Reconstitution of a heat shock effect in vitro: influence of GroE on the thermal aggregation of .alpha.-glucosidase from yeast

Self Cite

The prokaryotic molecular chaperone GroE is increasingly expressed under heat shock conditions. GroE protects cells by preventing the irreversible aggregation of thermally unfolding proteins. Here, the interaction of GroE with thermally unfolding citrate synthase (CS) was dissected into several steps that occur before irreversible aggregation, and the conformational states of the unfolding protein recognized by GroEL were determined. The kinetic analysis of CS unfolding revealed the formation of inactive dimeric and monomeric intermediates. GroEL binds both intermediates without affecting the unfolding pathway. Furthermore, the dimeric intermediates are not protected against dissociation in the presence of GroEL. Monomeric CS is stably associated with GroEL, thus preventing further irreversible unfolding steps and subsequent aggregation. During refolding, monomeric CS is encapsulated inside the cavity of GroEL⅐GroES complexes. Taken together our results suggest that for protection of cells against heat stress both the ability of GroEL to interact with a large variety of nonnative conformations of proteins and the active, GroES-dependent refolding of highly unfolded species are important.

Section: Discussionsupporting

confidence: 94%

“…Under heat shock condition in vitro, GroEL has been shown to interact with several unfolding proteins. These tightly bound polypeptides are protected against irreversible aggregation (21). Reactivation is possible by changing the folding environment to permissive conditions in the presence of GroES and ATP (21)(22)(23)(24).…”

mentioning

confidence: 99%

GroEL Traps Dimeric and Monomeric Unfolding Intermediates of Citrate Synthase

Grallert

Rutkat

Büchner

1998

Self Cite

“…AGLU recovered spontaneously from the denatured state by 10% compared with the activity of the native enzyme, as reported previously (11). In the absence of ATP, EC GroEL completely arrested the denatured AGLU, whereas CP GroEL1 did not, instead permitting the spontaneous refolding of the enzyme.…”

Section: Groel1 From C Pneumoniae Acts As a Chaperonin-supporting

confidence: 87%

The Intermediate Domain Defines Broad Nucleotide Selectivity for Protein Folding in Chlamydophila GroEL1

Okuda

Sakuhana

Yamamoto

et al. 2008

The chaperonin GroEL assists protein folding in the presence of ATP and magnesium through substrate protein capsulation in combination with the cofactor GroES. Recent studies have revealed the details of folding cycles of GroEL from Escherichia coli, yet little is known about the GroEL-assisted protein folding mechanisms in other bacterial species. Using three model enzyme assays, we have found that GroEL1 from Chlamydophila pneumoniae, an obligate human pathogen, has a broader selectivity for nucleotides in the refolding reaction. To elucidate structural factors involved in such nucleotide selectivity, GroEL chimeras were constructed by exchanging apical, intermediate, and equatorial domains between E. coli GroEL and C. pneumoniae GroEL1. In vitro folding assays using chimeras revealed that the intermediate domain is the major contributor to the nucleotide selectivity of C. pneumoniae GroEL1. Additional site-directed mutation experiments led to the identification of Gln 400 and Ile 404 in the intermediate domain of C. pneumoniae GroEL1 as residues that play a key role in defining the nucleotide selectivity of the protein refolding reaction.

“…All assays were performed in the presence of an ATP-regenerating system (3 mM phosphoenolpyruvate; 20 g/ml pyruvate kinase; 2 mM ATP). Determination of enzymatic activities followed published protocols (10,17,25,26). Refolding rates were calculated from the linear increase of substrate activities.…”

Section: Methodsmentioning

confidence: 99%

Refolding of Substrates Bound to Small Hsps Relies on a Disaggregation Reaction Mediated Most Efficiently by ClpB/DnaK

Mogk

Schlieker

Friedrich

et al. 2003

255

216

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that bind denatured proteins in vitro, thereby facilitating their subsequent refolding by ATP-dependent chaperones. The mechanistic basis of this refolding process is poorly defined. We demonstrate that substrates complexed to sHsps from various sources are not released spontaneously. Dissociation and refolding of sHsp bound substrates relies on a disaggregation reaction mediated by the DnaK system, or, more efficiently, by ClpB/DnaK. While the DnaK system alone works for small, soluble sHsp/substrate complexes, ClpB/DnaK-mediated protein refolding is fastest for large, insoluble protein aggregates with incorporated sHsps. Such conditions reflect the situation in vivo, where sHsps are usually associated with insoluble proteins during heat stress. We therefore propose that sHsp function in cellular protein quality control is to promote rapid resolubilization of aggregated proteins, formed upon severe heat stress, by DnaK or ClpB/DnaK.