Abstract
The present study describes the characterization of the unfolding and refolding pathways of the acidic human fibroblast growth factor (hFGF-1). The first chapter provides an updated, concise but in-depth introduction to the various topics which are relevant to the research elaborated in this study. The second chapter of the thesis reports the expression, purification and characterization of hFGF-1. A detailed account of the identification and characterization of an equilibrium intermediate is provided in Chapter-III of the thesis. It is found that hFGF-1 exists in a molten-globule-like intermediate at a guandinie hydrochloride concentration close to 1M. Using a variety of biophysical techniques including multidimensional NMR, the equilibrium intermediate is shown to have structural character(s) resembling that of the native state. The refolding kinetics of hFGF-1 is discussed in detail in Chapter-IV of the thesis. It is observed that hFGF-1 refolds from its urea unfolded state very slowly (> 50 seconds) but yet co-operatively. The refolding of the protein proceeds without hydrophobic collapse or formation of stable kinetic intermediates. The chronology of refolding events monitored by quenched-flow NMR revealed that hydrogen bond formation between the N- and C- terminal ends is the first detectable event in the refolding pathway of hFGF-1. Finally, in Chapter V, the role of osmolytes, such as proline in the prevention of aggregation during protein refolding is investigated. It is observed that proline effectively thwarts protein aggregation and helps the protein to regain its native state. The chaperone-like activity of proline is attributed to its ability to form a supramolecular assembly. It is envisaged that the supramolecular assemblies of proline provides the necessary hydrophobic contacts to the solvent exposed, non-polar surface(s) of the refolding protein and consequently prevents aggregation The research described herein offers several new leads which could be successfully exploited to formulate important and interesting research projects in the future.