| While a great many protein structures are now known, considerably less is known experimentally about how these molecules reversibly fold and unfold into nearly unique, active structures. For more than 50 years, the central hypothesis has been that micro-scale phase separation between oil-like non-polar and charged or polar amino acid residues drives the formation of a "hydrophobic" protein core. A great deal of evidence supports this hypothesis, but the unfolding of proteins as a response to pressure contradicts it. A possible solution is the suggestion that pressure induced unfolding is a different process from thermally or chemically driven unfolding. Separately, little is known about the structural response of proteins to pressure, although pressure has clear and sometimes large effects on protein activity and stability. For these reasons, we have chosen to study the cavity-containing mutant L99A of T4 Lysozyme, along with the wild-type protein, under pressures up to 2 kbar, a compression of about 0.1%. The protein is remarkably incompressible, and the presence of a cavity has almost no impact on the pressure response over the wild-type lysozyme. Instead, four water molecules cooperatively fill the cavity, interacting with each other and the protein roughly equally. We believe the cavity to be half full with water near 2 kbar, suggesting that despite its hydrophobic nature, the interior of a protein maintains strong electrostatic interactions. |
