Abstract
Interactions between charged residues are known to have significant effects on protein folding stability and binding properties. The contributions of different types of non-covalent interactions are altered by mutating one or more residues, resulting in change in the protein stability to considerable extent. The main goal of this dissertation is to understand contributions, specifically from electrostatic interactions, to the protein folding stability and also, to devise strategies in order to enhance protein stability. We have introduced single and double mutations in FKBP12 (FK506 binding protein) modeled after its close homolog FKBP12.6, and later accumulated the most stabilizing mutations in order to create a hyper stable mutant. Similar experimental study was carried out, where insertion, deletion and systematic introduction of ion-pair clusters in FKBP12 were modeled after the thermophilic homolog MtFKBP17 and the results suggest that charge residues can modulate the folding stability significantly. This experimental study has allowed rigorous testing of computational models developed by our group for predicting electrostatic contributions to protein folding stability, which does not match closely and need further refinement. To further broaden our research in understanding biophysical properties of proteins in living cells, we have investigated the effects of macromolecular crowding on the folding stability of one of the less stable mutant of FKBP. We show that there is an optimal size of crowder at which stability increase is maximum and also, the stabilization effect of mixture (different crowders) is greater than the sum of constituent crowding agents. These findings may have profound implications for understanding crowding effects inside cells. The main aim of my dissertation is to understand protein folding and stability, which are the fundamental problems in biophysics. Experiments are underway to understand effects of crowding on protein binding and their quantitative information will provide key insights to the biological relevance of experimental results obtained in vitro. Overall my research aims for understanding fundamental study on protein folding stability and binding which will serve as a valuable tool for designing therapies for human diseases.
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