The effect of pressure on amide 15 N chemical shifts was studied in uniformly 15 N-labeled basic pancreatic trypsin inhibitor~BPTI! in 90% 1 H 2 O010% 2 H 2 O, pH 4.6, by 1 H-15 N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15 N signals were low field shifted linearly and reversibly with pressure~0.468 6 0.285 ppm02 kbar!, indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups~0-1.5 ppm02 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 Å02 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15 N shifts is considered to reflect site-specific changes in f, c angles. For b-sheet residues, a decrease in c angles by 1-2802 kbar is estimated. On average, shifts are larger for helical and loop regions~0.553 6 0.343 and 0.519 6 0.261 ppm02 kbar, respectively! than for b-sheet~0.295 6 0.195 ppm02 kbar!, suggesting that the pressure-induced structural changes~local compressibilities! are larger in helical and loop regions than in b-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 b-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.

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