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Studies of Protein Conformation and Folding Using Biochemical and Biophysical Methods

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Title Studies of Protein Conformation and Folding Using Biochemical and Biophysical Methods
 
Creator Ghosh, Ranendu
 
Subject Structural Biology & Bioinformatics
 
Description ow a protein spontaneously folds into its biologically active folded structure is an extremely interesting and still an open question. In order to attain its final active structure, a protein needs to search an enormous number of possible conformations. This process may lead to some conformational defects and formation of partially folded intermediate states.These partially folded intermediates often contain exposed hydrophobic surface through which they can interact leading to aggregation. Protein aggregation has been implicated in several physiological disorders. In addition, aggregated proteins are often immunogenic and hence un –desirable in the formulation development of bio - therapeutics. An understanding of the conformational changes during protein folding is important to elucidate the mechanism of protein aggregation. It is also important to look for an appropriate agent or small molecule, which can prevent the protein aggregation. In this thesis, we have studied different aspects of protein folding, aggregation and explored the possible use of a chemical osmolyte to combat protein aggregation. We begin by investigating the thermodynamics of folding of a small, globular mycobacterial secretory protein, MPT63 using chemical and thermal denaturation. We observe significant similarities in the thermodynamic parameters obtained using different methods. We have also observed the presence of residual structures in the unfolded states. Our study demonstrates that key processes leading to protein folding can be understood through detailed structural studies using conventional biophysical methods (like fluorescence, circular dichroism etc) along with less conventional phosphorescence spectroscopy. Phosphorescence spectroscopy has been used to unravel valuable insights into the surroundings of individual tryptophan residues of this multi – tryptophan protein (containing four tryptophan residues). We have studied the conformational changes of MPT63 in different solution pH conditions and observed spatial rearrangement in two tryptophan environments. The other two tryptophan local environments are found unperturbed at low pH leading to protein aggregation. The study highlights the promise of phosphorescence spectroscopy for increasing our understanding of the link between partially folded intermediates and protein aggregation. Finally, we use arginine, as a small molecule stabilizer to inhibit protein aggregation and to increase refolding yield of the thermally unfolded proteins. We indicate that arginine inhibits the formation of partially folded intermediates during unfolding, which would otherwise lead to protein aggregation.
 
Date 2013
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/1841/1/RANENDU_GHOSH_FINAL.pdf
Ghosh, Ranendu (2013) Studies of Protein Conformation and Folding Using Biochemical and Biophysical Methods. PhD thesis, Calcutta University.
 
Relation http://www.eprints.iicb.res.in/1841/