Protein folding and Disulphide rich peptide(s) folds

Abstract

Protein folding and protein folds are two sides of a coin. The study of protein folding processes is crucial for advancing our knowledge in fundamental cellular processes while the study of protein folds is important for understanding their function. The work presented in this thesis describes the study of folding-unfolding dynamics of proteins and structures of peptides at atomic resolution. Nuclear Magnetic Resonance (NMR) spectroscopy was used as the principal method of investigation. Part-I of this thesis involves the detection, identification and characterization of intermediate states in the folding/unfolding pathway of ubiquitin using solution NMR spectroscopy as a probe. As a part of this study, the exchange dynamics of the folding/unfolding intermediate states were characterized using dynamic NMR spectroscopy. In addition, the structure elucidation of these intermediate states is also performed providing valuable insights into the folding-unfolding processes at atomic detail. Part-II of this thesis involves the study and characterization of two novel and rare three disulphide bonded conopeptides - As25a identified in Conus cancellatus and α-SII identified in Conus striatus.These conopeptides were successfully overexpressed and purified recombinantly in E.coli. As25a purified as a single conformation in solution while α-SII existed in solution as three different foldamers as was seen from RP-HPLC and NMR spectroscopy. Solution NMR spectroscopic methods were used to obtain information on structure and dynamics of both these conopeptides. Preliminary electrophysiology studies performed on rat DRG neurons have indicated that the peptide As25a blocked the sodium ion gated channels in the DRG neuronal cells of size ≈85 μm. Part-III of this thesis involves the study of an intrinsically disordered host defence peptide, Urumin identified from a frog, Hydrophylax bahuvistara, endemic to the Western Ghats of India. In literature, urumin was reported to show anti-virucidal effect against Influenza A virus specifically containing H1 subtype of hemagglutinin. This intrinsically disordered peptide was successfully overexpressed and purified recombinantly in E.coli. NMR spectroscopy is used as a primary tool of investigation to characterize the peptide in its oxidised and reduced forms and also obtain information on the dynamics of various conformational ensembles sampled by the peptide in solution.