Date

2022-5-12

Author

Rafiq, Azra

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The N-terminal domain (NTD) of small heat shock protein (sHSP) from archaea, Thermoplasma volcanium, is highly hydrophobic in the proximal and distal end. It is 32 amino acids long and has two highly conserved glutamic acid residues at positions 11 and 22. The 3-D model of the dimer generated by homology modelling predicted the NTD to be running away from each other and the dimer interface being formed mainly between β2 and β6 sheets of the Alpha Crystallin Domain (ACD). The decrease in hydrophobicity in the proximal region of the NTD decreased the chaperone activity of Tpv sHSP 14.3 while increasing hydrophilicity at the distal end increased the chaperone activity of the sHSP as compared to wild-type (WT). However, abolishing the negative charge at position 11 and 22 not only decreased the thermal stability of the resultant mutants but also decreased the chaperone activity. In addition, these mutations increased the propensity of the NTD to form fibril like structures as well. In the NTD, at position 12, when hydrophobicity was decreased by M12T mutation, not only the chaperone activity was decreased, but also, at high concentration the sHSP protein itself aggregated, at high temperature, along with the substrate. Moreover, M12T mutation also led to inclination of the NTD towards fibril structure formation. The length of the disordered region increased with the decrease in hydrophobicity in the proximal, as well as the distal NTD. However, abolishing the negative charge resulted in spatial rearrangements of the NTD, without affecting the disordered region. Tpv sHSP 14.3 WT protein possessed an oligomeric profile composed of 24-mer, in addition to 60-mer, dodecamer and the active dimeric forms. The I5T mutation alone, or in combination with F7SF8Y mutations, resulted in disappearance of 24-mer oligomer and a new species 36-meric form is formed. However, F7SF8Y double mutation resulted in disappearance of all species larger than 24-mer. In addition, for the first time, in this study it has been found that the whole NTD of Tpv sHSP 14.3, an archaeal sHSP, also possessed chaperone activity. Moreover, it was shown that a section of NTD, the initial 12 amino acids, possessed protection capacity for substrate against heat induced aggregation, equivalent to the intact NTD.

Subject Keywords

Tpv sHSP 14.3, N-terminal domain (NTD), Thermoplasma volcanium, Hydrophobicity, Mutagenesis, Chaperone activity, 3-D model structure analysis

URI

https://hdl.handle.net/11511/97360

Collections

Graduate School of Natural and Applied Sciences, Thesis