Abstract
ABSTRACT The present study describes the characterization of folding intermediates, dynamics properties and stability of the antitumor antibiotic chromoprotein neocarzinostatin (NCS). NCS is a potent antitumor drug consisting of an all-β sheet protein (11 k Da), aponeocarzinostatin (apoNCS), and a non-covalently bound, highly cytotoxic enediyne-structured NCS chromophore. The first chapter provides an elaborate introduction to the various topics, principles, theory which are relevant to the research described in the study. The second chapter describes the expression, purification and characterization of apoNCS and reconstitution of NCS chromophore into recombinant apoNCS to prepare holoneocarzinostatin (HoloNCS). Third chapter describes characterization and biological significance of a stable partially structured state, which accumulates in the equilibrium unfolding pathway(s) of apoNCS. In the presence of 1.2 M guanidine hydrochloride at pH 3 apoNCS forms a partially structured intermediate state. This intermediate is characterized using various biophysical techniques. Fourth chapter deals with the dynamical properties of apo- and holoNCS using 2D-NMR and computational analysis. In this chapter, we discussed how the apoNCS differs from holoNCS in dynamic properties, and its significance in drug delivery is also discussed. In fifth chapter, we investigated the stability of apoNCS using various techniques like guanidine hydrochloride-induced denaturation, thermal denaturation, and H/D exchange by 2D-NMR. Beta strands 2, 3 and 9 have higher protection factor than other strands and constitute the stability core of the protein. This provides protection to the chromophore. The loop regions between strands 7 & 8 and 9 & 10 in the opening of the binding pocket are more flexible and could be the regions responsible for chromophore release. Chapter six describes about interesting effects of fluorescence light on proteins and its influence on thermodynamic parameters. Fluorescence is an important instrument routinely used to characterize biophysical properties of proteins. Amazingly, the fluorescence light induces in situ photo reaction in proteins when the protein is exposed to fluorescence light during experiments like thermal- or chemical-induced unfolding monitored by fluorescence spectroscopy. This was proved by biophysical analyses of samples irradiated with fluorescence light.