In Situ ATR Spectroscopy Study of the Interaction of Acacia senegal Gum with Gold Nanoparticles Films at the Solid-Liquid Interface.

The adsorption process of Acacia gum (A. senegal), a complex heteropolysaccharide, was followed by using a spectroscopic method to unravel the relative contribution of the protein moieties and the carbohydrate blocks on the adsorption process. In situ ATR-FTIR was used to investigate the kinetics and conformational changes associated with the adsorption of A. senegal gum on gold nanoparticle films (Au-NPs) at different pHs. The results of this thorough study highlighted the adsorption of A. senegal gum through its protein moieties, in particular, AGPs of low molecular weight and high protein content, close to the Au-NPs surface. Isotherm experiments, by gradually increasing the concentration, showed that the gum adsorption was heterogeneous and followed the Freundlich model for the amide part, while the polysaccharide part followed the Langmuir model. In addition, the hydration and structural organization of the gum layer depended on the gum concentration. A. senegal gum adsorbed irreversibly on Au-NPs whatever the pHs, but the adsorbed layer presented a different behavior depending on pH. A more aggregated and less hydrated structure was observed at acidic pH, while a very hydrated and continuous layer was detected at higher pH. The secondary structure analysis through amide III band revealed a change in the gum secondary structure at high pH with the increase in ß-turn while random coil decreased.

Maturation of demineralized arabinogalactan-proteins from Acacia seyal gum in dry state: Aggregation kinetics and structural properties of aggregates.

The aggregation in dry state of mineral-loaded arabinogalactan-proteins (AGPs) from Acacia seyal gum (GA) generally occurs above 70 °C. This study focuses on the aggregation sensitivity of AGPs after their demineralization. The dry incubation in mild temperature (25 °C to 70 °C) of demineralized AGPs induced the formation of aggregates, not observed with GA. AGPs aggregated following a self-assembly mechanism for which temperature only modulated the aggregation rate. The activation energy was around 90-100 kJ·mol-1 that could correspond to chemical condensation reactions induced by the AGPs surface dehydration. The aggregation kinetics were characterized by the formation of soluble aggregates during the first times of incubation, whose molar mass increased from 1 · 106 g·mol-1 to 6.7 · 106 g·mol-1 (SEC MALS) or 12 · 106 g·mol-1 (AF4 MALS) after 1.66 days of dry heating at 40 °C. These soluble aggregates revealed they adopted a similar conformation to that of not aggregated AGPs with a νh value around 0.45. Above 1.66 days at 40 °C, the soluble aggregates grew up to form microparticles with sizes ranging from 10 to around 200 µm. This study highlighted the protective role of cations from AGPs whose demineralization increased their sensibility to dry heating and their chemical reactivity for aggregation.

Adsorption Behavior of Arabinogalactan-Proteins (AGPs) from Acacia senegal Gum at a Solid-Liquid Interface.

Adsorption of five different hyperbranched arabinogalactan-protein (AGP) fractions from Acacia senegal gum was thoroughly studied at the solid-liquid interface using a quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), and atomic force microscopy (AFM). The impact of the protein/sugar ratio, molecular weight, and aggregation state on the adsorption capacity was investigated by studying AGP fractions with different structural and biochemical features. Adsorption on a solid surface would be primarily driven by the protein moiety of the AGPs through hydrophobic forces and electrostatic interactions. Increasing ionic strength allows the decrease in electrostatic repulsions and, therefore, the formation of high-coverage films with aggregates on the surface. However, the maximum adsorption capacity was not reached by fractions with a higher protein content but by a fraction that contains an average protein quantity and presents a high content of high-molecular-weight AGPs. The results of this thorough study highlighted that the AGP surface adsorption process would depend not only on the protein moiety and high-molecular-weight AGP content but also on other parameters such as the structural accessibility of proteins, the molecular weight distribution, and the AGP flexibility, allowing structural rearrangements on the surface and spreading to form a viscoelastic film.

Acacia gums new fractions and sparkling base wines: How their biochemical and structural properties impact foamability?

Foam is the first attribute observed when sparkling wine is served. Bentonite is essentially used to flocculate particles in sparkling base wines but can impair their foamability. Gums from Acacia senegal and Acacia seyal improved the foamability of different bentonite-treated base wines. Our main goal was to see how the supplementation with new fractions separated from Acacia gums by Ion Exchange Chromatography affected foamability of sparkling base wines, deepening the relation between foam behavior and characteristics of wine and gums. High molar mass fractions increased the maximal foam height and the foam height during the stability period in, respectively, 11 out and 8 out of 16 cases (69% and 50%, respectively). The properties of the supplementing gums fractions obtained by IEC and, although to a minor extent, the wine characteristics, affected positively and/or negatively the foam behavior. Wine foamability also depended on the relationship between wine and gums fractions properties.

Emulsifying properties of Acacia senegal gum: Impact of high molar mass protein-rich AGPs.

The impact of high molar mass protein-rich arabinogalactan-proteins (AGPs) on emulsifying properties of Acacia senegal gums were studied using reconstituted gums obtained with two distinct fractions: one containing these specific high molar mass AGPs and the other protein-poor low molar mass AGPs. To produce and stabilize limonene emulsions, the experimental design emphasized not only the role of high molar mass protein-rich AGPs, but also the importance of high total concentration. At low protein contents, reconstituted gums required a slightly higher content in high molar mass protein-rich AGPs than original A. senegal gum, that confirmed the role of low molar mass protein-rich AGPs in the adsorption at interfaces. The comparison of the creaming index between original and reconstituted gums as well as the monitoring of instability phenomena by turbiscan up to 30 days clearly demonstrated the prevalent impact of the bulk apparent viscosity in the long-term stability of emulsions.

Improvement of the foamability of sparkling base wines by the addition of Acacia gums.

In sparkling wine, foam characteristics are one of the major attributes. The foam quality depends on wine components. Bentonite is added to the base wine to facilitate the riddling process, but causes a loss of foamability. Acacia gum can be used as additive in wine. We have studied if the addition of Acacia senegal gum (AsenG), Acacia seyal gum (AseyG) and different AsenG fractions could improve the foamability of different base wines treated with bentonite. The foamability differs depending on the gum or the gum fraction treatment but also on the wine, being these differences linked to some aspects of their respective compositions and molecular parameters. AsenG and AseyG increase the foamability (by Mosalux - sparging procedure), respectively, in five and seven out of eight base wines treated with bentonite. Therefore, AsenG and AseyG are potential treatments increasing the foamability of these wines.

Performances of different protocols for exocellular polysaccharides extraction from milk acid gels: Application to yogurt.

Dextran or xanthan were used as model exocellular polysaccharides (EPS) to compare the extraction efficiency of EPS from skim milk acid gels using three different protocols. Extraction yields, residual protein concentrations and the macromolecular properties of extracted EPS were determined. For both model EPS, the highest extraction yield (∼80%) was obtained when samples were heated in acidic conditions at the first step of extraction (Protocol 1). Protocols that contained steps of acid/ethanol precipitation without heating (Protocols 2 and 3) show lower extraction yields (∼55%) but allow a better preservation of the EPS macromolecular properties. Changing the pH of acid gels up to 7 before extraction (Protocol 3) improved the extraction yield of anionic EPS without effect on the macromolecular properties of EPS. Protocol 1 was then applied for the quantification of EPS produced during the yogurt fermentation, while Protocol 3 was dedicated to their macromolecular characterization.

The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 ß-Lactamase.

Metallo-ß-lactamases catalyze the hydrolysis of most ß-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus ß-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (ΔG(0)) of 32 ± 2 kJ·mol(-1) For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ΔG(0) value of 65 ± 1.4 kJ·mol(-1) These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity.

Hsp90 oligomerization process: How can p23 drive the chaperone machineries?

The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer that is able to self-associate in the presence of divalent cations or under heat shock. In a previous work, we focused on the Mg2+-induced oligomerization process of Hsp90, and characterized the oligomers. Combining analytical ultracentrifugation, size-exclusion chromatography coupled to multi-angle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we studied the interaction of p23 with both Hsp90 dimer and oligomers. Even if p23 predominantly binds the Hsp90 dimer, we demonstrated, for the first time, that p23 is also able to interact with Hsp90 oligomers, shifting the Hsp90 dimer-oligomers equilibrium toward dimer. Our results showed that the Hsp90:p23 binding stoichiometry decreases with the Hsp90 oligomerization degree. Therefore, we propose a model in which p23 would act as a "protein wedge" regarding the Hsp90 dimer closure and the Hsp90 oligomerization process.

Hsp90 Oligomers Interacting with the Aha1 Cochaperone: An Outlook for the Hsp90 Chaperone Machineries.

The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer able to self-associate in the presence of divalent cations or under heat shock. This study investigated the relationship between Hsp90 oligomers and the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase). The interactions of Aha1 with Hsp90 dimers and oligomers were evaluated by ultracentrifugation, size-exclusion chromatography coupled to multiangle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Hsp90 dimer was able to bind up to four Aha1 molecules, and Hsp90 oligomers are also able to interact with Aha1. The binding of Aha1 did not interfere with the Hsp90 oligomerization process. Except for Hsp90 dimer, the stoichiometry of the interaction remained constant, at 2 Aha1 molecules per Hsp90 dimer, regardless of the degree of Hsp90 oligomerization. Moreover, Aha1 predominantly bound to Hsp90 oligomers. Thus, the ability of Hsp90 oligomers to bind the Aha1 ATPase activator reinforces their role within the Hsp90 chaperone machineries.

Dynamic and supramolecular organisation of alpha-lactalbumin/lysozyme microspheres: A microscopic study.

Apo alpha-lactalbumin (apo alpha-LA) and lysozyme (LYS), two homologous globular proteins have been shown to be able to interact and self-assemble to form microspheres. We report on the organisation and the mechanism of such protein assembly process using a variety of microscopic techniques. We demonstrated that proteins involved into apo alpha-LA/LYS microspheres exchange with those free in solution. The exchange process takes place from the periphery to the centre of the microspheres. The formed spherical particles observed after fixed incubation time were found to be either individual or aggregated according to the total protein concentration leading to structures with different size and morphology. It appears that protein assembly occurs throughout successive steps of aggregated spherical particles that reorganise into biggest isolated microspheres. Direct microscopic observations over time confirm that microspheres resulted from a reorganisation of aggregated, clustered nanospheres. We propose that the formation of apo alpha-LA/LYS microspheres follows an "aggregation-reorganisation" mechanism.

Molecular interaction between apo or holo alpha-lactalbumin and lysozyme: formation of heterodimers as assessed by fluorescence measurements.

In a previous work, we reported that contrary to native calcium-loaded alpha-lactalbumin (holo alpha-LA), calcium-depleted form (apo alpha-LA) has the ability to self-assemble with lysozyme (LYS) to form different supramolecular structures in temperature-dependent manner. In this study, we examine what happens at molecular scale using fluorescence techniques. Fluorescence anisotropy coupled with fluorescence lifetime measurements provides a means to measure intermolecular interactions. We showed that LYS interacts with both apo alpha-LA and holo alpha-LA to form oligomers, assumed to be heterodimers, at 10 degrees C and 45 degrees C. The dissociation constants for dimerization were found to be in the muM range and increased significantly with increasing ionic strength from 39 to 124 mM. Although the binding constants of holo alpha-LA-LYS and apo alpha-LA-LYS complexes were of the same order of magnitude, the shape or conformation of formed heterodimers differed as assessed by fluorescence parameters in particular correlation time calculations. Such conformation differences could explain why holo alpha-LA-LYS complexes are trapped as heterodimers while the apo alpha-LA-LYS complexes have the ability to further self-assemble into various supramolecular structures.

Apo alpha-lactalbumin and lysozyme are colocalized in their subsequently formed spherical supramolecular assembly.

We have reported previously that the calcium-depleted form of bovine alpha-lactalbumin (apo alpha-LA) interacts with hen egg-white lysozyme (LYS) to form spherical supramolecular structures. These supramolecular structures contain an equimolar ratio of the two proteins. We further explore here the organization of these structures. The spherical morphology and size of the assembled LYS/apo alpha-LA supramolecular structures were demonstrated using confocal scanning laser microscopy and scanning electron microscopy. From confocal scanning laser microscopy experiments with labelled proteins, it was found that LYS and apo alpha-LA were perfectly colocalized and homogeneously distributed throughout the entire three-dimensional structure of the microspheres formed. The spatial colocalization of the two proteins was also confirmed by the occurrence of a fluorescence resonance energy transfer phenomenon between labelled apo alpha-LA and labelled LYS. Polarized light microscopy analysis revealed that the microspheres formed differ from spherulites, a higher order semicrystalline structure. As the molecular mechanism initiating the formation of these microspheres is still unknown, we discuss the potential involvement of a LYS/apo alpha-LA heterodimer as a starting block for such a supramolecular assembly.

Temperature affects the supramolecular structures resulting from alpha-lactalbumin-lysozyme interaction.

The interaction between alpha-lactalbumin and lysozyme, two globular proteins with highly homologous tertiary structures but opposite electric charges, was investigated. As assessed by isothermal titration calorimetry, lysozyme did not bind to the native form of alpha-lactalbumin but did interact with calcium-depleted alpha-lactalbumin (apo alpha-LA). This interaction leads to the formation of different supramolecular structures depending on the temperature. Heterogeneous, amorphous aggregates are formed at 5 degrees C, while droplets, coacervate-like structures, exist at 45 degrees C. The coacervates are formed by equimolar quantities of the two proteins, but their size and number depend on the initial protein molar ratio. These supramolecular structures are found to be stable when the temperature is decreased to 5 degrees C, while prolonged heating at 45 degrees C induces the formation of larger coacervates through a coalescence phenomenon. Surprisingly, interplay occurs between aggregates and coacervates when the temperature is increased from 5 to 45 degrees C. We discuss the results in terms of subtle heat-induced conformational changes in apo alpha-LA. In conclusion, our results show an association between globular proteins that leads to the formation of a variety of supramolecular structures in a temperature-dependent manner and confirm the primordial role of certain alpha-lactalbumin unfolding intermediates in protein-driven assembly.

Organization of human interferon gamma-heparin complexes from solution properties and hydrodynamics.

Heparan sulfate (HS) recognizes a variety of proteins, one of which is the pleiotropic cytokine IFN-gamma, and as such modulates many biological processes. IFN-gamma is a homodimer with a well-defined core and two flexible C-termini that constitute HS binding domains. We show here using molecular modeling that an extended IFN-gamma structure overlaps a HS fragment of 16 disaccharides (16 nm). Since a 21-24-disaccharide HS fragment was experimentally defined as the minimum size that interacts with IFN-gamma [Lortat-Jacob, H., Turnbull, J. E., and Grimaud, J. A. (1995) Biochem. J. 310 (Part 2), 497-505], this raises the question of the complexe organization. We combine analytical ultracentrifugation, size exclusion chromatography, and hydrodynamic bead modeling to characterize the complexes formed in solution with heparin oligosaccharides. For oligosaccharides of 14 and 20 nm, two types of complexes are formed with one IFN-gamma and one or two heparin molecules. Complexes consisting of two IFN-gamma and one or two heparin molecules are present for a fragment of 25 nm and aggregates for a fragment of 35 nm. The complexes are rather compact and can be formed without major conformational changes of the partners. The complex pattern of interaction is related to the size of the partners and their multiple binding possibilities. These various possibilities suggest networks of interactions at the crowded surface of the cells. Hydrodynamic methods used here proved to be very efficient tools for describing protein-HS complexes that, due to the intrinsic heterogeneity and flexibility of the partners, are otherwise very difficult to analyze.