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

Yagi, Masanori; Sakurai, Kazumasa; Kalidas, Chitkala; Batt, Carl A.; Goto, Yuji

doi:https://doi.org/10.1074/jbc.M308592200

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

Yagi, Masanori; Sakurai, Kazumasa; Kalidas, Chitkala et al.

Journal of Biological Chemistry, 2003, 278(47), 47009-47015

Number of Access:530（2025-08-30 13:29 Counts）

Identifier to cite or link to this item: https://hdl.handle.net/11094/71298

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Title	Reversible Unfolding of Bovine β-Lactoglobulin Mutants without a Free Thiol Group
Creator	CreatorName Yagi, Masanori
	NRID 1000010403015 1000010403015 CreatorName Sakurai, Kazumasa
	CreatorName Kalidas, Chitkala
	CreatorName Batt, Carl A.
	NRID 1000040153770 1000040153770 researchmap permalink read0014440 read0014440 CreatorName Goto, Yuji
Description	This research was originally published in the Journal of Biological Chemistry. Masanori Yagi, Kazumasa Sakurai, Chitkala Kalidas, Carl A. Batt and Yuji Goto. Reversible Unfolding of Bovine β-Lactoglobulin Mutants without a Free Thiol Group. J. Biol. Chem. 2003; 278, 47009-47015. © the American Society for Biochemistry and Molecular Biology
Abstract	Bovine β-lactoglobulin (β-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 β-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, reversed-phase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type β-lg. Urea-induced 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 β-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 β-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of β-lg more exactly under various conditions.
Publisher	American Society for Biochemistry and Molecular Biology
Source Title	Journal of Biological Chemistry
Volume (Issue)	278(47)
Page	47009-47015
Date of Issued	2003-11
Language	English
Handle URL	https://hdl.handle.net/11094/71298
eISSN	1083351X
NCID	AA1202467X
Relation.isIdenticalTo	DOI (Publisher Version) https://doi.org/10.1074/jbc.M308592200
Access Rights	open access
oaire:version	Version of Record
Category	学術雑誌論文 Journal Article

Resource Type	journal article
Local Type	Journal Article
DCMI Type	Text
DCTERMS.bibliographicCitation	Journal of Biological Chemistry. 2003, 278(47), p. 47009-47015
DC.title	Reversible Unfolding of Bovine β-Lactoglobulin Mutants without a Free Thiol Group
DC.creator	Yagi, Masanori
	Sakurai, Kazumasa
	Kalidas, Chitkala
	Batt, Carl A.
	Goto, Yuji
DC.publisher	American Society for Biochemistry and Molecular Biology
DC.language' scheme='DCTERMS.RFC1766	English
DC.type' scheme='DCTERMS.DCMIType	Text
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2003-11
DC.identifier	https://doi.org/10.1074/jbc.M308592200
DC.description	This research was originally published in the Journal of Biological Chemistry. Masanori Yagi, Kazumasa Sakurai, Chitkala Kalidas, Carl A. Batt and Yuji Goto. Reversible Unfolding of Bovine β-Lactoglobulin Mutants without a Free Thiol Group. J. Biol. Chem. 2003; 278, 47009-47015. © the American Society for Biochemistry and Molecular Biology
DCTERMS.abstract	Bovine β-lactoglobulin (β-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 β-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, reversed-phase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type β-lg. Urea-induced 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 β-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 β-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of β-lg more exactly under various conditions.
DC.format	71298
citation_title	Reversible Unfolding of Bovine β-Lactoglobulin Mutants without a Free Thiol Group
citation_author	Yagi, Masanori
	Sakurai, Kazumasa
	Kalidas, Chitkala
	Batt, Carl A.
	Goto, Yuji
citation_publisher	American Society for Biochemistry and Molecular Biology
citation_language	English
citation_date	2003-11
citation_journal_title	Journal of Biological Chemistry
citation_volume	278
citation_issue	47
citation_firstpage	47009
citation_lastpage	47015
citation_doi	https://doi.org/10.1074/jbc.M308592200
citation_public_url	https://hdl.handle.net/11094/71298

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

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