Study of protein folding under crowding and confined-space condition that mimics intracellular environments

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Hydration of biological macromolecules plays an important role in their structural stability and functions. Solvation of proteins is the key determinant for isothermal, concentration-dependent effects on protein equilibria, such as folding. On the other hand, the interior of a cell is very crowded with various macromolecules, and proteins must be designed to function in environments crowded by co-solutes. There is no doubt that crowding environments change protein equilibria, however, interpretations of the effect of crowding remain controversial since structural studies of protein folding and stability were conducted in dilute solutions in many cases. This project was executed to clarify how a protein folds into its native structure under a crowding condition created by high-macromolecular-weight co-solutes (polyvinylpyrrolidone (PVP)). The presence of PVP affords osmotic pressure to proteins. 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). We have found that the increase of osmotic pressure induced a change of hydration-shell density accompanying a suppression of the intramolecular fluctuation and also stabilized the intermediate unfolded state, so-called a molten globule state in the thermal unfolding process at high osmotic pressure.