Abstract
Conformational fluctuations of proteins are crucially important for their functions. However, changes in the location and dynamics of hydrated water in many proteins accompanied by the conformational transition have not been fully understood. Here, we used phase-modulated clean chemical exchange NMR approach to investigate pressure-induced changes in water-to-amide proton exchange occurring at sub-second time scale. With the transition of ubiquitin from its native conformation (N(1)) to an alternative conformation (N(2)) at 250 MPa, proton exchange rates of residues 32-35, 40-41, and 71, which are located at the C-terminal side of the protein, were significantly increased. These observations can be explained by the destabilization of the hydrogen bonds in the backbone and partial exposure of those amide groups to solvent in N(2). We conclude that phase-modulated clean chemical exchange NMR approach coupled with pressure perturbation will be a useful tool for investigations of more open and hydrated protein structures.