Thermal unfolding and refolding of protein under osmotic pressure clarified by wide-angle X-ray scattering

By using wide-angle X-ray scattering, we have studied the effect of osmotic pressure on protein unfolding and refolding of hen egg-white lysozyme (HEWL). The osmotic pressure was varied by adding polyvinylpyrrolidone (PVP). The increase of PVP concentration induced both the shortening of the intermolecular distance and the decrease of radius of gyration (Rg), indicating that PVP stabilizes HEWL to take another stable and compact structure. The decrement of Rg can be explained by a change of hydration-shell density accompanying a suppression of the intramolecular fluctuation by osmotic pressure. The increase of PVP concentration also stabilized the intermediate unfolded state, so-called a molten globule state in the thermal unfolding process at high osmotic pressure. The thermal reversibility of the structural transition was suppressed. The present results suggest that PVP works as a stabilizer for HEWL in the crowed solution through the changes both in the hydration shell and the intermolecular interaction.