Hyper-thermal stability and unprecedented re-folding of solvent-free liquid myoglobin

Isolating solvent effects by studying proteins in a liquid phase devoid of solvent has not been previously possible because freeze-dried protein solids do not melt but thermally degrade. Herein we circumvent this problem by modifying the interactions between myoglobin molecules via a polymer-surfactant coronal layer to produce a solvent-free liquid phase that is thermally stable over a wide temperature range. Using high-resolution synchrotron radiation circular dichroism and UV-Vis spectroscopies we determine the temperature-dependent structure and re-folding behaviour of cationized myoglobin under solvent-free conditions, and show that dehydration and subsequent melting of the nanoconstruct has no significant effect on the protein secondary structure at room temperature. Significantly, the solvent-free liquid myoglobin molecules exhibit hyper-thermophilic behaviour and can be reversibly re-folded by cooling from 155 degrees C. We attribute the abnormally high thermal stability and persistence of protein folding to entropic contributions associated with macromolecular crowding and confinement, and propose that re-folding in the absence of a solvent shell is facilitated by the configurational flexibility and molecular interactivity of the polymer surfactant coronal layer.