Role of free Cys121 in stabilization of bovine beta-lactoglobulin B

Burova, Tatiana V.; Choiset, Y; Tran, Cuong; Haertlé, Thomas

doi:10.1093/protein/11.11.1065

Cited by 68 publications

(55 citation statements)

References 2 publications

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“…Co-denaturation of b-lactoglobulin (Tm (fusion temperature) = 858C, [15]) and a-lactalbumin (Tm = 658C, [16]) led to aggregate formation as shown on chromatograms of samples heated at 85 and 95 8C (Figs. 6 and 7 before hydrolysis).…”

Section: Modification Of Susceptibility To Proteases By Thermal Treatmentioning

confidence: 97%

Thermal modifications of structure and codenaturation of α-lactalbumin and β-lactoglobulin induce changes of solubility and susceptibility to proteases

Bertrand-Harb

Baday

Dalgalarrondo

et al. 2002

Nahrung

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Study of heat denaturation of major whey proteins (beta-lactoglobulin or alpha-lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other codenaturation partner, and of existence of a previous thermal pretreatment (industrial whey). alpha-Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at > or = 85 degrees C. After its denaturation, alpha-lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys-X and Arg-X bonds). Heating over 85 degrees C of beta-lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co-denaturation of alpha-lactalbumin with beta-lactoglobulin increased their aggregation and resulted in complete exposure of beta-lactoglobulin peptic cleavage sites and partial unveiling of alpha-lactalbumin tryptic cleavage sites. The exposure of alpha-lactalbumin tryptic cleavage sites was slightly enhanced when the alpha-lactalbumin/beta-lactoglobulin mixture was heated at pH 7.5. Co-denaturation of fresh whey by heating at 95 degrees C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by beta-mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.

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Section: Modification Of Susceptibility To Proteases By Thermal Treatmentioning

confidence: 97%

Thermal modifications of structure and codenaturation of α-lactalbumin and β-lactoglobulin induce changes of solubility and susceptibility to proteases

Bertrand-Harb

Baday

Dalgalarrondo

et al. 2002

Nahrung

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“…37) Further, there are several lines of evidence that free cysteine residues contribute to protein thermostability. [38][39][40] On the other hand, there are reports that both buried and exposed cysteine residues increase irreversible unfolding to form incorrect disulfide bonds. 41,42) It is thought that Cys443 is buried to avoid exposure of its highly reactive free thiol group.…”

Section: )mentioning

confidence: 99%

A Single Free Cysteine Residue and Disulfide Bond Contribute to the Thermostability ofAspergillus saitoi1,2-α-Mannosidase

Tatara

Yoshida

Ichishima

2005

Bioscience, Biotechnology, and Biochemistry

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Aspergillus saitoi 1,2--mannosidase contains three conserved cysteine residues (Cys334, Cys363, and Cys443). We showed that Cys334 and Cys363 are involved in a disulfide bond, and that Cys443 contains a free thiol group. The cysteines were not essential for the activity analyzed by site-directed mutagenesis and kinetics. The substitution at each cysteine residue greatly destabilized the enzyme. The T m values of WT, C443A, C443G, C443S, and C443T were 55.8, 51.9, 50.2, 50.0, and 52.8 C respectively. The specific activity of these mutants was almost equal to that of WT. Introducing Asp, Leu, Met, or Val at position 443 caused partial denaturation, although the enzymes had some activity. C443F, C443I, C443N, and C443Y were not secreted. These results suggest that the hydrophilic and large side chain causes the destabilization. Molecular modelling showed that the Cys443 residue is buried and surrounded by a hydrophobic environment. Cys334 and Cys363 form a disulfide bond, and Cys443 is involved in a hydrophobic interaction to stabilize the enzyme.

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“…When the thiol group of Cys-121 was modified with thiol reagents, the structure of ␤-lg was affected, depending on the size and properties of the group introduced. Modification of ␤-lg by 2-mercaptoethanol or mercaptopropionic acid, which both are smaller molecules, resulted in the destruction of the dimer without a loss of the rigid native structure, although the stability of the native structure was decreased significantly (24). In the case of 5,5Ј-dithiobis(2-nitrobenzoic acid) (10) or tetramethylrhodamine (38), the dissociation into a monomer was coupled with significant disordering of the protein structure both at pH 7 and pH 3.…”

Section: Comparison With Chemical Modification Of Cys-121-wementioning

confidence: 99%

Reversible Unfolding of Bovine β-Lactoglobulin Mutants without a Free Thiol Group

Yagi

Sakurai

Kalidas

et al. 2003

Journal of Biological Chemistry

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Bovine ␤-lactoglobulin (␤-lg) has been used extensively as a model for studying protein folding. One of the problems preventing clarification of the folding mechanism is the incomplete reversibility from the unfolded state, probably caused by the thiol-disulfide exchange between a free thiol at Cys-121 and two disulfide bonds. We constructed and expressed three ␤-lg subtype A mutants in which Cys-121 was replaced by Ala, Ser, or Val (i.e. C121A, C121S, and C121V). We studied the reversibilities of these mutants from urea denaturation using circular dichroism, tryptophan fluorescence, reversedphase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type ␤-lg. Ureainduced unfolding at pH 7.0 and 3.0 showed that although the C121S mutation notably decreases the stability, the destabilizing effects of the C121A and C121V mutations are less severe. For all of the mutants, complete refolding from the unfolded state in 8 M urea at both pH 7.0 and 3.0 was observed. Kinetics of the formation of the irreversibly unfolded species of wild-type ␤-lg in 8 M urea at pH 7.0 indicated that, first, an intramolecular thiol-disulfide exchange occurs to produce a mixture of species with non-native disulfide bonds followed by the intermolecular thiol-disulfide exchange producing the oligomers. These results indicate that intramolecular and intermolecular thiol-disulfide exchange reactions cause the low reversibility of wild-type ␤-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of ␤-lg more exactly under various conditions.

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Role of free Cys121 in stabilization of bovine beta-lactoglobulin B

Cited by 68 publications

References 2 publications

Thermal modifications of structure and codenaturation of α-lactalbumin and β-lactoglobulin induce changes of solubility and susceptibility to proteases

Thermal modifications of structure and codenaturation of α-lactalbumin and β-lactoglobulin induce changes of solubility and susceptibility to proteases

A Single Free Cysteine Residue and Disulfide Bond Contribute to the Thermostability ofAspergillus saitoi1,2-α-Mannosidase

Reversible Unfolding of Bovine β-Lactoglobulin Mutants without a Free Thiol Group

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