The effect of pressure on amide 15N
chemical shifts was studied in uniformly 15N-labeled
basic pancreatic trypsin inhibitor (BPTI) in 90%1H2O/10%2H2O,
pH 4.6, by 1H-15N heteronuclear correlation
spectroscopy between 1 and 2,000 bar. Most 15N
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 Å/2 kbar for the bond between the amide nitrogen
atom and the oxygen atom of either carbonyl or water. The
variation of 15N shifts is considered to reflect
site-specific changes in φ, ψ angles. For β-sheet
residues, a decrease in ψ angles by 1–2°/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 β-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 β-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 β-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.