Effects of High Intensity Ultrasound on Physiochemical and Structural Properties of Goat Milk ß-Lactoglobulin.

This study aimed to compare the effects of high intensity ultrasound (HIU) applied at various amplitudes (20~40%) and for different durations (1~10 min) on the physiochemical and structural properties of goat milk ß-lactoglobulin. No significant change was observed in the protein electrophoretic patterns by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Deconvolution and second derivative of the Fourier transform infrared spectra (FTIR) showed that the percentage of ß-sheet of goat milk ß-lactoglobulin was significantly decreased while those of α-helix and random coils increased after HIU treatment The surface hydrophobicity index and intrinsic fluorescence intensity of samples was enhanced and increased with increasing HIU amplitude or time. Differential scanning calorimetry (DSC) results exhibited that HIU treatments improved the thermal stability of goat milk ß-lactoglobulin. Transmission electron microscopy (TEM) of samples showed that the goat milk ß-lactoglobulin microstructure had changed and it contained larger aggregates when compared with the untreated goat milk ß-lactoglobulin sample. Data suggested that HIU treatments resulted in secondary and tertiary structural changes of goat milk ß-lactoglobulin and improved its thermal stability.

Oxidative folding pathways of bovine milk ß-lactoglobulin with odd cysteine residues.

Bovine ß-lactoglobulin (BLG) is a major whey protein with unique structural characteristics: it possesses a free Cys thiol (SH) and two disulfide (SS) bonds and consists of a ß-barrel core surrounded by one long and several short α helices. Although SS-intact conformational folding has been studied in depth, the oxidative folding pathways and accompanying SS formation/rearrangement are poorly understood. In this study, we used trans-3,4-dihydroxyselenolane oxide, a water-soluble selenoxide reagent which undergoes rapid and quantitative SS formation, to determine the oxidative folding pathways of BLG variant A (BLGA) at pH 8.0 and 25 °C. This was done by characterizing two key one-SS intermediates, a particular folding intermediate having a Cys66-Cys160 SS bond (I-1) and a particular folding intermediate having a Cys106-Cys119 SS bond (I-2), which have a native Cys66-Cys160 and Cys106-Cys119 SS bond, respectively. In the major folding pathway, the reduced protein (R) with abundant α helices was oxidized to I-1, which was then transformed to I-2 through SS rearrangement. The native protein (N) was formed by oxidation of I-2. The redundant Cys121 thiol facilitates SS rearrangement. N is also generated from an ensemble of folding intermediates having two SS bonds (2SS) intermediates with scrambled SS bonds through SS rearrangement, but this minor pathway is deteriorative due to aggregation or overoxidation of 2SS. During oxidative folding of BLGA, αâ†’ß conformational transition occurred as previously observed in SS-intact folding. These findings are informative not only for elucidating oxidative folding pathways of other members of the ß-lactoglobulin family, but also for understanding the roles of a redundant Cys thiol in the oxidative folding process of a protein with odd Cys residues.

Confinement-induced liquid crystalline transitions in amyloid fibril cholesteric tactoids.

Chirality is ubiquitous in nature and plays crucial roles in biology, medicine, physics and materials science. Understanding and controlling chirality is therefore an important research challenge with broad implications. Unlike other chiral colloids, such as nanocellulose or filamentous viruses, amyloid fibrils form nematic phases but appear to miss their twisted form, the cholesteric or chiral nematic phases, despite a well-defined chirality at the single fibril level. Here we report the discovery of cholesteric phases in amyloids, using ß-lactoglobulin fibrils shortened by shear stresses. The physical behaviour of these new cholesteric materials exhibits unprecedented structural complexity, with confinement-driven ordering transitions between at least three types of nematic and cholesteric tactoids. We use energy functional theory to rationalize these results and observe a chirality inversion from the left-handed amyloids to right-handed cholesteric droplets. These findings deepen our understanding of cholesteric phases, advancing their use in soft nanotechnology, nanomaterial templating and self-assembly.

Mechanism of co-aggregation in a protein mixture with small additives.

Co-aggregation plays an important role in processing protein-rich food materials under heterogeneous conditions. The main cause of co-aggregation is an electrostatic attraction between oppositely charged molecules. This study investigated thermal aggregation of ß-lactoglobulin (BLG) (pI=5.1) and lysozyme (LYZ) (pI=10.7) as a model for the heterogeneous conditions of a protein solution. BLG and LYZ were more aggregated in the mixture than in the single solutions. Co-aggregation of the BLG-LYZ mixture was not observed below 60°C at which temperature BLG and LYZ retained their native structures. Adding sugars, salts, or amino acids to the BLG-LYZ mixture during the heat treatment revealed the co-aggregation process as follows. (i) All additives tested suppressed both the nucleation and growth of aggregates. (ii) Salts affected nucleation stage to the same degree, except arginine hydrochloride (Arg). (iii) Arg specifically suppressed both nucleation and growth of aggregates. These results indicate that co-aggregation in a protein mixture is more sensitive to the partial unfolding of proteins than that in a single protein solution, due to the presence of electrostatic attraction between different molecules. These results provide new insight into protein aggregation as well as the molecular mechanism of additives under heterogeneous conditions.

Influence of pH on viscoelastic properties of heat-induced gels obtained with a ß-Lactoglobulin fraction isolated from bovine milk whey hydrolysates.

A ß-Lactoglobulin fraction (r-ßLg) was isolated from whey hydrolysates produced with cardosins from Cynara cardunculus. The impact of the hydrolysis process on the r-ßLg structure and the rheological properties of heat-induced gels obtained thereafter were studied at different pH values. Differences were observed between r-ßLg and commercial ß-Lg used as control. Higher values for the fluorescence emission intensity and red shifts of the emission wavelength of r-ßLg suggested changes in its tertiary structure and more solvent-exposed tryptophan residues. Circular dichroism spectra also supported these evidences indicating that hydrolysis yielded an intermediate (non-native) ß-Lg state. The thermal history of r-ßLg through the new adopted conformation improved the microstructure of the gels at acidic pH. So, a new microstructure with better rheological characteristics (higher conformational flexibility and lower rigidity) and greater water holding ability was founded for r-ßLg gel. These results were reflected in the microstructural analysis by scanning electron microscopy.

Real-time observation of nonclassical protein crystallization kinetics.

We present a real-time study of protein crystallization of bovine ß-lactoglobulin in the presence of CdCl(2) using small-angle X-ray scattering and optical microscopy. From observing the crystallization kinetics, we propose the following multistep crystallization mechanism that is consistent with our data. In the first step, an intermediate phase is formed, followed by the nucleation of crystals within the intermediate phase. During this period, the number of crystals increases with time, but the crystal growth is slowed down by the surrounding dense intermediate phase due to the low mobility. In the next step, the intermediate phase is consumed by nucleation and slow growth, and the crystals are exposed to the dilute phase. In this stage, the number of crystals becomes nearly constant, whereas the crystals grow rapidly due to access to the free protein molecules in the dilute phase. This real-time study not only provides evidence for a two-step nucleation process for protein crystallization but also elucidates the role and the structural signature of the metastable intermediate phase in this process.

Optimizing the selective recognition of protein isoforms through tuning of nanoparticle hydrophobicity.

We demonstrate that ligand hydrophobicity can be used to increase affinity and selectivity of binding between monolayer-protected cationic gold nanoparticles and ß-lactoglobulin protein isoforms containing two amino acid mutations.

Modulating materials by orthogonally oriented ß-strands: composites of amyloid and silk fibroin fibrils.

Amyloid fibrils and silk fibroin (SF) fibrils are proteinaceous aggregates occurring either naturally or as artificially reconstituted fibrous systems, in which the constituent ß-strands are aligned either orthogonally or parallel to the fibril main axis, conferring complementary physical properties. Here, it is shown how the combination of these two classes of protein fibrils with orthogonally oriented ß-strands results in composite materials with controllable physical properties at the molecular, mesoscopic, and continuum length scales.

Inhibitory effects of ß-ionone on amyloid fibril formation of ß-lactoglobulin.

ß-Lactoglobulin (ß-LG) is the major constituent of whey food, which has been shown to interact with a wide range of aroma compounds. In the present work, a model aroma compound, ß-ionone, is used to investigate the influence of aroma compounds on the urea-induced unfolding of ß-LG at pH 7.0. ß-Ionone is observed to enhance the stability of ß-LG at pH 7.0. Moreover, the amyloid fibrils are observed when ß-LG at pH 7.0 is incubated for 12-20 days at 37 °C in the presence of 3-5M urea. However, the formation of amyloid fibrils is inhibited when ß-ionone is added into the samples and the inhibitory effects follow a concentration-dependent fashion. There is a clear correlation between Cm and lag time. The correlation demonstrates that protein stability affects the amyloid fibril formation of ß-LG. The results highlight the critical role of protein stability and provide an approach to prevent the formation of amyloid fibrils in vitro.

Determining the gelation temperature of ß-lactoglobulin using in situ microscopic imaging.

Evolution of microstructure during heat-induced gelation of ß-lactoglobulin (ß-LG) was investigated in situ using confocal laser scanning microscopy at various gel-preparation conditions: pH=2, 5, and 7; protein content=5, 10, and 15%; and salt (NaCl) content=0, 0.1, and 0.3 M. The number and area of evolving ß-LG clusters were observed as a function of time and temperature and the data were fitted to a log-normal model and sigmoid model, respectively. The gelation temperature (Tgel) of the ß-LG system was determined from both the number (Tgel/N) and total area (Tgel/A) of ß-LG clusters versus temperature data. The range of Tgel/N and Tgel/A values for all the cases was 68 to 87°C. The effect of pH was the most dominant on Tgel/N and Tgel/A, whereas the effects of ß-LG and salt contents were also statistically significant. Therefore, the combined effect of protein concentration, pH, and salt content is critical to determine the overall gel microstructure and Tgel. The Tgel/N and Tgel/A generally agreed well with Tgel determined by dynamic rheometry (Tgel/R). The correlations between Tgel/N and Tgel/A versus Tgel/R were 0.85 and 0.72, respectively. In addition, Tgel/N and Tgel/A values compared well with Tgel/R values reported in the literature. Based on these results, Tgel/N determined via in situ microscopy appears to be a fairly good representative of the traditionally measured gelation temperature, Tgel/R.

Direct AFM force mapping of surface nanoscale organization and protein adsorption on an aluminum substrate.

We investigate the nanoscale organization of a superficially hydroxylated Al substrate and its effect on subsequent protein adsorption using atomic force microscopy (AFM). For this purpose we used a mode which allows a direct mapping of a variety of surface properties (adhesion, elasticity, dissipation, etc.) to be probed simultaneously with topographical images. The hydroxylation treatment leads to a drastic modification of the surface morphology, owing to the formation of AlOOH compounds. In air, AFM images revealed the formation of regular nanorod-like structures randomly distributed, inducing the appearance of nanoporous domains on the surface. In buffer solution, prior to the adsorption of proteins, the surface nanoscale organization is preserved, mainly due to the chemical stability of AlOOH compounds under these conditions. The adsorption of proteins on the obtained nanostructured surface was performed using either a globular (ß-lactoglobulin) or a fibrillar (collagen) protein and by modulating the adsorbed amount through the incubation time or the concentration of proteins in solution. At low amounts, collagen adsorbs on the whole surface without preferential localization. The surface topography remains similar to the bare surface, while significant changes were evidenced on adhesion and elasticity maps. This is due to the fact that the surface became adhesive and less stiff, owing to the presence of a soft and hydrated protein layer. By contrast, ß-lactoglobulin tends to diffuse into the nanoporous domains, leading to their filling up, and the surface is blurred with a thick and dense protein layer upon increasing the amount of adsorbed molecules. Our findings demonstrate the interest in using AFM for surface mapping to investigate the mechanism of protein adsorption at the nanoscale on materials with high surface roughness.

Atomic force microscopy for protein nanotechnology.

This chapter introduces atomic force microscopy (AFM) as an important tool for protein nanotechnology. A short review of AFM-based imaging, mapping, and spectroscopy of protein samples is given. AFM imaging of ß-lactoglobulin nanofibrils in air is demonstrated. Basic concepts of AFM are described. Protocols for ß-lactoglobulin nanofibrils and multiwall carbon nanotubes (MWCNT) samples preparation are defined. The operation of the microscope is described using MWCNT and the NanoScope E instrument in contact mode as an example. Nanostructure manipulation based on AFM nano-sweeping is demonstrated.

Macromolecular crowding modulates the kinetics and morphology of amyloid self-assembly by ß-lactoglobulin.

The formation of amyloid fibrils by ß-lactoglobulin in the presence of GndHCl has been monitored by using thioflavin T (ThT) fluorescence, Congo Red and transmission electron microscopy (TEM). Large quantities of aggregated protein are formed by incubating ß-lactoglobulin in 2M GndHCl at room temperature and pH 7.0 for about 20 days. The kinetics of fibrillation process can be described by the lag time for formation of stable nuclei (nucleation) and the apparent rate constant for the growth of fibrils (elongation). Moreover, the effects of macromolecular crowding agents, Dextran 70 and polyethylene glycols (PEG), on the amyloid formation of ß-lactoglobulin at pH 7.0 are studied. The results show that the increase in macromolecular crowding agent concentrations results in shorter lag time and faster growth of fibrils. It proves that macromolecular crowding can effectively accelerate the fibril formation of ß-lactoglobulin at neutral pH. At the same time, it can be observed that the amplitude of the ThT fluorescence intensity decreases as the Dextran 70 concentration is increased. The observation suggests that the yield of amyloid fibrils is significantly reduced by the addition of macromolecular crowding agents. The conclusion is further confirmed by the transmission electron microscopy. In addition, the results of transmission electron microscopy also indicate that macromolecular crowding can alter the fibril morphology of ß-lactoglobulin. In brief, our findings demonstrate that the effects of macromolecular crowding are essential to the understanding of protein amyloid self-assembly occurring in vivo.

Stability of aqueous food grade fibrillar systems against pH change.

We report that the stability of an aqueous food grade fibril system upon pH change is affected by the presence of peptides that are formed during the process of fibril formation. We discuss several other relationships between food relevant properties and nano-scale characteristics, and compare these relationships for aqueous fibril systems to those of oil based fibril systems. In such fibril systems, dynamics, self-organisation, and sensitivity to external conditions, play an important role. These aspects are common to complex systems in general and define the future challenge in relating functional properties of food to molecular scale properties of their ingredients.

Relation between the gel structure and the mobility of tracers in globular protein gels.

Diffusion of fluorescent-labeled dextran with different molecular weights was investigated in ß-lactoglobulin (ß-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 0.2M 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 0.1M. The present investigation complements an earlier study of tracer diffusion of larger spherical probes in ß-lg gels using multi-particle tracking.

On the crucial importance of the pH for the formation and self-stabilization of protein microgels and strands.

Stable suspensions of protein microgels are formed by heating salt-free ß-lactoglobulin solutions at concentrations up to about C = 50 g·L(-1) if the pH is set within a narrow range between 5.75 and 6.1. The internal protein concentration of these spherical particles is about 150 g·L(-1) and the average hydrodynamic radius decreases with increasing pH from 200 to 75 nm. The formation of the microgels leads to an increase of the pH, which is a necessary condition to obtain stable suspensions. The spontaneous increase of the pH during microgel formation leads to an increase of their surface charge density and inhibits secondary aggregation. This self-stabilization mechanism is not sufficient if the initial pH is below 5.75 in which case secondary aggregation leads to precipitation. Microgels are no longer formed above a critical initial pH, but instead short, curved protein strands are obtained with a hydrodynamic radius of about 15-20 nm.

Understanding amyloid aggregation by statistical analysis of atomic force microscopy images.

The aggregation of proteins is central to many aspects of daily life, including food processing, blood coagulation, eye cataract formation disease and prion-related neurodegenerative infections. However, the physical mechanisms responsible for amyloidosis-the irreversible fibril formation of various proteins that is linked to disorders such as Alzheimer's, Creutzfeldt-Jakob and Huntington's diseases-have not yet been fully elucidated. Here, we show that different stages of amyloid aggregation can be examined by performing a statistical polymer physics analysis of single-molecule atomic force microscopy images of heat-denatured beta-lactoglobulin fibrils. The atomic force microscopy analysis, supported by theoretical arguments, reveals that the fibrils have a multistranded helical shape with twisted ribbon-like structures. Our results also indicate a possible general model for amyloid fibril assembly and illustrate the potential of this approach for investigating fibrillar systems.

Salt-induced gelation of globular protein aggregates: structure and kinetics.

Aggregates of the globular protein beta-lactoglobulin were formed by heating solutions of native proteins at pH 7, after which gels were formed by the addition of salt. The second step does not necessitate elevated temperatures and is therefore often called cold gelation. The structure of the gels was studied during their formation using light scattering and turbidity. Complementary confocal laser scanning microscopy measurements were done. We compared the structure with that of gels formed by heating native beta-lactoglobulin under the same conditions. Whereas in the latter case, microphase separation occurs above 0.2 M NaCl, no microphase separation was observed during cold gelation up to at least 1 M NaCl. The dependence of the kinetics and the final gel structure on the protein concentration, the temperature, the salt concentration, and the aggregate size was quantified. A few measurements on gels formed by adding CaCl(2) confirmed the higher efficiency of this bivalent cation but revealed no qualitative differences with gels formed by adding NaCl.

Native beta-lactoglobulin self-assembles into a hexagonal columnar phase on a solid surface.

Using electron scanning microscopy, we have studied the protein deposit left on silicon and mica substrates by dried droplets of aqueous solutions of bovine beta-lactoglobulin at low concentration and pH = 2-7. We have observed different self-assembled structures: homogeneous layers, hexagonal platelets and flower-shaped patterns laying flat on the surface, and rods formed by columns. Homogeneous layers covered the largest area of the droplet deposit. The other structures were found in small isolated regions, where the protein solution dried in the form of microdroplets. The presence of hexagonal platelets, flower-shaped patterns and columnar rods shows that beta-lactoglobulin self-assembles at the surface in a hexagonal columnar phase, which has never been observed in solution. A comparison with proteins showing similar aggregates suggests that beta-lactoglobulin structures grow from hexagonal germs composed of discotic nanometric building blocks, possibly possessing an octameric structure. We propose that discotic building blocks of beta-lactoglobulin may be produced by the anisotropic interaction with the solid surface.

Amyloid fibril-like structure underlies the aggregate structure across the pH range for beta-lactoglobulin.

The protein beta-lactoglobulin aggregates into two apparently distinct forms under different conditions: amyloid fibrils at pH values away from the isoelectric point, and spherical aggregates near it. To understand this apparent dichotomy in behavior, we studied the internal structure of the spherical aggregates by employing a range of biophysical approaches. Fourier transform infrared studies show the aggregates have a high beta-sheet content that is distinct from the native beta-lactoglobulin structure. The structures also bind the amyloidophilic dye thioflavin-T, and wide-angle x-ray diffraction showed reflections corresponding to spacings typically observed for amyloid fibrils composed of beta-lactoglobulin. Combined with small-angle x-ray scattering data indicating the presence of one-dimensional linear aggregates at the molecular level, these findings indicate strongly that the aggregates contain amyloid-like substructure. Incubation of beta-lactoglobulin at pH values increasingly removed from the isoelectric point resulted in the increasing appearance of fibrillar species, rather than spherical species shown by electron microscopy. Taken together, these results suggest that amyloid-like beta-sheet structures underlie protein aggregation over a much broader range of conditions than previously believed. Furthermore, the results suggest that there is a continuum of beta-sheet structure of varying regularity underlying the aggregate morphology, from very regular amyloid fibrils at high charge to short stretches of amyloid-like fibrils that associate together randomly to form spherical particles at low net charge.