Structural and spectroscopic studies of proteins in stress conditions

This thesis incorporates studies about the structural characterization of proteins in solution mainly by means of small angle X-ray scattering (SAXS). The major part of the thesis has as main focus the study of the effect of stress conditions on the conformation of Human Serum Albumin (HSA), the most abundant plasma protein. The consequences of the hypochlorite-induced oxidation at increasing doses were explored with SAXS in combination with UV-visible spectroscopy and fluorescence, together with additional characterization. Despite the chemical modification, the native shape was preserved up to oxidant/HSA molar ratio < 80, above which a structural transition occurred in the critical oxidant/HSA molar ratio range between 80-120. This conformational variation involved the drifting of one of the end-domains from the rest of the protein and corresponded to the loss of one third of the -helix and a net increase of the protein negative charge. The high reproducibility and well-defined nature of this transition suggested that it represents a structural response characteristic of this multi-domain protein that was never observed before. The unfolding in acid conditions is another typical conformational variation of serum albumin, the existence of which is well known in this case. We managed to implement an experimental setup in which the process could be followed in-situ as a function of a continuous decrease of pH in time. This allowed for the correlation between the structural and spectroscopic data as a function of pH together with an attempt to resolve the structural conformers involved in the transition. In the framework of a collaboration with a structural biology - molecular plant physiology group of the University of Bologna, this thesis also includes a structural study in solution of redox-sensitive proteins of photosynthetic organisms and of their regulatory complexes. The SAXS analysis aided by 3D-modeling methods provided an insight into the shape of the individual enzymes and the multi-protein constructs, and into the way these proteins assembly and disassembly in response to redox regulation.