Structural modelling and functional analysis of the engineered small heat-shock protein, tpv-hsp14.3 from thermoplasma volcanium

Sheraj, Ilir
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/L motif in the middle of the C-terminus. Computational results for secondary structure prediction showed a canonical class II sHSP with nine β-strands, which constitute the core α-Crystalline domain, flanked by an N-terminal α-helix and a short coiled coil secondary structure at the C-Terminus. These results were confirmed by the predicted three-dimensional (3D) of the tpv-Hsp14.3. 3D model analysis showed that sHSP protein monomers are interacting by means of β6-strand swapping to dimerize, and then form higher order oligomers which interact and protect the protein substrates under stress conditions. Sequence alignments at different levels and structure comparison with the sHSPs studies so far were carried out to determine important residues involved in dimer formation and chaperoning function. From these studies, three single-residue, and a double-residue were determined for mutagenesis. They were: R69, to be changed into R69K, R69E, and R69M; R81 to be changed into R81K, R81E and R81M; K87 to be changed into K87R, K87E and K87I; and QR(80-81) to be changed into QR(80-81)EL. To this end, site-directed mutagenesis was performed to change the selected residues into analogs, negatively-charged, and hydrophobic residues. Then the mutants and the wild-type proteins were expressed in E.coli and purified by affinity column chromatography. To see the change in the chaperoning function of the mutants, pig heart Citrate Synthase (CS) was used as model enzyme. CS was incubated with both, wild-type and each one of the sHSP mutant variants at denaturing temperature (47°C). The initial activity then was measured at 35°C which is the optimum temperature for CS. The enzyme assay results showed that the enzyme activities of mutant variants R69E, R69K, K87R and K87I, increased by 1.9-, 1.8-, 2.6- and 2.7-fold respectively, compared to CS activity incubated with the wild-type tpv-Hsp14.3. K87E and QR(80-81)EL mutants had a slight increased CS activity (1.2- and 1.3-fold respectively) as compared to the wild-type tpv-Hsp14.3. On the other hand, CS activities when incubated with R69M and R81K mutant variants were 0.9- and 0.6-fold lower, respecively than CS incubated with the wild-type chaperone. CS activity when the enzyme was incubated with R81E and R81M mutants did not significantly change as compared to CS activity when the enzyme was incubated alone at 47°C. Finally, Dynamic Light Scattering (DLS) Spectroscopy was used to study the oligomer dynamics of wild-type and mutant variants of tpv-Hsp14.3 in solution. DLS was carried out under two different conditions: In the first, the wild-type tpv-Hsp14.3 was incubated alone and with pig heart CS or Bovine Glutamate Dehydrogenase (GDH) substrates enzymes at temperatures where the two enzymes lose their activities rapidly. In the second case, the wild-type tpv-Hsp14.3 and the mutant variants were incubated at 20°C, 35°C, 45°C and 60°C for 10 minutes separately and DLS measurements were taken. Then, these data were converted into hydration radius (Rh) values which represent sizes of the oligomers in the solution and their distribution were recorded as peaks. According to these results, there was a general trend of decreasing in Rh size with increasing the temperature reducing in the number of peaks towards a single main peak.


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...
Improvement of holdase activity of a small heat shock protein from extremely acidophilic archaea
Kaçer, Yağmur; Kocabıyık, Semra; Department of Biochemistry (2019)
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 phys...
The role of small heat shock proteins of the thermoacidophilic archaeon thermoplasma volcanium in the stress response
Aygar, Sema; Kocabıyık, Semra; Department of Biological Sciences (2011)
In this study, possible involvement of the small heat shock proteins (sHsps) from a thermoacidophilic archaeon, Thermoplasma (Tp) volcanium in the stress response was investigated. Our results showed that heterologous, high level expression of TVN0775/sHsp gene in E.coli increased its thermotolerance at 53°C for two hours. But, the second sHsp of the Tp. volcanium, TVN0984/sHsp was not effective in improvement of the thermal resistance of the mesophilic bacterium (i. e., E.coli). The expression of the TVN07...
Investigation of structural and functional properties of OMPG mutants by FTIR spectroscopy
Yılmaz, İrem; Bek, Alpan; Korkmaz Özkan, Filiz; Department of Physics (2017)
The subject of the current study is a bacterial protein OmpG from Escherichia coli. OmpG-16S and OmpG-16SL are the mutants of OmpG that are genetically engineered for potential biotechnological applications. In this study, structural and functional similarities and/or differences of these mutants with respect to the wild type OmpG were revealed using IR spectroscopy. Also, a new spectrometer attachment enabling in situ 1H/2H exchange was developed. For the collection of mass amount of spectral data from eac...
Biochemical characterization of recombinant 20s proteasome from thermoplasma volcanium and cloning of it's regulatory subunit gene
Baydar, Gözde; Kocabıyık, Semra; Department of Biotechnology (2006)
In this study, we have characterized some biochemical and electrophoretic features of recombinant 20S Proteasome from a thermoacidophilic archaeon Thermoplasma volcanium. As revealed by SDS-PAGE the 20S Proteasome was composed of two subunits, ?- and β- subunits with estimated molecular masses of 24 kDa and 23 kDa, respectively. The highest chymotryptic activity was observed over an alkaline pH range (pH 8.0 ا pH 9.0) and the optimum temperature for the activity was determined as 85oC. The heat stability of...
Citation Formats
I. Sheraj, “Structural modelling and functional analysis of the engineered small heat-shock protein, tpv-hsp14.3 from thermoplasma volcanium,” M.S. - Master of Science, Middle East Technical University, 2014.