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The living cells show profoundly crowded condition, called as macromolecular crowding. Crowding essentially impacts on protein structure and lead to its aggregation. Protein aggregates have been involved in a wide range of diseases including Parkinson, Alzheimer’s, and Huntington’s. Increased in normal physiological macromolecular crowding because of increasing age can be implicated as one of the leading cause of proteopathies. In the present study, we have demonstrated the effect of macromolecular crowding on native structure of hemoglobin using bovine serum albumin as a crowding agent. Conformational changes of Hb at different concentrations of BSA were monitored using intrinsic fluorescence and ATR-FTIR spectroscopy. These results showed the transition of native Hb to a non-native form. Thermodynamic parameters were analyzed by isothermal titration calorimetry. The measurements of turbidity, thioflavin T, congo red and intrinsic fluorescence revealed the formation of significant protein aggregates with time. The kinetics of protein aggregation using relative ThT and 8-anilino-1-naphthalenesulphonic acid spectra clearly showed acceleration of the process with time and in concentration dependent manner. The spectra at 80 g/l of BSA incubated for 64 h showed formation of maximum Hb aggregates. Transmission electron microscopy results clearly showed the formation of amyloid aggregates structures, thus supporting the spectroscopic data.

This work investigated the effects of low-frequency ultrasound (US) induced conformational variation of duck liver globular proteins (DLGPs) on the binding behavior of n-alkenals based on multi-spectroscopic analysis combined with headspace solid phase microextraction technique. Results suggested that US-treatments, with the frequency of 40 kHz and the power density of 26.7 W/L, induced obvious tertiary and quaternary structure changes of DLGPs, as revealed by fluorescence and ultraviolet–visible absorption spectroscopy, whereas the secondary and primary structures of DLGPs remained almost unaffected, suggesting the presence of molten-globule (MG) intermediates due to US-treatment. Meanwhile, the particles size and absolute values of ζ-potential exhibited significant increases as the functions of ultrasonic durations. According to multivariate results, the conformational variations, involving natural, MG and post-MG states, could be differentiated successfully. The US-induced conformational changes of DGLPs allowed it easier to interact with volatile n-alkenals, attributed to the enhanced hydrophobic interaction and covalent binding. Correspondingly, the headspace fractions of n-alkenals were greatly reduced after ultrasound pretreatments, with different degrees ranging from 5.61 to 59.39%, depending on chemical structures and processing conditions. This work suggests the great potential of specific conformational variations of DLGPs induced by ultrasonic pretreatments to modulate flavor features of protein-based products.

The influence of different globular protein sources (soy, pea and whey proteins), casein-to-globular protein ratios (6:4 and 4:6), and initial heating pH values (6.6, 6.9 and 7.2) on the heat stability, creaming stability, and flow behavior of mixed protein stabilized emulsions (10% w/w protein and 10% w/w oil) was studied. Fine emulsions in the nanometric range (

We study in this paper the effect of small globular protein on the dynamic properties of long-chain NaPSS in semidilute regime using Dynamic Light Scattering and viscometry in three phases respecting the pH of the medium. The scaling concept of the heterogeneous system is compared with the De Gennes argument for homogeneous polymer solutions. The results showed a positive and negative deviations to the De Gennes approach of the correlation length scale of mixture defined by ( c c * ) δ . The macroscopic viscosity of protein and the potential electrostatic interaction are taken as principal factors affecting the cooperative motion of blobs. δ was discussed as the parameter responsible for the conformational change of polyelectrolyte chain subunit within blob. The theoretical analysis of the electrostatic interaction between protein and subunit gave one possible solution relating the deviation δ to the dimensionless coupling constant u as δ ~ u u + 1 and δ ~ − 1 3 u u + 1 for a swollen and shrunk subunit, respectively. A good accordance of experimental values of δ to theoretical approach was found.

The positions of water molecules have been analyzed in high quality protein X-ray crystal structures. About 70% of these water molecules are in contact with protein atoms at the protein surface and constitute the first hydration layer. About 20% of them are close to the first hydration layer but are not in contact with protein atoms and constitute the second hydration layer. The rest of the water molecules are either buried in the protein core or close to hetero-atoms (inorganic ions and small organic molecules). Upper layers (third, fourth, etc.) are not observed in the dataset of protein crystal structures examined here. Water molecules of both layers are not, in general, surrounded by a tetrahedral arrangement of atoms, as it should be expected on the basis of the electronic structure of water. Usually there are less than four atoms around water molecules and even when there are four atoms, the stereochemistry is often distorted. Water molecules are more mobile than protein atoms, more in the second hydration layer than in the first.

This contribution is concerned with the imidazolium-based ionic liquid surfactant being considered potential amphiphilic molecules. The interfacial dilational rheology of solutions of globular proteins (lysozyme and bovine serum albumin (BSA)) with imidazolium-based ionic liquid surfactant (1-dodecyl-3-methyl imidazolium bromide ([C12mim]Br)) have been measured as a function of the surfactant concentration, interface lifetime and interfacial pressure. The dynamic interfacial dilational modulus of lysozyme/[C12mim]Br solutions are always monotonous, but that of the BSA/[C12mim]Br solutions become nonmonotonous indicating the destruction of the protein structure. One can assume that some lysozyme molecules desorb from interface due to competitive of free [C12mim]Br molecules for lysozyme/[C12mim]Br solution with [C12mim]Br concentration increasing, however, BSA molecules unfolding of compact globule have been subject to conformational changes so that it can give space for more [C12mim]Br molecules to co-adsorb. This aimed to provide more theoretical information for practical applications of imidazolium-based ionic liquid surfactant.

Understanding globular protein adsorption to fluid interfaces, their interfacial assembly, and structural reorganization is not only important in the food industry, but also in medicine and biology. However, due to their intrinsic structural complexity, a unifying description of these phenomena remains elusive. Herein, we propose N-isopropylacrylamide microgels as a promising model system to isolate different aspects of adsorption, dilatational rheology, and interfacial structure at fluid interfaces with a wide range of interfacial tensions, and compare the results with the ones of globular proteins. In particular, the steady-state spontaneously-adsorbed interfacial pressure of microgels correlates closely to that of globular proteins, following the same power-law behavior as a function of the initial surface tension. However, the dilatational rheology of spontaneously-adsorbed microgel layers is dominated by the presence of a loosely packed polymer corona spread at the interface, and it thus exhibits a similar mechanical response as flexible, unstructured proteins, which are significantly weaker than globular ones. Finally, structurally, microgels reveal a similar spreading and flattening upon adsorption as globular proteins do. In conclusion, microgels offer interesting opportunities to act as powerful model systems to unravel the complex behavior of proteins at fluid interfaces., Colloids and Surfaces B: Biointerfaces, 217, ISSN:0927-7765, ISSN:1873-4367

High pressure technology is the outcome of consumer demand for better quality control of processed foods. There is great potential to apply HPP to condensed systems of globular proteins for the generation of industry-relevant biomaterials with advanced techno- and biofunctionality. To this end, research demonstrates that application of high hydrostatic pressure generates a coherent structure and preserves the native conformation in condensed globular proteins, which is an entirely unexpected but interesting outcome on both scientific and technological grounds. In microbiological challenge tests, high pressure at conventional commercial conditions, demonstrated to effectively reduce the concentration of typical Gram negative or Gram positive foodborne pathogens, and proteolytic enzymes in high-solid protein samples. This may have industrial significance in relation to the formulation and stabilisation of "functional food" products as well as in protein ingredients and concentrates by replacing spray dried powders with condensed HPP-treated pastes that maintain structure and bioactivity. Fundamental concepts and structural functionality of condensed matrices of globular proteins are the primary interest in this mini-review, which may lead to opportunities for industrial exploitation, but earlier work on low-solid systems is also summarised presently to put recent developments in context of this rapidly growing field.

There is still a debate about the effectiveness of globular proteins of several nanometers in stabilizing oil-in-water high internal phase emulsions (HIPEs). In this work, we report that a strong internal structural integrity and high refolding ability is one requirement for globular proteins to perform as soft particles to stabilize HIPEs. Using bovine serum albumin (BSA) as the model protein, it is demonstrated that BSA glycated with galactose (Gal), possessing a much stronger structural integrity and higher refolding ability, exhibited much improved HIPE emulsification performance and subsequent Pickering stabilization than native BSA. The underlying mechanism for the improvement is largely associated with the formation of a core-shell nanostructure with a hydrophilic Gal shell surrounding the protein core in glycated BSA. The ultra-stable HIPEs (gels) were stabilized by glycated BSA at a protein concentration in the aqueous phase as low as 0.1 wt%, much lower than that reported in the literature. These HIPEs exhibited a self-supporting gel network, an extraordinary stability upon long-term storage or against drastic heating as well as good freeze-thaw reversibility. The findings are of importance for the facile fabrication of biocompatible Pickering HIPE formulations with promising applications in the food, cosmetics and pharmaceutical fields.

The work prepared high-solid mixtures of whey protein or bovine serum albumin with an amorphous co-solute (glucose syrup) and examined their glass transition behaviour at subzero temperatures. The interest in these condensed matrices was in relation to what extent thermal denaturation and subsequent aggregation of the proteinaceous molecules affects vitrification and, therefore, they were subjected to distinct heating regimes followed by cooling. Small-deformation dynamic oscillation in shear is known to respond to changes in network formation as a function of thermal treatment, albeit published reports thus far focused on low-solid aqueous hydrocolloid samples, and it has been chosen presently to examine the viscoelasticity of their high-solid counterparts. Results were further compared with those from a micromolecular technique, i.e. modulated differential scanning calorimetry. It appears that thermally induced cross-linking is readily recorded in what is known in the literature as the mechanical or network glass transition temperature, whereas the calorimetric Tg is not affected by the extent of polymeric associations in these mixtures. Further, the thermal protocol employed presently results in considerable differences in predictions of the mechanical Tg, which should reflect distinct three dimensional morphologies in these systems of globular protein and co-solute.

In this study, we investigated the spectroscopic properties of the water-soluble globular protein bovine serum albumin (BSA) while interacting with zinc oxide (ZnO) semiconductor nanoparticles (NPs) in aqueous medium and in a ZnO/BSA layer-by-layer (LbL) self-assembled film fabricated on poly (acrylic acid) (PAA)-coated quartz or a Si substrate via electrostatic interactions. BSA formed a ground state complex due to its interaction with ZnO NPs, which was confirmed by ultraviolet–visible absorption, and steady state and time-resolved fluorescence emission spectroscopic techniques. However, due to its interaction with ZnO, the photophysical properties of BSA depend significantly on the concentration of ZnO NPs in the mixed solution. The quenching of the fluorescence intensity of BSA in the presence of ZnO NPs was due to the interaction between ZnO and BSA, and the formation of their stable ground state complex, as well as energy transfer from the excited BSA to ZnO NPs in the complex nano-bioconjugated species. Multilayer growth of the ZnO/BSA LbL self-assembled film on the quartz substrate was confirmed by monitoring the characteristic absorption band of BSA (280 nm), where the nature of the film growth depends on the number of bilayers deposited on the quartz substrate. BSA formed a well-ordered molecular network-type morphology due to its adsorption onto the surface of the ZnO nanostructure in the backbone of the PAA-coated Si substrate in the LbL film according to atomic force microscopic study. The as-synthesized ZnO NPs were characterized by field emission scanning electron microscopy, X-ray powder diffraction, and dynamic light scattering techniques.

In this work, a novel two-step method for preparing food globular protein-based hydrogel with transparent appearance and stable network was described. First, soy glycinin dispersion (6% w/v) was heated at 95 °C for 30 min in order to obtain partial unfolding conformation and expose more residues for the following enzymatic cross-linking. An anionic polysaccharide, dextran sulfate (DS), was added before glycinin was heated, forming highly charged complexes with glycinin and maintaining the transparency of the dispersion. Microbial transglutaminase (MTGase) was introduced for gelation, creating a covalent network. Transparent cold-set hydrogels could be obtained when the weight ratio of DS to glycinin was over 1/200. It was found that larger amounts of DS and lower ionic strength were favorable for increasing the transparency of the gels. At pH 7.0, effects of DS amounts and ionic strength on the rheological and mechanical properties of the hydrogels were investigated. Hydrogels with distinctive textural properties could be obtained by different combinations of DS amount and ionic strength. Scanning electron microscopy observation indicated that the addition of DS could promote the formation of regular and stable gel network.

Diffusion of fluorescent-labeled dextran with different molecular weights was investigated in β-lactoglobulin (β-lg) solutions and gels over a wide range of salt and protein concentrations at pH 7 by combining confocal laser scanning microscope (CLSM) with fluorescence recovery after photobleaching (FRAP). Effects of the protein concentration, the salt concentration and the tracer size were investigated in detail. Diffusion in turbid heterogeneous gels formed at 0.2 M NaCl depended weakly on the probe size and the protein concentration and remained close to that in unheated solutions. A strong decrease of the diffusion coefficient with increasing tracer size and protein concentration was observed in more homogeneous gels formed at lower salt concentrations. Larger dextran chains were trapped in transparent gels formed at NaCl concentration below 0.1 M. The present investigation complements an earlier study of tracer diffusion of larger spherical probes in β-lg gels using multi-particle tracking.

Gels of the globular protein β-lactoglobulin were made by heating solutions at pH 7 and different NaCI concentrations (C s ). The influence of the ionic strength on the gel structure was studied by confocal laser scanning microscopy (CLSM). For C s < 0.2 M the images were homogeneous, but at higher NaCI concentrations micro-phase separation was observed. The protein concentration in the two phases was determined from the images. It is shown how CLSM images can be quantitatively analysed in terms of the pair correlation function of the protein concentration fluctuations, yielding results that can be directly compared to those obtained from light scattering. The transition between so-called finely stranded and particulate gels is explained by a switch from net repulsive to net attractive interaction between growing protein aggregates.

The rheology of highly concentrated emulsions stabilized with bovine serum albumin and the same protein with the addition of two different cationic surfactants was studied. These materials demonstrate non-Newtonian behavior with zero-shear-rate Newtonian viscosity and rather abrupt decrease of viscosity in a narrow shear stress range. Emulsions also possess elastic properties and storage modulus does not depend on frequency. The addition of low-molecular-mass surfactant leads to the modification of all rheological properties: the viscosity, storage modulus, and yield stress decrease. Variation of the added surfactant in a very wide range shows that there is its threshold concentration, at which a surfactant substitutes protein in the interfacial layers. It was supposed that some peculiarities of emulsions under study are explained by their wide size distribution. As a result, these emulsions are possibly not “compressed” and their elasticity is mainly due to interlayer interactions rather than interfacial tension.

The potential of beet pectin for improving the physical and chemical stabilities of emulsions containing silk fibroin coated droplets was investigated. Five wt.% corn oil-in-water emulsions containing fibroin-coated droplets (0.5 wt.% fibroin) and anionic pectin (0.05 wt.%) were prepared at pH 7. The pH of these emulsions was then adjusted to pH 4, so that the anionic pectin molecules electrostatically deposited to the fibroin-coated droplets. The influence of pH (3 to 7) and sodium chloride concentrations (0 to 500 mM) on the properties of primary (0 wt.% pectin) and secondary (0.05 wt.% pectin) emulsions was studied. Pectin was deposited to the droplet surfaces at pH 3, 4, and 5, but not at pH 6 and 7. In addition, secondary emulsions were stable up to higher ionic strengths (

The objective of this study was to understand how cosolvents influence the molecular and functional properties of globular proteins in aqueous solutions. The ultrasonic velocity, density and adiabatic compressibility of cosolvent solutions (0–50 wt% sorbitol or glycerol) were measured in the absence and presence of a globular protein (1 wt% β-lactoglobulin) at 30 °C. These measurements were used to calculate the partial specific apparent volume and adiabatic compressibility of the protein. The protein's volume decreased and its compressibility increased in the presence of high cosolvent concentrations, which were attributed to changes in the properties of the protein interior and solvation layer. Sorbitol was more effective than glycerol at decreasing the protein volume at high cosolvent concentrations, which may be because glycerol has some surface activity and may therefore accumulate around hydrophobic regions on the protein surface. Our data were used to account for the observation that sorbitol is more effective than glycerol at increasing the thermal stability and self-association of the β-lactoglobulin. A better understanding of the influence of protein–cosolvent–solvent interactions on the functionality of globular proteins may facilitate the design of protein-based products.

The present investigation aimed at understanding how the type of cations (Na + and/or Ca 2+ ) influences heat-induced gelation of globular protein/LM pectin mixtures in comparison to the globular protein only. Two globular proteins (β-lactoglobulin or bovine serum albumin) were compared. On the basis of viscoelastic and microscopic observations (CLSM), it has been shown that the two cations play a major role in gelation of the individual biopolymers and, therefore, influence strongly the properties of the mixture. These differences are related to a competition between gelation of the protein (in absence of Ca 2+ ) or of both biopolymers (if Ca 2+ is added) and phase separation processes taking place before the mixed system is entirely gelled.

Interaction of acridinedione dyes with model transport proteins, bovine serum albumin (BSA) in aqueous solution were investigated by fluorescence spectral studies. A fluorescence enhancement was observed on the addition of BSA to photoinduced electron transfer (PET) based acridinedione dyes, which posses C 6 H 4 (p-OCH 3 ) in the 9th position of the basic acridinedione ring. On the contrary, the addition of BSA to non-PET based acridinedione dyes with methyl or phenyl substitution in the 9th position does not result in any fluorescence enhancement. The enhancement in the fluorescence intensity is attributed to the suppression of PET process through space between –OCH 3 group and the acridinedione moiety is elucidated by steady state fluorescence measurements. The fluorescence anisotropy value ( r ) of 0.40 reveals that the motion of the dye molecule is highly constrained and is largely confined to the rigid microenvironment of the protein molecule. The binding constant ( K ) was found to be in the order of 6.0×10 3 [M] −1 , which implies the existence of hydrophobic interaction between the PET based dye and BSA. Time resolved fluorescence lifetime measurements reveal that the PET based acridinedione dye preferably binds in the hydrophobic interior of BSA.

The influence of neutral cosolvents (polyols) on the stability of hydrocarbon oil-in-water emulsions stabilized by a globular protein was investigated. Glycerol (0–40 wt%) and sorbitol (0–35 wt%) were added to n -hexadecane oil-in-water emulsions stabilized by β-lactoglobulin (β-lg, pH 7.0, 150 mM NaCl), either before or after incubation at 30 °C for 24 h. The stability of the emulsions to flocculation and creaming improved when neutral cosolvents were added, with the effectiveness of the cosolvents depending on their type, concentration and time of addition. Emulsion stability was better for sorbitol than glycerol, improved with increasing cosolvent concentration, and was better when the cosolvents were added immediately after homogenization than when they were added 24 h later. The influence of the cosolvents on emulsion stability is interpreted in terms of their effect on the conformation and interactions of the adsorbed proteins, as well as on the droplet–droplet collision frequency. This study has implications for the development of protein stabilized oil-in-water emulsions for utilization in industrial products.

Hydrated soy-proteins display different macroscopic properties below and above approximately 25% moisture. This is relevant to the food industry in terms of processing and handling. Quasi-elastic neutron spectroscopy of a large globular soy-protein, glycinin, reveals that a similar moisture-content dependence exists for the microscopic dynamics as well. We find evidence of a transition analogous to those found in smaller proteins, when investigated as a function of temperature, at the so-called dynamical transition. In contrast, the glass transition seems to be unrelated. Small proteins are good model systems for the much larger proteins because the relaxation characteristics are rather similar despite the change in scale. For dry samples, which do not show the dynamical transition, the dynamics of the methyl group is probably the most important contribution to the QENS spectra, however a simple rotational model is not able to explain the data. Our results indicate that the dynamics that occurs above the transition temperature is unrelated to that at lower temperatures and that the transition is not simply related to the relaxation rate falling within the spectral window of the spectrometer.

The influence of a nonionic surfactant (polyoxyethylene(20)sorbitan monooleate, Tween 80) on the rheological behavior of concentrated toluene-in-water emulsions stabilized by globular protein (bovine serum albumin, BSA, C = 5 × 10−5 M) at 22 °C have been studied using different methods of shear rheology. The systems under investigation are non-Newtonian liquids while the yield stress is observed at low shear stresses. The dependencies of the yield stress and shear viscoelasticity on the total concentration and surfactant/protein molar ratio were discussed. The increase in concentration of a nonionic surfactant leads to the decrease in viscosity and suppression of emulsion elasticity. A sharp jump in the rheological properties appears at a minimal concentration of the added Tween 80, whereas elasticity practically disappears, viscosity abruptly decreases and emulsions become unstable against coalescence in approaching concentration of the nonionic surfactant to 1 × 10−3 M. The observed effects can be attributed to the gradual displacement of a high-molecular-mass component (protein), which is responsible for the emulsion elasticity, from the stabilizing (interfacial) layers by the nonionic surfactant.

The interactions and complexation process of the structurally related amphiphilic phenothiazines promazine and triflupromazine hydrochlorides with horse myoglobin in aqueous buffered solutions of pH 2.5, 5.5 and 9.0 have been examined by ζ-potential, isothermal titration calorimetry (ITC), UV–vis spectroscopy and dynamic light-scattering techniques with the aim of analyzing the effect of hydrophobic and electrostatic forces, the alteration of protein conformation and the effect of substituents in the drug molecular structure on the binding mechanism and structure of the resulting complexes. The energetics and stoichiometry of the binding process was derived from ITC. The enthalpies of binding obtained are small and exothermic, and the Gibbs energies of binding are dominated by large increases in entropy consistent with hydrophobic interactions. Binding isotherms were obtained from microcalorimetric data by using a theoretical model based on the Langmuir isotherm. ζ-Potential data showed a reversal in the sign of the protein charge at pH 9.0 as a consequence of drug binding. Gibbs energies of drug binding per mole of drug were also derived from ζ-potential data. On the other hand, binding of the phenothiazines causes a conformational transition on protein structure which was followed as a function of drug concentration by using UV–vis spectroscopy. These data were analyzed to obtain the Gibbs energy of the transition in water ( Δ G w ° ) and in a hydrophobic environment ( Δ G hc ° ) . Finally, the population distribution of the different species in solution and their size was analyzed through dynamic light scattering. The existence of an aggregation process of drug/protein complexes, mainly at pH 2.5, was observed. We think this is a consequence of the already expanded structure of the protein at this pH and the subsequent binding of drug molecules to the protein.

Heat capacities and enthalpy relaxation rates of completely anhydrous bovine serum albumin (BSA) and hydrous BSA with 1.85% (w/w) water were measured by using an adiabatic calorimeter to examine the thermal behavior at low temperatures of a small amount of water left within the globular protein molecule. The heat capacities of the hydrous BSA were larger a little on account of the presence of water molecules. In addition, two new phenomena appeared for the hydrous BSA while not for the anhydrous one: spontaneous exothermic and endothermic effects depending on the pre-cooling rates were observed and interpreted as due to a glass transition. Anomalous behavior of heat capacities was found in 60–140 K and recognized as potentially originating from a kind of phase transition. It is suggested that the water left a little within the BSA is responsible for both the phenomena occurring at low temperatures.

We describe a model that relates the optimal size of a globular protein domain to the ratio between hydrophilic and hydrophobic amino acid residues. This model represents a domain as a homogeneous spherical assembly of monodisperse spheres corresponding to the individual residues; the hydrophilic spheres are distributed on the assembly surface, and the hydrophobic spheres are buried in the core. The model predicts that a domain with a 1:1 ratio of hydrophilic and hydrophobic residues is composed of 156 residues. It also predicts that smaller protein domains have more hydrophilic than hydrophobic residues. These predictions are in agreement with the distribution of domain size and residue composition for the experimentally determined protein structures.

The adsorption of the globular protein β-lactoglobulin on hydrophilic and hydrophobic surfaces has been investigated. Spectroscopic ellipsometry and Fourier transform infrared spectroscopy in the attenuated total reflection mode were used in our study. β-lactoglobulin adsorbed in surface concentrations less than or equal to those corresponding to a closely packed monolayer of molecules. The amount of β-lactoglobulin removed from hydrophilic surfaces upon elution with buffer solution, as a fraction of the final amount reached upon adsorption, was observed to decrease at longer adsorption times. This provides evidence that the conformation of the adsorbed protein changes relatively slowly once adsorbed, leading to more irreversibly adsorbed states with stronger binding to the surface. The fraction of protein irreversibly adsorbed to the surface was generally higher on hydrophobic surfaces than hydrophilic surfaces, confirming the general principle that globular proteins exhibit stronger binding to hydrophobic surfaces than hydrophilic ones. The displacement of pre-adsorbed layers of the protein from hydrophobic and hydrophilic surfaces by the non-ionic surfactant octaethylene glycol monododecyl ether (C12E8) was also studied. We observed that the non-ionic surfactant caused partial displacement of adsorbed protein from hydrophobic surfaces. The displacement kinetics also reveal that the protein layers are more strongly bound to hydrophobic surfaces at longer than at shorter adsorption times, showing that slow protein binding changes and ageing effects are also important at hydrophobic surfaces.

The influence of the ionic strength on the structure of beta-lactoglobulin aggregates formed after heating at pH 7 has been studied using static and dynamic light scattering. The native protein depletion has been monitored using size exclusion chromatography. Above a critical association concentration (CAC) well-defined clusters are formed containing about 100 monomers. The CAC increases with decreasing ionic strength. The so-called primary aggregates associate to form self similar semi-flexible aggregates with a large scale structure that is only weakly dependent on the ionic strength. The local density of the aggregates increases with increasing ionic strength. At a critical gel concentration, Cg, the size of the aggregates diverges. Cg decreases from 100 g/l without added salt to 1 g/l at 0.4M NaCl. For C > Cg the system gels except at high ionic strength close to Cg where the gels collapse under gravity and a precipitate is formed.

Heat-set globular protein gel networks are discussed in relation to protein charge and screening. Homogeneous fine-stranded' gels form when electrostatic repulsion is high, with a transition to microphase-separated structures as repulsion falls. Fine-stranded networks showing density fluctuations occur close to this transition and probably arise from a combination of kinetic trapping and a drive towards only limited phase separation. For the most uniform structures (pH far from pI, and low salt content) network building appears to involve three main stages: initial protein unfolding, linear fibrillar aggregation, and random cross-linking of the fibrils. A mean field model is described which incorporates these features and includes the possibility of cooperative linear aggregation (nucleation and growth). Application of this to cure data for acid β-lactoglobulin gels was only partially successful, however, a higher order (n = 4) nucleation process being required to explain gel time-concentration data, while only a lower second order process could reproduce the shapes of the cure curves. As uniform gels give way to phase-separated structures network building becomes still more complex. Here solution demixing of unfolded monomers, and/or the initial aggregates, must be included in the model. This seems beyond the current mean-field approach, and simulation is likely to be required. This is true, even for the more homogeneous structures, when gel properties of interest extend from the linear elastic, to the time-dependent, and non-linear. La gelification induite par la chaleur des proteines globulaires est etudiee par rapport aux charges des proteines et a l'effet d'ecran. Des gels homogenes se forment quand la repulsion electrostatique est forte, avec une transition vers des structures separees en microphases quand la repulsion diminue.

Dynamic viscoelastic and steady flow properties of β-lactoglobulin, bovine serum albumin, ovalbumin, and α-lactalbumin aqueous solutions were investigated at 20°C. When a sinusoidal strain in the linear viscoelastic region was applied, the solutions of the globular proteins except for α-lactalbumin showed typical solid-like rheological behavior: the storage modulus G' was always larger than the loss modulus G in the entire frequency range examined (0.1-100 rad/s). Under a steady shear flow, strong shear thinning behavior was observed with increasing shear rate from 0.001 to 800 s -1 , for the globular proteins except for α-lactalbumin. The values of the steady shear viscosity η were lower than those of the dynamic shear viscosity η * at a comparable time scale of observation, violating the Cox-Merz rule, and thus suggesting that a solid-like structure in a globular protein solution was susceptible to a steady shear strain. During isothermal gelation of the protein colloids at 70°C, no crossover between G' and G was observed so that the gelation point was judged by an abrupt increase in the modulus or a sudden decrease in tanδ. Les proprietes d'ecoulement dynamique viscoelastique et permanent de solutions aqueuses de β-lactaglobuline, serumalbumine bovine, ovalbumine et α-lactalbumine sont etudiees a 20°C. Lorsqu'une deformation sinusoidale est appliquee dans la zone lineaire viscoelastique, les solutions de proteines globulaires a l'exception de l'α-lactalbumine presentent un comportement rhelogique typique d'un solide.

In prediction of a protein main-chain structure into which a query sequence of amino acids folds, one evaluates the relative stability of a candidate structure against reference structures. We developed a statistical theory for calculating the energy distribution over a main-chain structure ensemble, only with an amino acid composition given as a single argument. Then, we obtained a statistical formulae of the ensemble mean [E] and ensemble variance V[E] of the reference structural energies, as explicit functions of the amino acid composition. The mean [E] and the variance V[E] calculated from the formulae were well or roughly consistent with those resulting from a gapless threading simulation. We can use the formulae not, only to perform the high-through-put screening of sequences in the inverse folding problem, but also to handle the problem analytically. (C) 2003 Elsevier Science Ltd. All rights reserved.

In this paper, significant advances in the structure and rheological properties of globular protein gels have been described. The achievements of scattering methods, which have provided information over a range of distances from the monomer to the micron scale, are reviewed. The kinetics of gelation measured by rheological methods have also been discussed, including currently applied models for the gelation time, the gel elastic modulus and the critical gel concentration.

Experimental and theoretic studies of the adsorption behaviour for the mixture of globular protein (human serum albumin (HSA)), and non-ionic surfactant (decyl-dimethyl-phosphine-oxide C10DMPO) are performed. The experimental results for the mixtures agree well with a theoretical model which assumes significant differences between the partial molar areas of the protein and the surfactant, and takes into account large unbound charge of the protein molecules. An anomalous surface tension increase of the mixtures at low surfactant concentrations was found experimentally and explained on the basis of a thermodynamic model. The concentration range at which a comparable coverage of the mixed surface layer by protein and surfactant molecules appears is shown to be quite narrow.

An equilibrium statistical-thermodynamic model for the effect of volume exclusion arising from high concentrations of stable macromolecules upon the stability of a trace globular protein with respect to denaturation by heat and by chaotropes is presented. The stable cosolute and the native form of the trace protein are modeled by effective hard spherical particles. The denatured state of the trace protein is represented as an ensemble of substates modeled by random coils having the same contour length but different rms end-to-end distances (i.e., different degrees of compaction). The excess or nonideal chemical potential of the native state and of each denatured substate is calculated as a function of the concentration of stable cosolute, leading to an estimate of the relative abundance of each state and substate, and the ensemble average free energy of the transition between native and denatured protein. The effect of the addition of stable cosolute upon the temperature of half-denaturation and upon the concentration of chaotrope required to half-denature the tracer at constant temperature is then estimated. At high cosolute concentration (>100g/l) these effects are predicted to be large and readily measurable experimentally, provided that an experimental system exhibiting a fully reversible unfolding equilibrium at high total macromolecular concentration can be developed.

We have chemically crosslinked a globular protein, gamma IIIb-crystallin, to produce a system of well-defined oligomers: monomers, dimers, trimers and a mixture of higher n-mers. Gel electrophoresis, size exclusion chromatography, quasielastic light scattering spectroscopy, and electrospray ionization mass spectrometry were used to characterize the oligomers formed. The liquid-liquid phase separation boundaries of the various oligomers were measured. We find that at a given concentration the phase separation temperature strongly increases with the molecular weight of the oligomers. This phase behavior is very similar to previous findings for gamma II-crystallin, for which oxidation-induced oligomerization is accompanied by an increase in the phase separation temperature. These findings imply that for phase separation, the detailed changes of the surface properties of the proteins are less important than the purely steric effects of oligomerization.

A simple model for the phase behaviour of a globular protein and a flexible polymer in an aqueous medium is described, in which both the compact feature of the protein and the flexble feature of the polymer have been included. The phase diagrams calculated by using the model suggest that for a given protein, the behaviour depends strongly on the polymer molecular weight. Fluid-fluid-solid three-phase and fluid-fluid two-phase equilibria can be found only when the polymer molecular weight is sufficiently high; otherwise, the only two-phase region in the phase diagram is a fluid-solid two-phase region.