The small heat shock proteins are ubiquitous stress proteins proposed to increase cellular tolerance to heat shock conditions. We isolated IbpA, the Escherichia coli small heat shock protein, and tested its ability to keep thermally inactivated substrate proteins in a disaggregation competent state. We found that the presence of IbpA alone during substrate thermal inactivation only weakly influences the ability of the bi-chaperone Hsp70-Hsp100 system to disaggregate aggregated substrate. Similar minor effects were observed for IbpB alone, the other E. coli small heat shock protein. However, when both IbpA and IbpB are simultaneously present during substrate inactivation they efficiently stabilize thermally aggregated proteins in a disaggregation competent state. The properties of the aggregated protein substrates are changed in the presence of IbpA and IbpB, resulting in lower hydrophobicity and the ability of aggregates to withstand sizing chromatography conditions. IbpA and IbpB form mixed complexes, and IbpA stimulates association of IbpB with substrate.The proper conformation of proteins is challenged by stress conditions. Exposure to extreme heat shock conditions results in a massive aggregation of proteins inside both prokaryotic and eukaryotic cells (1-3). Chaperones from the Hsp100 family, that is ClpB in Escherichia coli and Hsp104 in the yeast Saccharomyces cerevisiae, were implicated in the disaggregation reaction, because aggregated proteins were not eliminated in either clpB or HSP104 deletion strains (1-3). Additionally, the clpB and HSP104 gene products were identified as factors conferring thermotolerance in E. coli and S. cerevisiae (4 -7). However, in vitro studies on the reactivation of aggregated proteins showed that chaperones from the Hsp100 family alone are not sufficient for disaggregation and refolding. Other chaperone proteins are also involved in this process. E. coli Hsp70 (DnaK) and its cochaperones (DnaJ and GrpE) cooperate with ClpB and form a bi-chaperone system capable of efficient disaggregation of aggregated proteins (3,8,9). Analogous Hsp100-Hsp70 bi-chaperone systems able to disaggregate denatured protein substrates in vitro were established using chaperones from other bacterial species (10), as well as from yeast cytosol (11) and mitochondria (12, 13). However, the efficiency of refolding reaction catalyzed by these bichaperone systems depends strongly on the physical properties of protein aggregates. It was proposed that small heat shock proteins (sHsps) 1 associate with aggregated proteins and change their physical properties in such a way that chaperone-mediated disaggregation and refolding become much more efficient (14 -19). However, little is known about the molecular mechanism of these processes.Small heat shock proteins are widely distributed both in prokaryotes and eukaryotes. Members of this diverse protein family are characterized by relatively low monomeric molecular masses (15-43 kDa) and a conserved stretch of ϳ100 amino acid residues (reviewed in Refs. 20 and ...

a b s t r a c tSmall heat shock proteins (sHsps) associate with aggregated proteins, changing their physical properties in such a way that chaperone mediated disaggregation becomes much more efficient. In Escherichia coli two small Hsps, IbpA and IbpB, exist. They are 48% identical at the amino acid level, yet their roles in stabilisation of protein aggregates are quite distinct. Here we analysed the biochemical properties of IbpA. We found that IbpA assembles into protofilaments which in turn form mature fibrils. Such fibrils are atypical for sHsps. Interaction of IbpA with either its cochaperone IbpB or an aggregated substrate blocks IbpA fibril formation. Structured summary:MINT-7876715: ibpA (uniprotkb:P0C054) and ibpA (uniprotkb:P0C054) bind (MI:0407) by molecular sieving (MI:0071) MINT-7888427: ibpB (uniprotkb:P0C058) and ibpB (uniprotkb:P0C058) bind (MI:0407) by molecular sieving (MI:0071) MINT-7888448: ibpA (uniprotkb:P0C054) and ibpA (uniprotkb:P0C054) bind (MI:0407) by electron microscopy (MI:0040) MINT-7888434: ibpB (uniprotkb:P0C058) and ibpB (uniprotkb:P0C058) bind (MI:0407) by electron microscopy (MI:0040) MINT-7888459: ibpA (uniprotkb:P0C054) and ibpA (uniprotkb:P0C054) bind (MI:0407) by fluorescence microscopy (MI:0416)

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