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PubMed1997

Identification of a conserved hydrophobic cluster in partially folded bovine beta-lactoglobulin at pH 2.

BACKGROUND: NMR studies of denatured states, both fully unfolded and partially folded, give insight into the conformations and interactions formed during folding. Although the complete structural characterization of partially folded proteins is a very difficult task, the identification of structured subsets, such as hydrophobic clusters, is of value in understanding the structural organization of such states. Here, we report the NMR characterization, in acidic conditions (pH 2), of a well-defined hydrophobic cluster localized in the core of bovine beta-lactoglobulin. RESULTS: The existence of a small hydrophobic cluster present in the lipocalin protein family has been assessed on the basis of structural alignment and NRM data obtained for the partially folded bovine beta-lactoglobulin. The presence of the cluster had been predicted identifying those residues that are highly conserved in most members of the family. An NMR study conducted at pH 2, where the protein exhibits a very stable beta-core together with disordered regions, reveals the presence of NOEs among sidechains of 11 hydrophobic residues centered around Trp19 and pointing towards the interior of the protein. This buried cluster is found to be unusually stable at pH 2, not only at room temperature but also at 323K. Furthermore, conserved hydrophobic residues pointing towards the surface of the protein define a hydrophobic surface patch located in a groove between the strands and the helix. CONCLUSIONS: The detected buried cluster most likely plays an important role in bovine beta-lactoglobulin stability. The analysis of five structurally related proteins reveals that the same extended cluster is present in these structures. We propose that the buried cluster may represent the internal binding site as well and that the hydrophobic surface patch is involved in a second external binding site.

Algorithms↗
PubMed2000

Effect of partial hydrolysis with an immobilized proteinase on thermal gelation properties of beta-lactoglobulin B.

We have investigated the influence of partial hydrolysis with an immobilized proteinase from Bacillus licheniformis on the thermal gelation of isolated beta-lactoglobulin B. Gelation behaviour was determined by dynamic rheological measurements (small deformation) and the gels were characterized with respect to microstructure and water-holding properties. A fine-stranded gel with a complex modulus of approximately 2000 Pa was formed from beta-lactoglobulin (50 g/l in 75 mM-Tris-HCl, pH 7.5). Limited hydrolysis prior to thermal gelation resulted in coarser gels with thicker protein strands and larger pores. Gel structure correlated with its permeability, proton mobility and water-holding capacity. Total stiffness gel increased with low degrees of hydrolysis, but decreased after prolonged hydrolysis. Maximal gel stiffness was 1.5-fold that gels made from of unhydrolysed beta-lactoglobulin. This was much lower than the stiffening effect obtained after partial hydrolysis of whey protein isolate, showing that the gel strengthening effect of partial hydrolysis was depedent on the protein composition and/or the hydrolysis and gelatin conditions. A mechanism to explain the observed effects of hydrolysis on gelation and gel properties is presented.

Bacillus↗
PubMed2001

Effect of beta-lactoglobulin and precipitation of calcium phosphate on the thermal coagulation of milk.

The effect of beta-lactoglobulin and heat-induced precipitation of calcium phosphate on the pH dependence and mechanism of thermal coagulation of milk throughout the pH range 6.3-7.3 was studied using serum protein-free milk and sodium caseinate as models for micellar and non-micellar milk protein systems respectively. It appears that the specific effect of beta-lactoglobulin at the pH of maximum stability may be related to its ability to chelate calcium. The effect of beta-lactoglobulin at the pH of minimum stability does not appear to be directly related to heat-induced dissociation of K-casein or micellar integrity but may be due to its ability to sensitize casein micelles to heat-induced precipitation of calcium phosphate, by increasing micellar hydrophobicity. The extent of heat-induced precipitation of calcium phosphate, as a function of pH, is an inverse reflection of the pH dependence of heat stability. Micellar integrity appears to play a critical role in the heat stability of milk but for reasons not previously appreciated.

Animals↗
PubMed2001

Inactivation kinetics of alkaline phosphatase and lactoperoxidase, and denaturation kinetics of beta-lactoglobulin in raw milk under isothermal and dynamic temperature conditions.

A detailed kinetic study of alkaline phosphatase, lactoperoxidase and beta-lactoglobulin was carried out in the context of identifying intrinsic time-temperature indicators for controlling the heat processing of milk. The heat inactivation or denaturation of alkaline phosphatase, lactoperoxidase and beta-lactoglobulin under isothermal conditions was found to follow first order kinetics. Experimental results were analysed using both a two step linear regression and a one step non-linear regression method. Results obtained using the two statistical techniques were comparable, but the 95% confidence interval for the predicted values was smaller when the one step non-linear regression method was used, indicating its superiority for estimating kinetic parameters. Thermal inactivation of alkaline phosphatase and lactoperoxidase was characterized by z values of 5.3 deg C (D60 degrees C = 24.6 min) and 4.3 deg C (D71 degrees C = 38.6 min) respectively. For the denaturation of beta-lactoglobulin we found z values of 7.9 deg C (D7.5 degrees C = 49.9 min) in the temperature range 70-80 degrees C and 24.2 deg C (D85 degrees C = 3.53 min) in the range 83-95 degrees C. Dref and z were evaluated under dynamic temperature conditions. To estimate the statistical accuracy of the parameters, 90% joint confidence regions were constructed.

Alkaline Phosphatase↗
PubMed1992

Heat-related changes to the hydrophobicity of cheese whey correlate with levels of native beta-lactoglobulin and alpha-lactalbumin.

Correlations were identified between levels of the native whey proteins, beta-lactoglobulin and alpha-lactalbumin and the surface and total hydrophobicities of cheese whey in response to different heat treatments. Heat-induced changes in the native beta-lactoglobulin content and surface hydrophobicity of whey exhibited the most significant linear relationship while correlations between total hydrophobicity and the native proteins were less significant because of an atypical rise in the n-heptane-binding capacity of whey after high-temperature treatment. The content of native beta-lactoglobulin in whey was more sensitive to heating than the content of native alpha-lactalbumin, while heat-related changes in the total hydrophobicity of whey were generally greater than similar changes in surface hydrophobicity.

Cheese↗
PubMed1994

Hormonal induction of alpha-lactalbumin and beta-lactoglobulin in cultured mammary explants from pregnant pigs.

Mammary tissue from pigs on days 60, 80, 90, 100 and 100+ (days 106-111) of pregnancy has been cultured in vitro as explants. The total accumulation in tissue and culture medium of the whey proteins alpha-lactalbumin and beta-lactoglobulin has been measured using specific radioimmunoassays. The control, uncultured tissue showed progressive morphological development from sparse, non-secretory epithelial tissue on day 60 to full lobulo-alveolar development with some accumulated secretion from day 100. In uncultured explants beta-lactoglobulin could be detected consistently from day 90 (13 +/- 12 ng/micrograms DNA, n = 4) and alpha-lactalbumin from day 100 (1.3 +/- 0.5 ng/micrograms DNA, n = 11). At all stages of pregnancy, both whey proteins increased markedly during the period of culture (up to 7 d). Stimulation of alpha-lactalbumin appeared to be primarily under prolactin control. Prolactin increased alpha-lactalbumin accumulation to a similar extent alone, or in the presence of insulin and/or corticosterone. The response to prolactin was dose-dependent over the range 0.4-20 nM (10-500 ng/ml). Porcine prolactin was more potent than ovine prolactin. There was no effect of porcine growth hormone and no synergism detected between prolactin and tri-iodothyronine. By contrast, no specific hormonal requirements were established for accumulation of beta-lactoglobulin, which appeared to increase in vitro if tissue remained viable in various combinations of insulin, corticosterone and prolactin. It was not stimulated by growth hormone. There was some indication of a prolactin-sensitive component in longer term cultures after day 4.

Animals↗
PubMed1995

Hormone-dependent expression of the ovine beta-lactoglobulin gene.

The minimal hormonal requirements for inducing the ovine beta-lactoglobulin gene have been investigated using mammary gland explants from ewes in the first half of pregnancy. Quantification of beta-lactoglobulin mRNA showed that a combination of insulin, cortisol and prolactin was required to stimulate the expression of the gene and that this response could not be enhanced by the addition of oestrogen and thyroid hormone to the culture medium. Explants cultured in the presence of insulin, cortisol and prolactin also demonstrated the capacity to synthesize the protein. Progesterone did not inhibit the induction of the gene, which is consistent with the increase in beta-lactoglobulin mRNA observed in vivo in the mammary gland during the final 2 months of pregnancy when the circulating level of progesterone is elevated.

Animals↗
PubMed2005

Polymorphisms in the goat beta-lactoglobulin gene.

Beta-lactoglobulin polymorphisms have been reported in the milk of different goat breeds, although no genetic variants affecting the protein have been characterized. In the present study, we amplified and sequenced the proximal promoter and the first six exons containing the entire coding region for the beta-lactoglobulin gene in eleven goat breeds from Spain, France, Italy, Switzerland, Senegal and Asia to identify genetic variants. Fifteen polymorphisms were detected, nine in the promoter region and six in the exons of the beta-lactoglobulin gene. All polymorphisms were single nucleotide substitutions with the exception of one deletion/insertion in the promoter region. The polymorphisms in the coding region did not produce any amino acid change. In addition, pyrosequencing technology was used to genotype four polymorphisms in the promoter region in 200 goats belonging to eleven breeds. Differences in allelic frequencies for these polymorphisms between breeds are described and a specific polymorphism for the Italian populations was identified. Finally, the analysis of association between these four promoter point mutations was investigated resulting in five haplotypes, GCGC being the most frequent haplotype in all breeds analysed.

Alleles↗
PubMed1989

Protein aggregation in high-performance liquid chromatography: hydrophobic interaction chromatography of beta-lactoglobulin A.

Aggregation of beta-lactoglobulin A under acidic buffer conditions was studied in hydrophobic interaction chromatography. At high ammonium sulfate concentrations, pH 4.5 and 4 degrees C, UV chromatograms revealed a maximum of three peaks for beta-lactoglobulin A concentrations greater than 5 mg/mL, suggesting three distinct aggregate species. The size of the smallest aggregate (tetramer) and its stoichiometric relationship to the other two aggregates (octamer and dodecamer) were determined from the chromatographic data and a simple mass balance model. These stoichiometries agreed with those determined in a separate study by on-line low-angle laser light scattering. In addition, the association constants describing the formation of octamer from two tetramer molecules and the formation of dodecamer from the octameric and tetrameric species were found to be (2.4 +/- 0.5) X 10(4) M-1 and (3.3 +/- 0.8) X 10(3) M-1, respectively. Analysis of the beta-lactoglobulin A system is based on a model in which aggregates form in solution upon injection before adsorbing to the column matrix. The column retains those species formed in solution and induces little change in the relative amounts of each species. These results illustrate another example by which multiple peaks can arise in high-performance liquid chromatography, beyond the previously described studies of protein conformational changes during chromatography.

Chromatography, High Pressure Liquid↗
PubMed2000

Structural changes accompanying pH-induced dissociation of the beta-lactoglobulin dimer.

We have used NMR spectroscopy to determine the three-dimensional (3D) structure, and to characterize the backbone dynamics, of a recombinant version of bovine beta-lactoglobulin (variant A) at pH 2. 6, where the protein is a monomer. The structure of this low-pH form of beta-lactoglobulin is very similar to that of a subunit within the dimer at pH 6.2. The root-mean-square deviation from the pH 6.2 (crystal) structure, calculated for backbone atoms of residues 6-160, is approximately 1.3 A. Differences arise from the orientation, with respect to the calyx, of the A-B and C-D loops, and of the flanking three-turn alpha-helix. The hydrophobic cavity within the calyx is retained at low pH. The E-F loop (residues 85-90), which moves to occlude the opening of the cavity over the pH range 7.2-6.2, is in the "closed" position at pH 2.6, and the side chain of Glu89 is buried. We also carried out measurements of (15)N T(1)s and T(2)s and (1)H-(15)N heteronuclear NOEs at pH 2.6 and 37 degrees C. Although the residues of the E-F loop (residues 86-89) have the highest crystallographic B-factors, the conformation of this loop is reasonably well defined by the NMR data, and its backbone is not especially mobile on the pico- to nanosecond time scale. Several residues (Ser21, Lys60, Ala67, Leu87, and Glu112) exhibit large ratios of T(1) to T(2), consistent with conformational exchange on a micro- to millisecond time scale. The positions of these residues in the 3D structure of beta-lactoglobulin are consistent with a role in modulating access to the hydrophobic cavity.

Animals↗
PubMedDate not supplied

Multiple steps during the formation of beta-lactoglobulin fibrils.

In this study, the heat induced fibrilar aggregation of the whey protein beta-lactoglobulin is investigated at low pH and at low ionic strength. Under these circumstances, tapping mode atomic force microscopy results indicate that the fibrils formed have a periodic structure with a period of about 25 nm and a thickness of one or two protein monomers. Fibril formation is followed in situ using light scattering and proton NMR techniques. The dynamic light scattering results show that the fibrils that form after short heating periods (up to a few hours) disintegrate upon slow cooling, whereas fibrils that form during long heating periods do not disintegrate upon subsequent slow cooling. The NMR results show that even after prolonged heating an appreciable fraction of the protein molecules is incorporated into fibrils only when the beta-lactoglobulin concentration is above approximately 2.5 wt %. The data imply multiple steps during the heat induced formation of beta-lactoglobulin fibrils at low pH and at low ionic strength: (partly) denatured protein monomers are either incorporated into fibrils or form instead a low molecular weight complex that is incapable of forming fibrils. Fibril formation itself also involves (at least) two steps: the reversible formation of linear aggregates, followed by a slow process of "consolidation" after which the fibrils no longer disintegrate upon slow cooling.

Animals↗
PubMedDate not supplied

Fibrillar beta-lactoglobulin gels: Part 2. Dynamic mechanical characterization of heat-set systems.

Oscillatory shear rheometry (mechanical spectroscopy) has been used to study the heat-set gelation of beta-lactoglobulin at pH 2. Modulus-concentration relationships were obtained by extrapolating cure data to infinite time. In terms of theory, these fail to provide a clear distinction between the fractal description of biopolymer gels and the classical random f-functional polycondensation branching theory (cascade) approach, though the latter is preferred. Critical exponents for the sol-gel transition, derived from these data, are also discussed. Where gel time-concentration results are concerned the fractal model makes no predictions, and the cascade approach in its simplest form must be rejected in favor of a more sophisticated version involving delivery of fibrils by nucleation and growth into the random aggregation process. Over the limited concentration range accessed experimentally, cure data for the different beta-lactoglobulin solutions, reduced to the universal form G'/G'inf versus t/tgel, superimposed well for samples heated both at 80 and 75 degrees C and for different batches of protein. Studies of the frequency responses of the fully cured gels confirm the validity of the gel description given to these materials, and a study of the temperature dependence of the frequency spectrum suggests a fall in the elastic component of the modulus as temperature decreases. This contrasts with what has been found for other heat-set globular protein gels such as those from serum albumin where the gel modulus increases at lower temperatures. The present results are in good agreement with more limited amounts of pH 2 beta-lactoglobulin data published earlier, though some differences arise through a previous neglect of measurement "dead time".

Amyloid↗
PubMed2002

Nuclear magnetic resonance spectroscopic study of beta-lactoglobulin interactions with two flavor compounds, gamma-decalactone and beta-ionone.

Interactions between a well-characterized protein, beta-lactoglobulin, and two flavor compounds, beta-ionone and gamma-decalactone, were studied by 2D NMR spectroscopy. NMR spectra were recorded in aqueous solution (pH 2.0, 12 mM NaCl, 10% D(2)O) under conditions such that beta-lactoglobulin is present in a monomeric state. TOCSY and NOESY spectra were recorded on the protein and the complexes between protein and ligands. The spectra of the NH-CH(alpha) region showed the cross-signals due to the coupling between N- and C-bonded protons in the polypeptide backbone. The observed chemical shift variations in the presence of ligands can be assigned to changes in the protein conformation. It appears that the side chains of several amino acids are affected by binding of gamma-decalactone point into the central cavity (Leu46, Ile56, Met107, and Gln120), whereas binding of beta-ionone affects amino acids located in a groove near the outer surface of the protein (Leu104, Tyr120, and Asp129), as illustrated by molecular visualization. This NMR study provides precise information of the location of binding and confirms the existence of two different binding sites for aroma compounds on beta-lactoglobulin, which was suggested in previous competition studies by fluorometry or affinity chromatography and by structural information obtained from infrared spectroscopy.

Binding Sites↗
PubMed1999

Formation of stable covalent dimer explains the high solubility at pH 4.6 of lactose-beta-lactoglobulin conjugates heated near neutral pH.

The solubility of lactose-beta-lactoglobulin conjugates at pH 4.6, after heating near neutral pH in phosphate buffer/0.116 M NaCI, was investigated by size exclusion chromatography and compared with unmodified protein. Heated conjugates in the temperature range 65-90 degrees C showed greater solubility at pH 4.6. The proportion of soluble protein increased with the number of bound lactose molecules. Total solubility was obtained for conjugates with nine lactose residues attached per monomer of beta-lactoglobulin. The protective effect of bound sugar toward precipitation was associated with the formation of soluble disulfide cross-linked dimers, highly accessible to trypsin digestion. These results suggested that bound lactose, through steric hindrance and high surface hydrophilicity, prevents the thiol-disulfide exchange reactions of the polymerization-aggregation process of lactose-beta-lactoglobulin conjugates.

Chromatography, Gel↗
PubMed2000

Interactions between methyl ketones and beta-lactoglobulin: sensory analysis, headspace analysis, and mathematical modeling.

Interaction of flavor compounds with proteins is known to have an influence on the release of flavor from food. Hydrophobic interactions were found between beta-lactoglobulin and methyl ketones; the affinity constant increases by increasing the hydrophobic chain. Addition of beta-lactoglobulin (0.5 and 1%) to aroma solutions (12.5, 50, and 100 microL L(-)(1)) of three methyl ketones induces a significant decrease in odor intensity. The chosen methyl ketones were 2-heptanone (K(b) = 330), 2-octanone (K(b) = 950), and 2-nonanone (K(b) = 2440). The release of these flavor compounds (50 microL L(-)(1)) was studied by static headspace in water solution (50 mM NaCl, pH 3) with different concentrations of beta-lactoglobulin (0, 0.5, 1, 2, 3, and 4%). Increasing the concentration of protein increases the retention of volatiles, and this effect is greatest for 2-nonanone, the compound with the highest affinity constant, and lowest for 2-heptanone. A mathematical model previously developed to describe flavor release from aqueous solutions containing flavor-binding polymers (Harrison, M.; Hills, B. P. J. Agric. Food Chem. 1997, 45, 1883-1890) was used to interpret the data. The model assumes that the polymer-flavor interaction is reversible and the rate-limiting step for release is the transfer of volatiles across the macroscopic gas-liquid interface. This model was used to predict the equilibrium partitioning properties and the rate of release of the three methyl ketones. Increasing the affinity constant leads to decreased release rates and a lower final headspace aroma concentration.

Ketones↗
PubMed2006

Penetration of beta-lactoglobulin into monoglyceride monolayers. dynamics, interactions, and topography of mixed films.

In this work we have analyzed the penetration of betalactoglobulin into a monoglyceride monolayer (monopalmitin or monoolein) spread at the air-water interface and its effects on the structural, dilatational, and topographical characteristics of mixed films. Dynamic tensiometry, surface film balance, Brewster angle microscopy (BAM), and surface dilatational rheology have been used, maintaining the temperature constant at 20 degrees C and the pH and ionic strength at 7 and 0.05 M, respectively. The initial surface pressure (mN/m) of the spread monoglyceride monolayer (pii(MONOGLYCERIDE)) at 10, 20, and the collapse point is the variable studied. Beta-lactoglobulin can penetrate into a spread monoglyceride monolayer at every surface pressure. The penetration of beta-lactoglobulin into the monoglyceride monolayer with a more condensed structure, at the collapse point of the monoglyceride, requires monoglyceride molecular loss by collapse and/or desorption. However, the structural, topographical, and dilatational characteristics of monoglyceride penetrated by beta-lactoglobulin mixed monolayers are essentially dominated by the presence of monoglyceride (either monopalmitin or monoolein) in the mixed film. In fact, monoglyceride molecules have the capacity to re-enter the monolayer after expansion and recompression of the mixed monolayer. Thus, monoglyceride molecular loss by collapse and/or desorption is reversible. The topography of the monolayer under dynamic conditions corroborates these conclusions.

Air↗
PubMed2005

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

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

Adsorption↗
PubMed2005

Effect of time on the interfacial and foaming properties of beta-lactoglobulin/acacia gum electrostatic complexes and coacervates at pH 4.2.

The electrostatic complexation between beta-lactoglobulin and acacia gum was investigated at pH 4.2 and 25 degrees C. The binding isotherm revealed a spontaneous exothermic reaction, leading to a DeltaHobs = -2108 kJ mol(-1) and a saturation protein to polysaccharide weight mixing ratio of 2:1. Soluble electrostatic complexes formed in these conditions were characterized by a hydrodynamic diameter of 119 +/- 0.6 nm and a polydispersity index of 0.097. The effect of time on the interfacial and foaming properties of these soluble complexes was investigated at a concentration of 0.1 wt % at two different times after mixing (4 min, referred as t approximately 0 h and t = 24 h). At t approximately 0 h, the mixture is mainly made of aggregating soluble electrostatic complexes, whereas after 24 h these complexes have already insolubilize to form liquid coacervates. The surface elasticity, viscosity and phase angle obtained at low frequency (0.01 Hz) using oscillating bubble tensiometry revealed higher fluidity and less rigidity in the film formed at t approximately 0 h. This observation was confirmed by diminishing bubble experiments coupled with microscopy of the thin film. It was thicker, more homogeneous and contained more water at t approximately 0 h as compared to t = 24 h (thinner film, less water). This led to very different gas permeability's of Kt approximately 0 h = 0.021 cm s(-1) and Kt=24 h) = 0.449 cm s(-1), respectively. Aqueous foams produced with the beta-lactoglobulin/acacia gum electrostatic complexes or coacervates exhibited very different stability. The former (t approximately 0 h) had a stable volume, combining low drainage rate and mainly air bubble disproportionation as the destabilization mechanism. By contrast, using coacervates aged for 24 h, the foam was significantly less stable, combining fast liquid drainage and air bubble destabilization though fast gas diffusion followed by film rupture and bubble coalescence. The strong effect of time on the air/water interfacial properties of the beta-lactoglobulin/acacia gum electrostatic complexes can be understood by their reorganization at the interface to form a coacervate phase that is more fluid/viscous at t approximately 0 h vs rigid/elastic at t = 24 h.

Air↗