In vitro reconstitution of the 24-meric E2 inner core of bovine mitochondrial branched-chain α-keto acid dehydrogenase complex: Requirement for chaperonins GroEL and GroES

R. Max Wynn, James R. Davie, Wang Zhi, Rody P. Cox, David T. Chuang

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Abstract

We have investigated the in vitro reconstitution of the 24-meric inner core domain (E2c) of the transacylase (E2) component of bovine branched-chain α-keto acid dehydrogenase complex. The yield of recombinant E2c (amino acid residues 161-421 of bovine E2) expressed in Escherichia coli was markedly increased by fusing the bacterial maltose-binding protein (MBP) to the amino terminus of bovine E2c. Following factor Xa digestion to remove the MBP moiety, E2c was completely unfolded in 4.5 M guanidine HCl (Gdn·HCl). The denatured E2c monomers (apparent Mr = 27 000) were diluted 100-fold at 25°C into a refolding buffer containing 5 mM Mg-ATP and a 4-fold molar excess of chaperonins GroEL and GroES. Full E2 activity was recovered in 45 min. Omission of the chaperonins in the refolding buffer failed to recover any E2 activity. Recovery of E2 activity obeyed hyperbolic kinetics as a function of the chaperonin-to-E2c molar ratio and showed a requirement for hydrolysis of Mg-ATP. A stable GroEL-E2c complex was isolated which, in the presence of GroES and Mg-ATP, generated active E2c 24-mers. Dissociation of recombinant E2c 24-mers into active trimers was achieved by incubation in 1.5 M Gdn·HCl at 25°C. The E2c trimers with an apparent Mr of 84 000 were isolated by sucrose density gradient centrifugation in the presence of the chaotropic reagent. Removal of 1.5 M Gdn·HCl resulted in the spontaneous reassembly of trimers into the native 24-mer structure independent of chaperonins. Our results indicate that in vitro refolding of bovine E2c is a chaperonin-mediated process and that spontaneous assembly of the 24-meric structure of E2 proceeds through active trimeric intermediates.

Original languageEnglish (US)
Pages (from-to)8962-8968
Number of pages7
JournalBiochemistry
Volume33
Issue number30
StatePublished - 1994

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3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
Chaperonins
Guanidine
Maltose-Binding Proteins
Adenosine Triphosphate
Buffers
Factor Xa
Bacterial Proteins
Density Gradient Centrifugation
Centrifugation
Escherichia coli
Sucrose
Digestion
Hydrolysis
Monomers
In Vitro Techniques
Amino Acids
Recovery
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "In vitro reconstitution of the 24-meric E2 inner core of bovine mitochondrial branched-chain α-keto acid dehydrogenase complex: Requirement for chaperonins GroEL and GroES",
abstract = "We have investigated the in vitro reconstitution of the 24-meric inner core domain (E2c) of the transacylase (E2) component of bovine branched-chain α-keto acid dehydrogenase complex. The yield of recombinant E2c (amino acid residues 161-421 of bovine E2) expressed in Escherichia coli was markedly increased by fusing the bacterial maltose-binding protein (MBP) to the amino terminus of bovine E2c. Following factor Xa digestion to remove the MBP moiety, E2c was completely unfolded in 4.5 M guanidine HCl (Gdn·HCl). The denatured E2c monomers (apparent Mr = 27 000) were diluted 100-fold at 25°C into a refolding buffer containing 5 mM Mg-ATP and a 4-fold molar excess of chaperonins GroEL and GroES. Full E2 activity was recovered in 45 min. Omission of the chaperonins in the refolding buffer failed to recover any E2 activity. Recovery of E2 activity obeyed hyperbolic kinetics as a function of the chaperonin-to-E2c molar ratio and showed a requirement for hydrolysis of Mg-ATP. A stable GroEL-E2c complex was isolated which, in the presence of GroES and Mg-ATP, generated active E2c 24-mers. Dissociation of recombinant E2c 24-mers into active trimers was achieved by incubation in 1.5 M Gdn·HCl at 25°C. The E2c trimers with an apparent Mr of 84 000 were isolated by sucrose density gradient centrifugation in the presence of the chaotropic reagent. Removal of 1.5 M Gdn·HCl resulted in the spontaneous reassembly of trimers into the native 24-mer structure independent of chaperonins. Our results indicate that in vitro refolding of bovine E2c is a chaperonin-mediated process and that spontaneous assembly of the 24-meric structure of E2 proceeds through active trimeric intermediates.",
author = "Wynn, {R. Max} and Davie, {James R.} and Wang Zhi and Cox, {Rody P.} and Chuang, {David T.}",
year = "1994",
language = "English (US)",
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pages = "8962--8968",
journal = "Biochemistry",
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T1 - In vitro reconstitution of the 24-meric E2 inner core of bovine mitochondrial branched-chain α-keto acid dehydrogenase complex

T2 - Requirement for chaperonins GroEL and GroES

AU - Wynn, R. Max

AU - Davie, James R.

AU - Zhi, Wang

AU - Cox, Rody P.

AU - Chuang, David T.

PY - 1994

Y1 - 1994

N2 - We have investigated the in vitro reconstitution of the 24-meric inner core domain (E2c) of the transacylase (E2) component of bovine branched-chain α-keto acid dehydrogenase complex. The yield of recombinant E2c (amino acid residues 161-421 of bovine E2) expressed in Escherichia coli was markedly increased by fusing the bacterial maltose-binding protein (MBP) to the amino terminus of bovine E2c. Following factor Xa digestion to remove the MBP moiety, E2c was completely unfolded in 4.5 M guanidine HCl (Gdn·HCl). The denatured E2c monomers (apparent Mr = 27 000) were diluted 100-fold at 25°C into a refolding buffer containing 5 mM Mg-ATP and a 4-fold molar excess of chaperonins GroEL and GroES. Full E2 activity was recovered in 45 min. Omission of the chaperonins in the refolding buffer failed to recover any E2 activity. Recovery of E2 activity obeyed hyperbolic kinetics as a function of the chaperonin-to-E2c molar ratio and showed a requirement for hydrolysis of Mg-ATP. A stable GroEL-E2c complex was isolated which, in the presence of GroES and Mg-ATP, generated active E2c 24-mers. Dissociation of recombinant E2c 24-mers into active trimers was achieved by incubation in 1.5 M Gdn·HCl at 25°C. The E2c trimers with an apparent Mr of 84 000 were isolated by sucrose density gradient centrifugation in the presence of the chaotropic reagent. Removal of 1.5 M Gdn·HCl resulted in the spontaneous reassembly of trimers into the native 24-mer structure independent of chaperonins. Our results indicate that in vitro refolding of bovine E2c is a chaperonin-mediated process and that spontaneous assembly of the 24-meric structure of E2 proceeds through active trimeric intermediates.

AB - We have investigated the in vitro reconstitution of the 24-meric inner core domain (E2c) of the transacylase (E2) component of bovine branched-chain α-keto acid dehydrogenase complex. The yield of recombinant E2c (amino acid residues 161-421 of bovine E2) expressed in Escherichia coli was markedly increased by fusing the bacterial maltose-binding protein (MBP) to the amino terminus of bovine E2c. Following factor Xa digestion to remove the MBP moiety, E2c was completely unfolded in 4.5 M guanidine HCl (Gdn·HCl). The denatured E2c monomers (apparent Mr = 27 000) were diluted 100-fold at 25°C into a refolding buffer containing 5 mM Mg-ATP and a 4-fold molar excess of chaperonins GroEL and GroES. Full E2 activity was recovered in 45 min. Omission of the chaperonins in the refolding buffer failed to recover any E2 activity. Recovery of E2 activity obeyed hyperbolic kinetics as a function of the chaperonin-to-E2c molar ratio and showed a requirement for hydrolysis of Mg-ATP. A stable GroEL-E2c complex was isolated which, in the presence of GroES and Mg-ATP, generated active E2c 24-mers. Dissociation of recombinant E2c 24-mers into active trimers was achieved by incubation in 1.5 M Gdn·HCl at 25°C. The E2c trimers with an apparent Mr of 84 000 were isolated by sucrose density gradient centrifugation in the presence of the chaotropic reagent. Removal of 1.5 M Gdn·HCl resulted in the spontaneous reassembly of trimers into the native 24-mer structure independent of chaperonins. Our results indicate that in vitro refolding of bovine E2c is a chaperonin-mediated process and that spontaneous assembly of the 24-meric structure of E2 proceeds through active trimeric intermediates.

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