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

Abstract

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.