Universal Convergence of the Specific Volume Changes of Globular Proteins Upon Unfolding

Abstract

Both pressure and temperature are important environmental variables, and in order to obtain a complete understanding of the mechanisms of protein folding, it is necessary to determine how protein stability is dependent on these fundamental thermodynamic parameters. Although the temperature dependence of protein stability has been widely explored, the dependence of protein stability on pressure is not as well studied. In this paper, we report the results of the direct thermodynamic determination of the change in specific volume (ΔV/V) upon protein unfolding, which defines the pressure dependence of protein stability, for five model proteins (ubiquitin, eglin c, ribonuclease A, lysozyme, and cytochrome C). We have shown that the specific volumetric changes upon unfolding for four of the proteins (ubiquitin, eglin c, ribonuclease A, lysozyme) appear to converge to a common value at high temperature. Analysis of various contributions to the change in volume upon protein unfolding allowed us to put forth the hypothesis that the change in volume due to hydration is very close to zero at this temperature, such that ΔV/V is defined largely by the total volume of cavities and voids within a protein, and that this is a universal property of all small globular proteins without prosthetic groups. To test this hypothesis, additional experiments were performed with variants of eglin c that had site-directed substitutions at two buried positions, in order to create an additional cavity in the protein core. The results of these experiments, coupled with the structural analysis of cytochrome c showing a lower packing density compared to the other four proteins, provided further support for the hypothesis. The deviation of the high-temperature ΔV value from the convergence value can be used to experimentally determine the size of the excess cavities in proteins.