Pressure response of protein backbone structure.: Pressure-induced amide 15N chemical shifts in BPTI

The effect of pressure on amide N-15 chemical shifts was studied in uniformly N-15-labeled basic pancreatic trypsin inhibitor (BPTI) in 90%(H2O)-H-1/10%(H2O)-H-2, pH 4.6, by H-1-N-15 heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most N-15 signals were low field shifted linearly and reversibly with pressure (0.468 +/- 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0-1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three-dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 Angstrom/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of N-15 shifts is considered to reflect site-specific changes in phi, psi, angles. For beta-sheet residues, a decrease in psi angles by 1-2 degrees/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 +/- 0.343 and 0.519 +/- 0.261 ppm/2 kbar, respectively) than for beta-sheet (0.295 +/- 0.195 ppm/2 kbar), suggesting that the pressure-induced structural changes (local compressibilities) are larger in helical and loop regions than in beta-sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in beta-sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place.