Effect of pressure on individual hydrogen bonds in proteins. Basic pancreatic trypsin inhibitor.

By performing two-dimensional 1H NMR measurements at 750 MHz at varying hydrostatic pressure (1-2000 bar) in an aqueous environment (90% 1H2O/10% 2H2O), we found that the signals of the peptide NH protons of basic pancreatic trypsin inhibitor (BPTI) in the folded state shift their positions linearly and reversibly with pressure. The strong tendency for low-field shifts of these protons indicates that most of the amide groups form hydrogen bonds either with carbonyls or with water and that these hydrogen bonds are shortened by pressure. The NH protons interacting favorably with solvent water tend to exhibit larger pressure-induced shifts than others, showing that the shift can be used as a diagnostic probe for the hydrogen bonding state of an NH group with water. Furthermore, we estimated shortening of individual H...O distances of the NH...O=C hydrogen bonds at 2000 bar on the basis of the empirical shift-distance correlation for BPTI. The estimated shortened distances varied considerably from site to site in the range of 0-0.11 A, larger in the turn but smaller in the interiors of secondary structures. These variations suggest that the volume fluctuation is heterogeneous within BPTI and that high-pressure NMR at high field can offer a unique opportunity for detecting microscopic structural fluctuation in proteins.