It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavitycontaining variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.energy landscape | fluorescence | volume change T he first observation that pressure unfolds proteins was made in 1914 by Bridgman (1). Despite numerous studies since then, the physical basis of the pressure-induced unfolding of proteins has not been explained. This difference in volume underlying pressure effects has been rationalized previously in terms of (i) increases in solvent density concomitant with solvation of exposed surfaces upon unfolding (2), (ii) modifications in the structure of bulk water leading to weakened hydrophobic interactions (3), and (iii) cavities in the folded state that are not present in the unfolded state (4-7). The goal of this study was to examine the structural origins of pressure unfolding of proteins. Twenty-five years ago Walter Kauzmann stressed the importance of understanding pressure effects (8): "Enthalpy and volume are equally fundamental properties of the (protein) unfolding process, and no model can be considered acceptable unless it accounts for the entire thermodynamic behavior." He also noted important discrepancies between the volumetric properties of hydrophobic interactions and the pressure unfolding of proteins.To date, no conclusive explanation for the origins of pressure unfolding of proteins has been proposed. In the present work, 10 cavity-containing variants of staphylococcal nuclease (SNase) were engineered by substitution of internal core residues to Ala. Crystal structures of the variants were obtained to verify the exi...

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