The Effect of Quaternary Structure on Small Heat Shock Proteins: Chaperone Activity of GST-Induced Dimers of Human HspB1 and HspB5
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Small heat shock proteins (sHsps) are a class of ATP-independent molecular chaperones that maintain protein homeostasis by preventing denatured proteins from aggregating and causing cell damage. When cells are stressed (whether by heat, oxidation, infection, or a variety of other stressors), proteins denature and expose hydrophobic surfaces. In the aqueous cellular environment, this denaturation promotes protein aggregation linked to many diseases such as Alzheimer’s or cataracts. sHsps bind to and form complexes with these unfolded client proteins in a way that prevents their aggregation and can promote their refolding or degradation with the help of other cellular chaperones. sHsps exist in vivo as polydisperse oligomers composed of numerous subunits that can mix and exchange with other subunits, and this dynamic nature has made the determination of a specific mechanism of sHsp action difficult. A given amount of sHsps can bind up to an equal amount of denatured proteins in terms of molecular weight; in order to have enough surface area available to bind an equal amount of denatured proteins, it has been suggested that dissociated sHsp subunits are responsible for observed chaperone activity, while large oligomers merely act as molecular storage for the smaller active chaperone subunits. X-ray crystallography and mass spectrometry studies have identified the dimer as the most-likely dissociated subunit of large oligomers. HspB1 and HspB5 are both widely produced in human tissues as a stress response to prevent client protein aggregation. In order to pin down the role of the dimer in chaperone activity, glutathione s-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both proteins in order to constrain HspB1 and HspB5 forms to a dimer. Using purified GST fusion dimers and wild type HspB1 and HspB5, the in vitro chaperone activity of dimers as compared to polydisperse oligomers was measured through the use of a UV-Vis aggregation assay. The results suggest the fusion proteins function as active molecular chaperones, and furthermore, the two different fusion proteins demonstrate different chaperone activity in relation to multiple different substrate proteins.