Water Penetration in the Low and High Pressure Native States of Ubiquitin

Theoretical studies on the solvation of methane molecules in water have shown that the effect of increased pressure is to stabilize solvent separated contacts relative to direct contacts. This suggests that high pressure stabilizes waters that have penetrated into a protein's core, indicating a mechanism for the high pressure denaturation of proteins. We test this theory on a folded protein by studying the penetration of water into the native state of ubiquitin at low and high pressures, using molecular dynamics. An ensemble of conformations sampled in the folded state of ubiquitin has been determined by NMR at two pressures below the protein's denaturation pressure, 30 atm and 3000 atm. We find that 1-5 more waters penetrate the high pressure conformations than the low pressure conformations. Low volume configurations of the system are favored at high pressures, but different components of the system may experience increases or decreases in their specific volumes. We find that penetrating waters have a higher volume per water than bulk waters, but that the volume per protein residue may be lowered by solvation. Furthermore, we find that penetration of the protein by water at high pressures is driven by the difference in the pressure dependence of the probability of cavity opening in the protein and pressure dependence of the probability of cavity opening in the bulk solvent. The volume changes associated with cavity opening and closing indicate that each penetrating water reduces the volume of the system by about 12 mL/mol. The experimental volume change going from the low pressure to the high pressure native state of ubiquitin is 24 mL/mol. Our results indicate that this volume change can be explained by penetration of the protein by two water molecules.