Protein Aggregates May Differ in Water Entrapment but Are Comparable in Water Confinement.

Aggregate size and density are related to gel morphology. In the context of the water distribution in complex food systems, in this study, it was aimed to investigate whether protein aggregates varying in size and density differ in entrapped and confined water. Heat-set soy protein aggregates (1%, v/v) prepared in the presence of 3.5 mM divalent salts increased in size and decreased in apparent density following the salt type order MgSO4, MgCl2, CaSO4, and CaCl2. In the absence of applied (centrifugal) forces, larger and less dense aggregates entrap more water. When force is applied from larger and more deformable aggregates, more water can be displaced. Entrapped water of ∼8-13 g of water/g of protein is associated with (pelleted) aggregates, of which approximately 4.5-8.5 g of water/g of protein is not constrained in exchangeability with the solvent. The amount of confined water within aggregates was found to be independent of the aggregate density and accounted for ∼3.5 g of water/g of protein. Confined water in aggregates is hindered in its diffusion because of physical structure constraints and, therefore, not directly exchangeable with the solvent. These insights in the protein aggregate size and deformability in relation to water entrapment and confinement could be used to tune water holding on larger length scales when force is applied.

Origin of water loss from soy protein gels.

Water holding (WH) of soy protein gels was investigated to identify which length scales are most contributing to WH when centrifugal forces are applied. More specifically, it was attempted to differentiate between the contributions of submicron and supramicron length scales. MgSO4 and MgCl2 salt specificities on soy protein aggregation (submicron contribution) were used to create different gel morphologies (supramicron contribution). Obtained results showed that the micrometer length scale is the most important contribution to WH of gels under the applied deformation forces. WH of soy protein gels correlated negatively with Young's modulus and positively with recoverable energy. The occurrence of rupture events had only a limited impact on WH. The ease by which water may be removed from the gel, but not the total amount, seemed to be related to the initial building block size. These insights could be exploited in product development to predict and tune oral perception properties of (new) products.

Tribological properties of neutral polysaccharide solutions under simulated oral conditions.

Predictability of the perception of foods thickened by polysaccharides is only poor. Therefore, the effect of saliva on the lubrication properties of 2 types of neutral polysaccharides, cross-linked starch and locust bean gum, was studied. Despite the similar bulk rheological behavior of the 2 polysaccharides, the starch solution exhibited a significantly lower friction coefficient. Although starch viscosity was strongly decreased upon 10 s incubation with human saliva, a low friction coefficient was retained. The presence of remaining granules is held partly responsible for this. Addition of starch granules to locust bean gum also resulted in a decrease in the friction coefficient, but the effect was smaller compared to starch solutions digested by saliva. Smaller contact angles were measured for (digested) starch compared to locust bean gum solutions. This points to other parameters that assist in lubrication, such as the interaction of starch solution constituents with the rubbing surfaces. In addition, the importance of bulk viscosity for spreadability on surfaces was demonstrated. This study illustrates that the type of starch will determine not only the viscosity change but also the presence of intact granules upon digestion by saliva in the oral cavity; the combination of these 2 properties is regarded to be responsible for the poor predictability of sensory responses of starch containing foods.

Effects of pH and heat treatments on the structure and solubility of potato proteins in different preparations.

The soluble potato proteins are mainly composed of patatin and protease inhibitors. Using DSC and both far-UV and near-UV CD spectroscopy, it was shown that potato proteins unfold between 55 and 75 degrees C. Increasing the ionic strength from 15 to 200 mM generally caused an increase in denaturation temperature. It was concluded that either the dimeric protein patatin unfolds in its monomeric state or its monomers are loosely associated and unfold independently. Thermal unfolding of the protease inhibitors was correlated with a decrease in protease inhibitor activities and resulted in an ionic strength dependent loss of protein solubility. Potato proteins were soluble at neutral and strongly acidic pH values. The tertiary structure of patatin was irreversibly altered by precipitation at pH 5. At mildly acidic pH the overall potato protein solubility was dependent on ionic strength and the presence of unfolded patatin.

Adsorption properties of proteins at and near the air/water interface from IRRAS spectra of protein solutions.

A detailed study is performed using infrared reflection absorption spectroscopy (IRRAS) to characterize the molecular behaviour of proteins at and near the air/water interface of protein solutions. IRRAS spectra of beta-casein solutions in H2O and D2O show spectral shifts and derivative-like features not commonly observed in monomolecular layer systems. They can be fully understood using optical theory. Fair agreement between experimental and simulated IRRAS spectra over a broad spectral range (4000-1000 cm(-1)) is obtained using a stratified layer model. An attenuated total reflection and transmission spectrum is used to represent the protein extinction coefficient in H2O and D2O, respectively. It is shown that the derivative-like features observed result from the reflective properties of the proteins themselves. Furthermore, both concentration and film thickness could be fitted. At high protein concentrations (100 mg/mL) the spectrum is that of a single homogeneous protein solution. At 0.1 mg/mL, beta-casein is accumulated at the surface in a thin layer of approximately 10 nm thickness, with a concentration about 2500 times higher than in the sub-phase. At an initial concentration of 10 mg/mL, the concentration in the surface layer is about 15 times higher than in the subphase, while the thickness is about 30 nm.

Mild isolation procedure discloses new protein structural properties of beta-lactoglobulin.

To explore the potentially available functional properties of beta-lactoglobulin in, for example, the processing of food products, it is important to isolate the protein by a procedure that avoids all possible denaturing conditions, such as low pH, high ionic strength, or low or elevated temperatures that could cause the protein to undergo irreversible conformational changes. In this work, a mild isolation protocol for beta-lactoglobulin from bovine milk is presented, applicable to semi large-scale isolations (50 to 200 g). The protein could be isolated with a high efficiency (>80%) and a good purity (>98%). Biochemical characterization of the material demonstrated no lactosylation of the protein, nor the formation of irreversibly associated dimers. Also, no proteose peptones could be detected. The ability of beta-lactoglobulin to undergo conformational changes is studied by far and near-ultraviolet circular dichroism and differential scanning calorimetry. A "global" unfolding of the protein is detected around 72 (tertiary level) and 77 degrees C (secondary level). The dimer-monomer dissociation occurring around 52 degrees C could also be monitored at a secondary structural level. Remarkably, a low temperature transition around 30 degrees C was observed, where approximately 10 beta-stranded residues unfold cooperatively, not been reported previously. This low temperature transition is irreversible at temperatures higher than 35 degrees C or upon freezing the material at -20 degrees C. The addition of 20% glycerol could prevent this irreversible conformational change. The effect of the low temperature transition on the protein's functionality remains to be investigated.

On the galactosyl distribution of commercial galactomannans.

A simple method was developed that enabled the enzymatic determination of the galactose distribution in galactomannans. endo-Mannanase of Aspergillus niger was used to degrade the galactomannan polymers and the degradation products were determined with high-performance anion-exchange chromatography. A whole range of commercial high-to-low substituted galactomannans was analyzed in this way. It was found that differences in the anion-exchange chromatograms reflected dissimilarities in the distribution of galactose and could be used directly to discern these dissimilarities. The differences among the various elution profiles were used to construct a similarity distance tree. In addition to this approach, the absolute amount of non-substituted mannose released by the enzyme was found to be a good discriminating factor. In this way, galactomannans with regular, blockwise, and randomly distributed galactose could be discerned. All guars and the highly substituted gum of Prosopis juliflora were found to have a blockwise distribution of galactose. For different batches of tara gum both random and blockwise distributions were found. Among batches of locust bean gum the greatest variation was observed: both random, blockwise, and ordered galactose distributions were present. Cassia gum was found to have a highly regular distribution of galactose.

The helix nucleation site and propensity of the synthetic mitochondrial presequence of ornithine carbamoyltransferase.

This study describes the helix nucleation site and helix propagation of the amphiphilic helical structure of the mitochondrial presequence of rat ornithine carbamoyltransferase. We investigated this property of the 32-residue synthetic presequence using CD and 2D-HR NMR techniques by determining the structure as a function of the concentration of trifluoroethanol. It was found that the hydrophobic cluster Ile7-Leu8-Leu9 forms the helix nucleation site, expanding to include residues Asn4 to Lys16 when the concentration of trifluoroethanol is increased from 10 to 30%. At higher trifluoroethanol concentrations an increased 'stiffening' of the polypeptide backbone (to Arg26) is observed. In addition, by recording CD spectra at different trifluoroethanol concentrations as a function of temperature, it was found that the equilibrium constant between helix and random coil formation for this peptide exhibits a strong temperature dependence with maximum values between 20 and 30 degrees C. Comparison of these equilibrium constants with those of homopolymers stressed the unique character of the mitochondrial presequence. The findings are discussed in relation to the molecular recognition events at different stages of the transport process of this protein into mitochondria.

Soy glycinin: influence of pH and ionic strength on solubility and molecular structure at ambient temperatures.

This study describes the relationship between the solubility of glycinin, a major soy protein, and its structural properties at a quaternary, tertiary, and secondary folding level under conditions representative for food products. When the ionic strength is lowered from 0.5 to 0.2 or 0.03, the basic polypeptides shift more to the exterior of the glycinin complex, as determined at pH 7.6 by labeling solvent-exposed lysines, supported by the study of the proteolytic action of clostripain on glycinin. This structural reorganization caused the pH of minimal solubility to shift to higher values. Ultracentrifugational analysis shows that at pH 7.6 and an ionic strength of 0.5 glycinin forms hexameric complexes (11S), whereas at pH 3.8 and at an ionic strength of 0.03 glycinin exists as trimers (7S). Intermediate situations are obtained by modulation of pH and ionic strength. The observed quaternary dissociation correlates with an increased amount of nonstructured protein at a secondary level and with changes in tertiary folding as determined using circular dichroism. Tryptophan fluorescence shows no significant structural changes for different ionic strengths but demonstrates a more tightly packed fluorophore environment when the pH is lowered from 7.6 to 3.8.

Heat denaturation of soy glycinin: influence of pH and ionic strength on molecular structure.

The 7S/11S glycinin equilibrium as found in Lakemond et al. (J. Agric. Food Chem. 2000, 48, xxxx-xxxx) at ambient temperatures influences heat denaturation. It is found that the 7S form of glycinin denatures at a lower temperature than the 11S form, as demonstrated by a combination of calorimetric (DSC) and circular dichroism (CD) experiments. At pH 7.6, at which glycinin is mainly present in the 11S form, the disulfide bridge linking the acidic and the basic polypeptides is broken during heat denaturation. At pH 3.8, at which glycinin has dissociated partly into the 7S form, and at pH 5.2 this disruption does not take place, as demonstrated by solubility and gel electrophoretic experiments. A larger exposure of the acidic polypeptides (Lakemond et al., 2000) possibly correlates with a higher endothermic transition temperature and with the appearance of an exothermic transition as observed with DSC. Denaturation/aggregation (studied by DSC) and changes in secondary structure (studied by far-UV CD) take place simultaneously. Generally, changes in tertiary structure (studied by near-UV CD) occur at lower temperatures than changes in secondary structure.

The effect of pH on heat denaturation and gel forming properties of soy proteins.

This study is focussed on the influence of pH on the gel forming properties of soy protein isolate and purified glycinin in relation to denaturation and aggregation. At pH 7.6 more fine-stranded gels were formed characterised by low G' values, and a smooth, slightly turbid appearance, whereas at pH 3.8 coarse gels were obtained with a high stiffness and a granulated, white appearance. Low G' values, as found at pH 7.6, correlate with a high solubility of glycinin and soy protein isolate (ca. 50%) after heating at low protein concentration. At pH 3.8 all protein precipitated upon heating, which correlates with relatively high G' values. The role of beta-conglycinin during gelation of SPI seems to be minor at pH 7.6, which is indicated by the fact that, in contrast to pH 3.8, notable gel formation did not start upon heat denaturation of beta-conglycinin. Furthermore, the mechanism of gel formation seems to be affected by pH, because at pH 7.6, in contrast to pH 3.8, the disulphide bridge between the acidic and the basic polypeptide of glycinin is broken upon heating.

Kinetic modeling of the thermal aggregation of patatin.

A kinetic model of the thermal aggregation of patatin is presented based on chromatographic analysis of the proportions of nonaggregated and aggregated patatin. It was observed that the decrease of the amount of nonaggregated patatin proceeded initially quickly and was followed by slower aggregation at longer incubation times. It was shown that this behavior was not due to heterogeneity of the starting material. It was noted that overestimation of the amount of native molecules after a heat treatment, caused by refolding of the unfolded protein during the cooling step prior to the analysis, was significant and could not be neglected. Hence, corrections based on information on the structural properties of patatin were applied. Taking this into account, a model was proposed consisting of a first-order formation of reactive particles, followed by a second-order aggregation reaction. This model described the thermal aggregation of patatin rather accurately and was confirmed by experiments at various protein concentrations.

The adsorption-induced secondary structure of beta-casein and of distinct parts of its sequence in relation to foam and emulsion properties.

Changes in the secondary structure upon adsorption of beta-casein (betaCN) and of distinct parts of its sequence were investigated by far-ultraviolet circular dichroism in order to find suggested relationships with foam and emulsion-forming and -stabilising properties of the same protein/peptides. A teflon/water interface was used as a model system for foam and emulsion interfaces. The maximum surface loads of beta-casein and its derived peptides were investigated. The main secondary structure element of all samples in solution was the unordered random coil, but upon adsorption ordered structure, especially alpha-helix, was induced. At lower pH more ordered structure was induced, just as at lower ionic strength. Apparently, both hydrophobic and hydrophilic groups influence the change of secondary structure induced at a hydrophobic interface. The results suggest that the hydrophobic C-terminal half of betaCN accounted for the high maximum surface load on teflon, while the N-terminal half of betaCN seemed to be responsible for the secondary structure induction upon adsorption. A relation between the maximum surface load and the foam-stabilising properties was found, but an influence of the secondary structure properties on the foam and emulsion-forming and -stabilising properties was not observed.

Heat-induced conformational changes of Ara h 1, a major peanut allergen, do not affect its allergenic properties.

Ara h 1, a major peanut allergen was isolated, and its structure on secondary, tertiary, and quaternary level at ambient temperature was investigated using spectroscopic and biochemical techniques. Ara h 1 appeared to be a highly structured protein on a secondary level, possesses a clear tertiary fold, and is present as a trimeric complex. Heat treatment of purified Ara h 1 results in an endothermic, irreversible transition between 80 and 90 degreesC, leading to an increase in beta-structures and a concomitant aggregation of the protein. Ara h 1 from peanuts that were heat-treated prior to the purification procedure exhibited a similar denatured state with an increased secondary folding and a decreased solubility. The effect of heat treatment on the in vitro allergenic properties of Ara h 1 was investigated by means of a fluid-phase IgE binding assay using serum from patients with a clinically proven peanut allergy. Ara h 1 purified from peanuts heated at different temperatures exhibited IgE binding properties similar to those found for native Ara h 1, indicating that the allergenicity of Ara h 1 is heat-stable. We conclude that the allergenicity of Ara h 1 is unaffected by heating, although native Ara h 1 undergoes a significant heat-induced denaturation on a molecular level, indicating that the recognition of conformational epitopes of Ara h 1 by IgE either is not a dominant mechanism or is restricted to parts of the protein that are not sensitive to heat denaturation.

Heat-induced conformational changes of patatin, the major potato tuber protein.

This paper presents a first structural characterization of isolated patatin, the major potato tuber protein, at ambient and elevated temperatures. Isolated patatin at room temperature is a highly structured molecule at both secondary and tertiary levels. It is estimated from far-ultraviolet circular dichroism data that about 33% of the residues adopts an alpha-helical and 46% a beta-stranded structure. Patatin is thermally destabilized at temperatures exceeding 28 degrees C, as was indicated by near-ultraviolet circular dichroism. It was shown that parts of the alpha-helical contributions unfold in the 45-55 degrees C region, whereas the beta-stranded parts unfold more gradually at temperatures of 50-90 degrees C. This was confirmed with Fourier-transform infrared spectroscopy. Differential scanning calorimetry indicated a cooperative transition between 50-60 degrees C, most likely reflecting the unfolding of alpha-helical parts of the molecule. Furthermore, fluorescence spectroscopy confirmed a global unfolding of the protein between 45-55 degrees C. The observed unfolding of the protein coincides with the inactivation of the patatin enzyme activity and with the precipitation as occurs in the potato fruit juice upon heating. At high temperatures, patatin still contains some helical and stranded structures. Upon cooling the protein partly refolds, it was observed that mainly alpha-helical structures were formed.

Comparison of the conformational state and in vitro refolding of yeast chaperonin protein cpn10 with bacterial GroES.

Gel filtration studies demonstrate that the heptameric complex of yeast cpn10 (pI around 8.8) reversibly disassembles into monomers when lowering the pH to 4.5, whereas its secondary structure is retained as demonstrated by circular dichroism. Monomeric yeast cpn10 does not bind to GroEL in the presence of nucleotides, whereas under identical conditions E. coli cpn10 (GroES), having a strong sequence homology to the yeast form but a pI of 5.2, shows no pH-dependent dissociation and is able to complex with GroEL at both pH 7.5 and 4.5. Using circular dichroism it is shown that, unlike E. coli cpn10, yeast cpn10 is not able to refold spontaneously after first being fully unfolded in 8 M urea. However, refolding of yeast cpn10 to a complex that can be recognised by GroEL depends on the presence of a lipid-water interface with a specificity for negatively charged lipids. We suggest that the requirements for refolding of yeast cpn10 are related to its post-translational transport and subcellular localization.

Monitoring structural stability of trypsin inhibitor at the submolecular level by amide-proton exchange using Fourier transform infrared spectroscopy: a test case for more general application.

Combining the information on the secondary structure content as present in the shape of a protein amide I infrared band with the approach of monitoring amide-proton exchange using infrared spectroscopy, we have been able to investigate the structural stability of different components present in a protein, which are shown to be correlated to the different classes of secondary structures. For this purpose, the changes in intensity in different regions of the amide I have been detected upon exposure of the protein to a 2H2O environment, revealing four separate classes of exchanging components. As a test case for the approach described in this work, the amide-proton exchange of hydrated protein films of bovine pancreatic trypsin inhibitor has been studied using infrared spectroscopy, and is compared to literature data obtained by other techniques. A slow amide-proton exchange is observed for a class correlated to the beta-strands present in the protein, with protection of amide-protons for more than 19 h. Another class, which has been assigned to mainly helical residues, shows much less protection from exchange. The distribution function of the exchange rates of a class linked to the beta-turns displays five times faster exchange rates compared to those found for the majority of the helical residues, but they are still ten times slower compared to a class which we defined to represent the nonstructured parts of the protein.

Amide-proton exchange of water-soluble proteins of different structural classes studied at the submolecular level by infrared spectroscopy.

For eleven films of various water-soluble alpha-, beta-, alpha-/beta-, and alpha-+beta-proteins, the amide-proton exchange, initiated by exposure of the protein film to 2H2O, has been monitored using infrared spectroscopy. The approach to obtain the kinetics of exchange for four different classes of amide protons, correlating to the different secondary structure types, has been described in detail in the preceding paper. In this work the more general applicability of the approach is illustrated by testing it for different types of proteins. The results obtained are shown not only to be comparable to reported time-resolved nuclear magnetic resonance data (as in the case of myoglobin, phospholipase A2, lysozyme, and cytochrome c), or to the more qualitative data obtained by neutron diffraction (trypsin, ribonuclease S, papain, and subtilisin BPN'), but the infrared approach us also provides with quantitative detailed insight on the distribution of exchange rate constants at the submolecular level of proteins, too complex to be studied by other techniques, as for tetrameric hemoglobin, and of proteins in which exchange is too fast to be detected by these other techniques, as is shown in this work for alpha-casein and apocytochrome c.

The different molar absorptivities of the secondary structure types in the amide I region: an attenuated total reflection infrared study on globular proteins.

Differences in molar absorptivity of the various secondary structures in the amide I region of infrared protein spectra would have a great impact on the interpretation of the data published thus far on protein films studied by attenuated total reflection infrared spectroscopy. In this work, representative values for amide I absorptivities are obtained for 15 different films of globular proteins spread from H2O solutions. The observed intensities are corrected for variations in film thickness and for contributions of hydration water, atmospheric water, and side chains. These absorptivities, together with the reported secondary structure of the proteins investigated, are used to deduce the molar absorptivities of the individual secondary structure types. It is found that the molar absorptivity of beta-strands is 1.4-1.6 times larger than that of alpha-helices, which in turn is 1.3-2.1 times larger than those found for beta-turns or random coiled structures. The implications of our findings for spectral analysis currently used in literature are discussed.

PhoE signal peptide inserts into micelles as a dynamic helix-break-helix structure, which is modulated by the environment. A two-dimensional 1H NMR study.

Proteins that are destined for export out of the cytoplasm of Escherichia coli cells are synthesized as precursor proteins with N-terminal extensions or signal sequences, which are essential for translocation of the protein across the inner membrane. Signal sequences contain very little primary sequence homology, and therefore recognition of these sequences is thought to involve specific folding. To assess the conformational flexibility of signal sequences, we have studied the signal peptide of PhoE (MKKSTLALVVMGIVASASVQA) by two-dimensional nuclear magnetic resonance and circular dichroism in different membrane mimetic environments. The secondary structure of the PhoE signal peptide was analyzed via interresidue nuclear Overhauser enhancement measurements, chemical shifts of backbone protons, and by measuring amide proton exchange. The membrane mimetic environments studied were trifluoroethanol (TFE) and micelles of sodium dodecyl sulfate (SDS) or dodecylphosphocholine (DPC). In all systems alpha-helix formation was observed. In TFE, the alpha-helix stretches from the positively charged N-terminus to Ser18. In SDS and DPC micelles, the N- and C-terminal alpha-helical half are separated from each other by a kink at the Gly12 position, with the helical content being higher at the N-terminus and lower at the C-terminus. In zwitterionic DPC micelles, the C-terminal region has a less regular or more flexible structure compared to SDS. The insertion of the PhoE signal peptide into the hydrophobic environment of the micelles was demonstrated by the effect of spin-labeled 12-doxylstearate on the line widths of the peptide proton resonances.(ABSTRACT TRUNCATED AT 250 WORDS)