Date

2014

Author

Sheraj, Ilir

Metadata

Show full item record

Item Usage Stats

240
views

136
downloads

Cite This

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.

Subject Keywords

Proteins., Morphology., Heat shock proteins., Thermoplasma volcanium.

URI

http://etd.lib.metu.edu.tr/upload/12617162/index.pdf
https://hdl.handle.net/11511/23537

Collections

Graduate School of Natural and Applied Sciences, Thesis