Folding And Conformation Studies Of Therapeutically Rel Kunduevant Proteins Using Biophysical And Biochemical Methods

Kundu, Amrita (2017) Folding And Conformation Studies Of Therapeutically Rel Kunduevant Proteins Using Biophysical And Biochemical Methods. PhD thesis, C U.


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    Chattopadhyay, Krishnananda


    landscape to achieve the correct physical conformation at a minimal energy state. A series of interactions between different amino acid residues in a regulated manner ultimately lead to a stable conformation. However, proteins sometimes fail to maintain their correct folded state under some circumstances. Misfolded or aggregated proteins are found to be associated with different diseases.In this study, we studied different conformational switches in the folding pathway of proteins that can lead to correct folding or misfolding. We chose three model systems of different proteins. These proteins have different secondary and tertiary structures in their native forms. Interestingly, all these three proteins are shown to be implicated in pathogenesis of three different diseases. In this thesis, we investigated the equilibrium unfolding transition of a Mycobacterium protein MPT63. MPT63 has been shown to raise humoral responses in tuberculosis patients. Our study showed that in spite of being a complete β-sheet protein, MPT63 has a prominent propensity towards helix structures in its early intermediates. Far UV-CD and FTIR spectra suggested that the low pH intermediate of MTP63 has enhanced helical contents, while fluorescence correlation spectroscopy suggested a significant compaction. Furthermore, molecular dynamicssimulation complemented the experimental results. Our data suggested that the secondary structure preferences of the local interactions in early folding pathway of a protein might not always follow the native conformation. Moreover, we showed that this non-native intermediate has a propensity to form amyloid aggregates. Our next model protein system was alpha-synuclein which is the major component in the amyloid plaques found in Parkinson’s disease. We used spectroscopic measurements at ensemble and single molecule resolution to study how the late and early events of -syn aggregation modulate each other. At the early stage, the protein fluctuated between conformers of different hydrodynamic radii and forms oligomers. The late stage followed through the formation of different sized iv intermediate species eventually generating amyloid fibrils. Additionally, we used two small molecules and investigated their effect on -syn folding/aggregation landscape. We chose an inhibitor arginine, and a facilitator glutamate based on their respective abilities to delay or accelerate the late stage of aggregation (amyloidosis) in solution and in live neuroblastoma cell line (SH-SY5Y). In the presence of the inhibitor molecule, the formation of a compact conformer was favored, while the addition of the facilitator sped up oligomerization. Our data also suggested that when arginine and glutamate were present together in a solution, they cancelled each other’s effects. Our last model system was a Saccharomyces serevisiae protein Sup35. Sup35 is the yeast version of translation termination factor. Incorrect folding due to mutation or other external triggers lead to Sup35 aggregation. Aggregated Sup35 showed human dentified and characterized the aggregation of the GFP-tagged Sup35 inside yeast cells. In this study, we chose a small molecule trehalose and investigated its effect on the Sup35 aggregation. Trehalose is the most studied chemical osmolyte which is naturally synthesized in yeast and reported to protect proteins from misfolding in stressed conditions. Additionally, we investigated the effect of different heatshock treatments on Sup35 expression pattern and aggregation inside live yeast cells in the absence and presence of trehalose. Our studies suggested that trehalose is able to successfully prevent the heatshock-induced enhanced aggregation of Sup35 in live yeast cells.

    Item Type: Thesis (PhD)
    Subjects: Structural Biology & Bioinformatics
    Divisions: Indian Institute of Chemical Biology
    Depositing User: Ms Sutapa Ganguly
    Date Deposited: 31 Jul 2017 15:28
    Last Modified: 31 Jul 2017 15:28

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