Cavities in the Hydrophobic Core Govern Pressure Unfolding of Proteins

Abstract

Since Bridgman's seminal experiments on high-pressure denaturation of albumen in 19141, the origin of pressure unfolding of proteins has remained unresolved. We report here a systematic study of the contribution of cavities to the volume difference between unfolded and folded states (ΔVu), using 10 single point variants of staphylococcus nuclease (SNase). Each mutation is localised in a strategic position and was designed to change a large buried hydrophobic side chain into alanine, thus opening tunable cavities in the SNase structure. For every variant, a crystal structure confirmed the presence of the designed cavity with no detectable presence of water molecules. High-pressure fluorescence experiments show significantly larger ΔVu values for the cavity mutants in comparison to the reference protein. This demonstrates that solvent-excluded cavities make a major contribution to ΔVu. Thus, pressure effects have their origin in a property of the folded states of proteins, unlike temperature and chemical denaturants, whose effects are governed by exposed surface area in the unfolded state. High-pressure NMR experiments on 4 cavity mutants, recording HSQCs peak intensities up to 2500 bar, allowed precise estimations of the apparent ΔVu monitored by more than two-thirds of the residues. An innovative combination of the site-specific NMR data and Go-model simulations revealed significant departures from the apparent two-state folding process for the SNase reference protein and the cavity mutants. This study opens up highly promising perspectives on the use of high pressure for characterization of folding landscapes inaccessible by other methods.1. Bridgman, P. W. J. Biol. Chem. 1914.19, 511–512.