Improvement of holdase activity of a small heat shock protein from extremely acidophilic archaea

2019
Kaçer, Yağmur
In this study, a small heat shock protein gene (po-sHSP20) of archaeon Picrophilus oshimae which is the most thermoacidophilic organism known, was cloned and overexpressed in E.coli for the first time. The recombinant po-sHSP20 protein that was resistant to high temperature was purified to homogeneity by affinity chromatography. Oligomeric structure analysis of the po-sHSP20 by Size Exclusion Chromatography showed that it exists mostly as 12- and 18-mer oligomers with some dimeric/monomeric units under physiological conditions. Highly conserved K99 residue of the po-sHSP20 was substituted by glutamic acid and glycine. Mutant proteins were successfully expressed in E.coli. Chaperone activities of the po-sHSP20 variants were studied by using pig heart citrate synthase as the client protein. The K99E and K99G variants better protected citrate synthase (2.3- and 2-fold, respectively) from heat inactivation at 47ºC, as compared to wild-type. All chaperone variants could prevent thermal aggregation of citrate synthase at a molar ratio of 1:7 and 1:35. The analysis of the 3D model structure of the po-sHSP20 showed that its monomer consists of an alpha crystallin domain flanked by an N-terminal α-helix and C-terminal coiled structure. Dimerization occurs via interactions of β6-strand of one monomer with β2-strand of other monomer. According to model structure analyses of K99G and K99E mutations resulted in loss of the intermolecular hydrogen bond, together with the some intramolecular hydrogen and hydrophobic bonds that K99 residue participates.

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Citation Formats
Y. Kaçer, “Improvement of holdase activity of a small heat shock protein from extremely acidophilic archaea,” Thesis (M.S.) -- Graduate School of Natural and Applied Sciences. Biochemistry., Middle East Technical University, 2019.