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

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.


Improvement of protein stability and enzyme recovery under stress conditions by using a small HSP (tpv-HSP 14.3) from Thermoplasma volcanium
Kocabıyık, Semra (2012-11-01)
In this study we cloned and expressed a small heat shock protein, tpv-HSP 14.3, from thermoacidophilic archaeon Thermoplasma volcanium. This novel recombinant small heat shock protein was purified to homogeneity and produced a protein band of 14.3 kDa on SDS-polyacrylamide gel. Transmission electron microscopy images of the negatively stained tpv-EISP 14.3 samples showed spherical particles of 13 nm diameter. E. coli cells over expressing tpv-HSP 14.3 endowed the cells with some degree of thermotolerance. A...
Essential Structural and Functional Features of Small Heat Shock Proteins in Molecular Chaperoning Process
Kocabıyık, Semra (2009-06-01)
Small heat shock proteins are ubiquitously found in all three domains of life, although they are the most poorly conserved family of molecular chaperones. Their involvement in anti-stress mechanisms of the cells have been clearly demonstrated by induction of their expression in response to various environmental and pathological stresses. Small heat shock proteins comprise the most effective chaperone family concerning their unusual capacity of substrate binding. It is well documented that small heat shock p...
Structural modelling and functional analysis of the engineered small heat-shock protein, tpv-hsp14.3 from thermoplasma volcanium
Sheraj, Ilir; Kocabıyık, Semra; Department of Biology (2014)
In this study, a small heat shock protein tpv-Hsp14.3 from a thermoacidophilic Archaeon, Thermoplasma volcanium was studied. sHSPs are low molecular weight proteins involved in different stress responses to protect the cellular proteome and enhance survival of the host organism. For structure-function analysis of the protein, both experimental and computational tools were used. Sequence alignments with closely-related sHSPs showed high sequence conservation at the core α-Crystalline domain and a V/I/L-X-V/I...
Induced CYP1A mRNA, protein and catalytic activity in the liver of feral fish, leaping mullet, Liza saliens
Arinc, E; Kocabıyık, Semra; Su, E (2001-02-01)
In this study, we examined whether levels of P4501A mRNA expression were naturally induced in feral fish, Liza saliens, and whether CYP1A protein levels and associated enzyme activity, EROD, were also increased. Induction of mRNA was measured using a nucleic acid hybridization technique. For the hybridization studies, a new 33-mer oligonucleotide probe 5 ' -dCTC ATC CAG CTT CCT GTC CTC GCA GTG ATC AAT-3 ' was designed, which corresponded to the totally conserved amino acid motif of CYP1A protein from positi...
Predicting Protein-Protein Interactions from the Molecular to the Proteome Level
Keskin, Ozlem; Tunçbağ, Nurcan; Gursoy, Attila (2016-04-27)
Identification of protein protein interactions (PPIs) is at the center of molecular biology considering the unquestionable role of proteins in cells. Combinatorial interactions result in a repertoire of multiple functions; hence, knowledge of PPI and binding regions naturally serve to functional proteomics and drug discovery. Given experimental limitations to find all interactions in a proteome, computational prediction/modeling of protein interactions is a prerequisite to proceed on the way to complete int...
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.