Date

2022-6-23

Author

Zabcı, Sema

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Small heat shock proteins (sHSPs) are characterized by a conserved, α-crystallin domain (ACD) which is the structural hallmark and considered as main functional unit in their chaperone activity. In this study, targeted residues in the ACD of Tpv sHSP 14.3 were replaced by amino acids of different biochemical features by site directed mutagenesis to analyze their effects on structure and function of the Tpv sHSP14.3. Results from chaperone activity assays showed that Tpv sHSP 14.3 ACD mutants and wild type prevented the thermal inactivation of the model substrates; pig heart citrate synthase (CS) and yeast alcohol dehydrogenase (ADH). The extent of the protection against the heat induced inactivation was found to be different among the mutants and specific to the substrate proteins. Increasing hydrophobicity at the junction of ACD and N-terminal domain by Y34F mutation enhanced protection efficiency of the sHSP against heat induced inactivation of the ADH and CS. To investigate the importance of the charge residues in the ACD for the chaperone function, when the negative charge at position 45 (E45G) of the β3 strand was eliminated, the chaperone activity was compromised. On the other hand, introduction of charged groups in place of hydrophobic residues (i.e., G48E, I108K, Y34FG48E, G48EI108K) resulted in higher chaperone activities against heat induced inactivation of ADH. This result indicated that both hydrophobic and electrostatic interactions can be crucial for the chaperone activity of Tpv sHSP 14.3. In accordance with the previous reports for other sHSPs, this study provided additional evidences for the critical roles of two conserved motifs; P/A-G doublet and G-x-L motif. Single point mutations, A47D, G107A and G107D, on those motifs, altered the stability and activity of the resultant mutant proteins. As deduced from changes in Rosetta Energy levels, substitution with the negatively charged residues more reduced than the hydrophobic amino acids the propensity of the sHSP variant for fibrillation. The contacts between alpha carbon atoms, as well as the RR distances, also varied as a result of the mutations. The extent of this variation was depended on the substituted amino acid, and the hydrophobic ones mostly preferred long-range contacts. On the other hand, there was a correlation between long range contacts and thermodynamic stability of the mutant proteins. Thermodynamic stabilities of the all mutant sHSP variants were reduced, except E43V mutant protein that had slight increase. The Tpv sHSP WT and most of the mutant variants possessed 24-mer oligomeric form which is typical for the sHSPs as revealed by BN-PAGE analysis. As an exception, two mutants (Y34F and I108K) instead formed 36-meric oligomers. The equilibrium of the oligomeric specie shifted toward larger species which was particularly obvious for the K87E mutant variant. Our results also indicated that the novel mini chaperones we designed regarding their superior chaperone activities as compared to WT Tpv sHSP14.3 can be considered promising for chaperone therapy.

Subject Keywords

Archaea, Thermoplasma volcanium, Small Heat Shock Protein, Site Directed Mutagenesis, Mini Chaperone, Structural Modifications

URI

https://hdl.handle.net/11511/98168

Collections

Graduate School of Natural and Applied Sciences, Thesis