Role of Internal Cavities as Determinants of Pressure Unfolding of Proteins

Abstract

Pressure unfolds many proteins. In contrast to acid, heat and chemical denaturation, which have been studied with many proteins and which are relatively well understood, the molecular determinants of pressure unfolding of proteins are not known. Volume is the conjugate variable of pressure; therefore, the pressure unfolding of proteins is governed by differences in volume between the native and the unfolded ensembles. What is less obvious is where the differences in volume originate. Among the possibilities that have been considered are volume changes related to conformational fluctuations, volume changes related to hydration, and volume changes related to the loss of cavities present in the folded state. We have examined the role of cavities as determinants of pressure unfolding systematically, using staphylococcal nuclease as a model system. Ten different variants of a highly stable form of nuclease were engineered with single site substitutions in the hydrophobic core, designed to create cavities in the interior of the protein. High resolution crystal structures of all proteins were obtained to examine the state of the cavities and as starting structures for calculations of internal void volume with MD and MC methods. Pressure unfolding monitored with Trp fluorescence was used to measure volume changes upon unfolding. Our ultimate goal was to determine if volume changes measured in the equilibrium thermodynamic experiments correlated with the size of the cavities observed in the proteins. The mapping of experimentally determined cavity persistence ratios, combined with simulation data, provides new insight into the subtle interplay between local dynamics, water penetration, and structural properties of cavities in determining volume changes upon unfolding. Overall, the data suggest that internal cavities are one of the main determinants of the pressure-induced unfolding of proteins.