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Title:	Structural and functional characterisation of glaciozyma antarctica molecular chaperones
Authors:	Nur Athirah Yusof (P58673)
Supervisor:	Abdul Munir Abdul Murad, Dr.
Keywords:	Glaciozyma antarctica Molecular chaperones Proteins Characterisation
Issue Date:	20-Aug-2015
Description:	Glaciozyma antarctica is a psychrophilic yeast that has the ability to thrive in extreme cold environment. The acclimation of G. antarctica to low temperature involves the induction of several groups of molecular chaperones that appear to be involved in proper protein folding. The objectives of this study were to identify possible genes that encode for molecular chaperones from G. antarctica genome as well as elucidate the structure and characterise the function of selected G. antarctica molecular chaperones. From G. antarctica genome mining, a total of 89 genes encoding molecular chaperones were identified. Using reverse transcriptase-polymerase chain reaction, a total of 54 cDNA were amplified from the mRNA extracted from cells grown at 4°C. All recombinant G. antarctica molecular chaperones were produced in Escherichia coli expression system. However, only two proteins, namely TCP-1 Ring Complex 5/ε-subunit (GaTRiCε) and Suppressor of G2 (Two) allele of skp1 (GaSgt1) were produced in soluble monomeric form whereas the others were produced in inclusion bodies and aggregates. The gene expression analysis of all eight GaTRiC subunits showed that all subunits were constitutively expressed at temperatures -12°C, 0°C and 20°C. Chaperone activity assays showed that GaTRiC was able to refold denatured luciferase to functional state with the highest luciferase activity at 4°C followed by 12°C, 0°C and 30°C. As for GaSGT1, the gene expression showed higher induction at 20°C which was 16.3-fold followed by 12.5-fold at -12°C, 2.9-fold at 0°C and 1.7-fold at 4°C when compared to G. antarctica optimal growth temperature which was 12°C. Thermotolerance experiment of GaSgt1 in E. coli managed to protect the bacterial cells from lethal growth temperatures which were at 50°C for 75 minutes and 0°C for 8 days. Luciferase activity assay incubated with GaSgt1 showed perpetuation of 60% of its activity at 40°C. Crystallisation of the GaTRiCε and GaSgt1 recombinant proteins and native complex of GaTRiC was done in order to elucidate the structure and function of G. antarctica molecular chaperones. However, several attempts to get a good diffraction for all protein crystals failed to generate satisfactory diffraction data. Therefore, protein homology modeling was used to construct the protein structure of GaTRiC and GaSgt1 since the protein sequences showed more than 30% identity to known structures in the Protein Data Bank (PDB). Based on the developed models, GaTRiC is a hetero-oligomeric chaperonin that consists of eight homologous subunits that have three domains: substrate binding apical domain, intermediate domain and ATP-binding domain. Majority of the variations in the sequences were found in the substrate binding apical domains advocating the important for substrate recognition. Interestingly, three categories of residue substitutions were found in α, β and δ subunits: (i) bulky/polar side chains to alanine or valine, (ii) charged residues to alanine, and (iii) isoleucine to valine that would be expected to increase intramolecular flexibility within the GaTRiC. Observation of alanine substitution on the apical domain extrapolated from this structure model unveil that hydrophobicity and polarity contribute to substrate recognition. Additionally, superimposed of GaSgt1 structure showed high alanine substitution in place of charged side chains and bulky polar residues on its template structure. The residue substitutions observed in the build structures possibly affect the hydrophobic, hydrogen bonds, ionic, aromatic-aromatic, aromatic-sulphur and cation-pi interactions which lead to increase in structure flexibility. In conclusion, the molecular chaperones GaTRiC and GaSgt1 demonstrated chaperone activities in vitro and the protein structure flexibility is expected to contribute towards cold adaptation of these proteins.,Certification of Master's/Doctoral Thesis" is not available
Pages:	344
Call Number:	QP552.M64N87 2016 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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