Understanding the physical determinants of pressure denaturation: Temperature switches the pressure-induced unfolding pathway

Abstract

AbstractProteins unfold under different environmental insults, among which heat, cold, high pressure and chaotropic agents. Understanding the mechanisms that determine unfolding under each of these condition is an important problem that directly relates to the physical forces that determine the three-dimensional structure of a protein. Here, we studied the mechanism of pressure unfolding of the marginally stable yeast protein Yfh1 using high-pressure nuclear magnetic resonance as this is the most appropriate technique to obtain a residue-specific description of the folding/unfolding transitions of a protein. We demonstrate that the pressure-unfolded spectrum shares features in common with that at low but not at high temperature and room pressure, suggesting a tighter similarity between the two processes that could be explained by a similar role of hydration in the process. This is the first time that comparison between the three infolded states could be tested experimentally, and confirms something that up to now has been only suggested as an hypothesis. By recording the phase diagram of the protein, we also show that temperature switches the pressure-induced unfolding pathway suggesting a synergic mechanism between pressure- and temperature-induced denaturation: at moderate pressures, Yfh1 unfolding at low temperature starts at a patch of negatively charged residues that we have previously demonstrated to induce electrostatic frustration and cause cold denaturation. Heat unfolding involves instead a cavity created by insufficient protection of the hydrophobic core. These observations help us to reconstruct the structural events determining unfolding and distinguish the mechanisms that rule the different processes of unfolding.