Assessing the Contribution of Cavity Density to Protein Partial Molar Expansivity by High-Pressure NMR

Abstract

Staphylococcal nuclease (SNase) protein is a model protein folding system. We recently reported that the magnitude of internal void volumes primarily determines proteins sensitivity to pressure (1) and that the tridimensional position of cavities can modulate the folding free-energy landscape (2). Here we characterize the effect of temperature on pressure denaturation of these previously studied cavity containing variants of SNase (L125A and I92A) to address the determinants of thermal expansivity using high-pressure NMR. The experiments show that thermal expansivity is sensitive to changes in the degree of internal packing. We corroborate these observations by measuring the coefficient of thermal expansion using pressure perturbation calorimetry. We also analyze the effect of temperature on the activation volume using pressure-jump kinetics NMR. We find that the transition state ensemble presents structurally heterogeneous expansion with significant differences among the mutants tested. We take advantage of the single residue resolution analysis to map the differences of expansivity on the tridimensional structure of proteins tested. We propose that the network of interactions in the protein interior is responsible for the degree of thermal expansion. Assessing this unexplored thermodynamic parameter will provide unique insight into protein behavior in the pressure-Temperature plane.