Functional and structural characterization of HspB1/Hsp27 from Chinese hamster ovary cells

Small heat shock proteins (sHsps) endow cells with stress tolerance. Of the various sHsps in mammals, HspB1, also known as Hsp27, is the most ubiquitous. To examine the structure and function of HspB1, we expressed, purified, and characterized HspB1 from Chinese hamster (Cricetulus griseus) ovary cells (CgHspB1). CgHspB1 forms a large oligomeric structure. We observed a monodisperse 16‐mer with an elongated sphere, but this is affected by changes in various conditions, including temperature. Under dilute conditions, CgHspB1 dissociates into small oligomers at elevated temperatures. The dissociated conformers interacted with the gel filtration column through hydrophobic interactions. In contrast, dissociation of the oligomer was not observed by small‐angle X‐ray scattering at 55 °C. The result partially coincides with the results of size exclusion chromatography, showing that dissociation did not occur at high protein concentrations. However, a significant structural change in the oligomeric conformations appears to occur between room and higher temperatures. Reflecting their status as homeotherms, mammalian sHsps are regulated by phosphorylation. A phosphorylation mimic mutant of CgHspB1 with the replacement of Ser15 to Asp exhibited relatively lower oligomer stability and greater protective ability against thermal aggregation than the wild‐type protein. The result clearly shows a correlation between oligomer dissociation and chaperone activity. A single phosphorylation mimic mutation, S15D, significantly increased chaperone activity and decreased oligomer stability of HspB1 from Chinese hamster ovary cells (CgHspB1). Even at room temperature, the mutant partially dissociated into small oligomers, most likely dimers. Heat stress and phosphorylation change oligomer assembly equilibrium to activate CgHspB1.