 1,2-PB, NaNO(3) and phosphate buffer, and lysozyme, bovine serum albumin, beta-lactoglobulin, and fibrinogen. On the hydrophilic surface the coating reduces protein adsorption >90% in 0.1 M phosphate buffer, whereas the reduction on the coated hydrophobic surface is much lower. Reduction is better in phosphate buffer than in NaNO(3), except for the positively charged lysozyme, where the effect is reversed.     

Structural changes in apolipoproteins bound to nanoparticles

Nanoparticles are widely used in the pharmaceutical and food industries, but the consequences of exposure to the human body have not been thoroughly investigated. Apolipoprotein A-I (apoAI), the major protein in high-density lipoprotein (HDL), and other lipoproteins are found in the corona around many nanoparticles, but data on protein structural and functional effects are lacking. Here we investigate the structural consequences of the adsorption of apoAI, apolipoprotein B100 (apoB100), and HDL on polystyrene nanoparticles with different surface charges. The results of circular dichroism, fluorescence spectroscopy, and limited proteolysis experiments indicate effects on both secondary and tertiary structures. Plain and negatively charged nanoparticles induce helical structure in apoAI (negative net charge) whereas positively charged nanoparticles reduce the amount of helical structure. Plain and negatively charged particles induce a small blue shift in the tryptophan fluorescence spectrum, which is not noticed with the positively charged particles. Similar results are observed with reconstituted HDL. In apoB100, both secondary and tertiary structures are perturbed by all particles. To investigate the generality of the role of surface charge, parallel experiments were performed using human serum albumin (HSA, negative net charge) and lysozyme (positive net charge). Again, the secondary structure is most affected by nanoparticles carrying an opposite surface charge relative to the protein. Nanoparticles carrying the same net charge as the protein induce only minor structural changes in lysozyme whereas a moderate change is observed for HSA. Thus, surface charge is a critical parameter for predicting structural changes in adsorbed proteins, yet the effect is specific for each protein.