Multi-state thermal unfolding and aggregation of β-lactoglobulin A

Qi, Xiao Lin; Brownlow, Sharon; Holt, Carl; Sellers, Peter

doi:10.1042/bst023074s

Cited by 6 publications

(4 citation statements)

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“…22,23 BLG is a globular protein widely analyzed for investigation of heatinduced aggregation. [24][25][26] The thermal denaturation process of BLG was described as a multistep mechanism and highly dependent on the protein concentration and pH. [27][28][29] At neutral and alkaline pH values and physiological concentrations (<5 wt %), the dissociation of the dimer is coupled with a conformational transition to an R-type state, around 40-55 °C.…”

Section: Introductionmentioning

confidence: 99%

“…In contrast, β-lactoglobulin (BLG) is mainly composed of β-sheets (54%) and α-helices (17%) . At native pH, this protein of 162 residues is found in a dimeric conformation, while, at acidic pH, it dissociates into monomers, recognized as more stable than the native state. , BLG is a globular protein widely analyzed for investigation of heat-induced aggregation. − The thermal denaturation process of BLG was described as a multistep mechanism and highly dependent on the protein concentration and pH. − At neutral and alkaline pH values and physiological concentrations (<5 wt %), the dissociation of the dimer is coupled with a conformational transition to an R-type state, around 40−55 °C. The native (N) to the R-state transition was associated with a single anomalous carboxyl group which appears to be buried in the hydrophobic interior of the protein in conformation N, and becomes exposed to the surface in conformation R .…”

Section: Introductionmentioning

confidence: 99%

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Thermal Denaturation of Beta-Lactoglobulin and Stabilization Mechanism by Trehalose Analyzed from Raman Spectroscopy Investigations

et al. 2010

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The thermal denaturation process of beta-lactoglobulin has been analyzed in the 20-100 degrees C temperature range by Raman spectroscopy experiments simultaneously performed in the region of amide modes (800-1800 cm(-1)) and in the low-frequency range (10-350 cm(-1)). The analysis of amide modes reveals a two-step thermal denaturation process in the investigated temperature range. The first step corresponds to the dissociation of dimers associated with an increase of flexibility of the tertiary structure. In the second step, large conformational changes are detected in the secondary structure and described as a loss of alpha-helix structures and a concomitant formation of beta-sheets. Raman investigations in the low-frequency range provide important information on the origin of the denaturation process through the analysis of the solvent dynamics and its coupling with that of the protein. The softening of the tetrahedral structure of water induces the dissociation of dimers and makes the tertiary structure softer, leading to the water penetration in the protein interior. The methodology based on Raman investigations of amide modes and in the low-frequency region was used to analyze the mechanism of beta-lactoglobulin thermostabilization by trehalose. The main effect of trehalose is determined to be related to its capabilities to distort the tetrahedral organization of water molecules.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Thermal Denaturation of Beta-Lactoglobulin and Stabilization Mechanism by Trehalose Analyzed from Raman Spectroscopy Investigations

et al. 2010

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“…β-LG represents approximately 50% of the whey proteins and is a small globular water-soluble protein consisting of 162 amino acids. The molecular structure and functional properties of β-LG are the most studied among the whey proteins . Particularly, a lot of data on TAG oil emulsions stabilized by β-LG have been accumulated.…”

Section: Introductionmentioning

confidence: 99%

Adsorption and Structural Change of β-Lactoglobulin at the Diacylglycerol−Water Interface

et al. 2008

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Diacylglycerol (DAG)/water and triacylglycerol (TAG)/water emulsions were prepared using beta-lactoglobulin (beta-LG) as an emulsifier. The oil phase (20% in emulsion) was mixed with beta-LG solution (1% beta-LG in water, pH 7) to prepare the emulsions. A fine oil-in-water emulsion was produced from both DAG and TAG oils. The interfacial protein concentration of the TAG emulsion was higher than that of the DAG emulsion. The zeta potential of the DAG oil droplet was higher than that of the TAG oil droplet. The front-surface fluorescence spectroscopy results revealed that tryptophan residues in beta-LG moved to the more hydrophobic environment during the adsorption of protein on the oil droplet surfaces. Changes in secondary structure of beta-LG during the adsorption were determined by FT-IR spectroscopy. Decreases in the beta-sheet content concomitant with increases in the alpha-helix content were observed during the adsorption to the oil droplets, and the degree of structural change was greater for beta-LG in the TAG emulsion than in the DAG emulsion, indicating the increased unfolding of adsorbed beta-LG on the TAG oil droplet surface. Results of interfacial tension measurement supported this speculation, that is, the increased unfolding of the protein at the TAG-water interface. Trypsin- and proteinase K-catalyzed proteolysis was used to probe the topography of the adsorbed beta-LG on the oil droplet surface. SDS-PAGE analyses of liberated peptides after the proteolysis indicated the higher susceptibility of beta-LG adsorbed on the DAG oil droplet surface than on the TAG oil droplet surface. On the basis of all the results, we discussed the conformation of the adsorbed beta-LG on the two oil droplet surfaces.

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“…When heated at neutral pH to above about 60 °C, BLG is irreversibly denatured, due to its polymerization through inter-and intramolecular disulfide bond formation and exchange promoted by C121 (4,5). The molecular detail of the heat-induced denaturation and polymerization process of BLG has not been fully elucidated yet, but it clearly involves several successive reactions and intermediates (6)(7)(8) leading to irreversible modifications of BLG structure at different levels (9)(10)(11). C121 plays a crucial role in this heat-induced polymerization of BLG: chemical blocking of the free thiol group prevents the protein from aggregating and renders its denaturation reversible (4,5,10,12,13).…”

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A Recombinant C121S Mutant of Bovine β-Lactoglobulin Is More Susceptible to Peptic Digestion and to Denaturation by Reducing Agents and Heating

et al. 2004

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The lipocalin beta-lactoglobulin (BLG) is the major whey protein of bovine milk and is homodimeric at physiological conditions. Each monomer contains two disulfide bonds and one cysteine at position 121 (C121). This free thiol plays an important role in the heat-induced aggregation of BLG and, possibly, in its conformational stability. We describe here the expression in the yeast Pichia pastoris of a mutant bovine BLG, in which C121 was changed into Ser (C121S). Circular dichroism and high-performance liquid chromatography experiments, together with the X-ray crystal structure, show that the C121S mutant retains a nativelike fold at both neutral and acid pH. The mutation completely blocks the irreversible aggregation induced by heat treatment at 90 degrees C. Compared to the recombinant wild-type protein, the mutant is less stable to temperature and disulfide reducing agents and is much more sensitive to peptic digestion. Moreover, its affinity for 1-anilino-8-naphthalenesulfonate is increased at neutral and acid pH. We suggest that the stability of the protein arising from the hydrophobic effect is reduced by the C121S mutation so that unfolded or partially unfolded states are more favored.

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Multi-state thermal unfolding and aggregation of β-lactoglobulin A

Cited by 6 publications

References 0 publications

Thermal Denaturation of Beta-Lactoglobulin and Stabilization Mechanism by Trehalose Analyzed from Raman Spectroscopy Investigations

Thermal Denaturation of Beta-Lactoglobulin and Stabilization Mechanism by Trehalose Analyzed from Raman Spectroscopy Investigations

Adsorption and Structural Change of β-Lactoglobulin at the Diacylglycerol−Water Interface

A Recombinant C121S Mutant of Bovine β-Lactoglobulin Is More Susceptible to Peptic Digestion and to Denaturation by Reducing Agents and Heating

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