Reversible and irreversible denaturation processes in globular proteins: from collective to molecular spectroscopic analysis

Different spectroscopic techniques were applied for studying the structural properties of lysozyme in salt-free aqueous solutions. The results of vibrational and Brillouin scattering measurements were compared to obtain both single-molecule and collective properties of the solutions. The characterization of the protein system, from the conformation of the polypeptide chain to the exposure of side chains to the solvent and the arrangement of the solution network, was then achieved in the range 25-85 degrees C. Through the analysis of the indole breathing mode, a different environment for the six tryptophan residues of an unfolded lysozyme could be evidenced. Short and long exposures to high temperatures were used to modulate the competition between the thermally induced reversible and irreversible denaturation processes. These different thermal treatments were applied to distinguish between the effects of global unfolding of the single molecule from those of self-aggregation and gel formation. It has been observed that clusterization occurs at melting temperatures with slow kinetics; also, aggregates evolve from the completely unfolded state of the protein and lead to a sensitive increase in viscosity. This effect probably hinders any further conformational rearrangement of the molecules in the aggregate; thus as a consequence, the disordered structure of clusters does not change to give the beta-sheet organization, characteristic of filaments or fibrils.