Internal Cavities and their Role as Determinants of Pressure Unfolding of Proteins

Abstract

The molecular mechanisms of chemical and heat denaturation of proteins are relatively well established; those of pressure unfolding are not. Volume is the conjugate variable of pressure; it is the fundamental thermodynamic variable governing the pressure sensitivity of proteins. By generating artificial cavities by substitution of internal hydrophobic residues in staphylococcal nuclease (SNase) with Ala we have shown previously that cavities that are present in the native state and absent in the unfolded state contribute significantly to the change in volume upon unfolding (ΔV). We have extended these studies to variants of SNase with very large cavities achieved with multiple Ala substitutions, variants with substitutions of small residues with large ones that eliminate the natural cavity, and variants with substitutions to polar residues to affect the state of hydration of cavities to study its role in pressure unfolding. For 27 variants studied we obtained (a) crystal structures, (b) thermodynamic stabilities using chemical denaturation, and (c) ΔV of unfolding measured by pressure denaturation monitored with Trp fluorescence. ΔV of unfolding were also measured using NMR spectroscopy for select variants. The cavities generally did not affect the structure. Although large enough to hold several waters, water molecules were only detected in the cavities when lined with polar groups. The measured ΔV of variants was always larger than for the wild-type. A near-linear correlation between the ΔV measured experimentally and the one calculated from structures illustrates the importance of cavities in pressure sensitivity. A correlation between measured ΔV and thermodynamic stability (ΔG) suggests that 1 kcal/mol is lost per 12 mL/mol of increased void volume. This study demonstrates irrefutably the significant contributions cavities make towards the pressure sensitivity of proteins and their effects on internal hydration and structural fluctuations of proteins.