title: The activation mechanism of Hsp26 does not require dissociation of the oligomer.
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.
authors.affiliation: Department Chemie, Technische Universität München, 85747 Garching, Germany.
authors.forename: Titus M
authors.initials: TM
authors.lastname: Franzmann
authors.forename: Martin
authors.initials: M
authors.lastname: Wühr
authors.forename: Klaus
authors.initials: K
authors.lastname: Richter
authors.forename: Stefan
authors.initials: S
authors.lastname: Walter
authors.forename: Johannes
authors.initials: J
authors.lastname: Buchner
mesh_terms: D019943:Amino Acid Substitution; D003545:Cysteine; D019281:Dimerization; D004220:Disulfides; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004220:Disulfides; C499528:HSP26 protein, S cerevisiae; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; D003545:Cysteine