Characterization of low-lying excited states of proteins by high-pressure NMR.

Hydrostatic pressure alters the free energy of proteins by a few kJ mol, with the amount depending on their partial molar volumes. Because the folded ground state of a protein contains cavities, it is always a state of large partial molar volume. Therefore pressure always destabilises the ground state and increases the population of partially and completely unfolded states. This is a mild and reversible conformational change, which allows the study of excited states under thermodynamic equilibrium conditions. Many of the excited states studied in this way are functionally relevant; they also seem to be very similar to kinetic folding intermediates, thus suggesting that evolution has made use of the 'natural' dynamic energy landscape of the protein fold and sculpted it to optimise function. This includes features such as ligand binding, structural change during the catalytic cycle, and dynamic allostery.