The activation mechanism of Hsp26 does not require dissociation of the oligomer

Titus M. Franzmann, Martin Helmut Wuehr, Klaus Richter, Stefan Walter, Johannes Buchner

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

Small heat shock proteins (sHsps) are molecular chaperones that specifically bind non-native proteins and prevent them from irreversible aggregation. A key trait of sHsps is their existence as dynamic oligomers. Hsp26 from Saccharomyces cerevisiae assembles into a 24mer, which becomes activated under heat shock conditions and forms large, stable substrate complexes. This activation coincides with the destabilization of the oligomer and the appearance of dimers. This and results from other groups led to the generally accepted notion that dissociation might be a requirement for the chaperone mechanism of sHsps. To understand the chaperone mechanism of sHsps it is crucial to analyze the relationship between chaperone activity and stability of the oligomer. We generated an Hsp26 variant, in which a serine residue of the N-terminal domain was replaced by cysteine. This allowed us to covalently crosslink neighboring subunits by disulfide bonds. We show that under reducing conditions the structure and function of this variant are indistinguishable from that of the wild-type protein. However, when the cysteine residues are oxidized, the dissociation into dimers at higher temperatures is no longer observed, yet the chaperone activity remains unaffected. Furthermore, we show that the exchange of subunits between Hsp26 oligomers is significantly slower than substrate aggregation and even inhibited in the presence of disulfide bonds. This demonstrates that the rearrangements necessary for shifting Hsp26 from a low to a high affinity state for binding non-native proteins occur without dissolving the oligomer.

Original languageEnglish (US)
Pages (from-to)1083-1093
Number of pages11
JournalJournal of molecular biology
Volume350
Issue number5
DOIs
StatePublished - Jul 29 2005

Fingerprint

Small Heat-Shock Proteins
Disulfides
Cysteine
Proteins
Molecular Chaperones
Serine
Saccharomyces cerevisiae
Shock
Hot Temperature
Temperature

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Structural Biology

Keywords

  • Aggregation
  • Alpha crystallin
  • Disulfide bond
  • Molecular chaperone
  • Small heat shock protein

Cite this

Franzmann, Titus M. ; Wuehr, Martin Helmut ; Richter, Klaus ; Walter, Stefan ; Buchner, Johannes. / The activation mechanism of Hsp26 does not require dissociation of the oligomer. In: Journal of molecular biology. 2005 ; Vol. 350, No. 5. pp. 1083-1093.
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The activation mechanism of Hsp26 does not require dissociation of the oligomer. / Franzmann, Titus M.; Wuehr, Martin Helmut; Richter, Klaus; Walter, Stefan; Buchner, Johannes.

In: Journal of molecular biology, Vol. 350, No. 5, 29.07.2005, p. 1083-1093.

Research output: Contribution to journalArticle

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T1 - The activation mechanism of Hsp26 does not require dissociation of the oligomer

AU - Franzmann, Titus M.

AU - Wuehr, Martin Helmut

AU - Richter, Klaus

AU - Walter, Stefan

AU - Buchner, Johannes

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N2 - Small heat shock proteins (sHsps) are molecular chaperones that specifically bind non-native proteins and prevent them from irreversible aggregation. A key trait of sHsps is their existence as dynamic oligomers. Hsp26 from Saccharomyces cerevisiae assembles into a 24mer, which becomes activated under heat shock conditions and forms large, stable substrate complexes. This activation coincides with the destabilization of the oligomer and the appearance of dimers. This and results from other groups led to the generally accepted notion that dissociation might be a requirement for the chaperone mechanism of sHsps. To understand the chaperone mechanism of sHsps it is crucial to analyze the relationship between chaperone activity and stability of the oligomer. We generated an Hsp26 variant, in which a serine residue of the N-terminal domain was replaced by cysteine. This allowed us to covalently crosslink neighboring subunits by disulfide bonds. We show that under reducing conditions the structure and function of this variant are indistinguishable from that of the wild-type protein. However, when the cysteine residues are oxidized, the dissociation into dimers at higher temperatures is no longer observed, yet the chaperone activity remains unaffected. Furthermore, we show that the exchange of subunits between Hsp26 oligomers is significantly slower than substrate aggregation and even inhibited in the presence of disulfide bonds. This demonstrates that the rearrangements necessary for shifting Hsp26 from a low to a high affinity state for binding non-native proteins occur without dissolving the oligomer.

AB - Small heat shock proteins (sHsps) are molecular chaperones that specifically bind non-native proteins and prevent them from irreversible aggregation. A key trait of sHsps is their existence as dynamic oligomers. Hsp26 from Saccharomyces cerevisiae assembles into a 24mer, which becomes activated under heat shock conditions and forms large, stable substrate complexes. This activation coincides with the destabilization of the oligomer and the appearance of dimers. This and results from other groups led to the generally accepted notion that dissociation might be a requirement for the chaperone mechanism of sHsps. To understand the chaperone mechanism of sHsps it is crucial to analyze the relationship between chaperone activity and stability of the oligomer. We generated an Hsp26 variant, in which a serine residue of the N-terminal domain was replaced by cysteine. This allowed us to covalently crosslink neighboring subunits by disulfide bonds. We show that under reducing conditions the structure and function of this variant are indistinguishable from that of the wild-type protein. However, when the cysteine residues are oxidized, the dissociation into dimers at higher temperatures is no longer observed, yet the chaperone activity remains unaffected. Furthermore, we show that the exchange of subunits between Hsp26 oligomers is significantly slower than substrate aggregation and even inhibited in the presence of disulfide bonds. This demonstrates that the rearrangements necessary for shifting Hsp26 from a low to a high affinity state for binding non-native proteins occur without dissolving the oligomer.

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