Solvent exchange of buried water and hydrogen exchange of peptide NH groups hydrogen bonded to buried waters in bovine pancreatic trypsin inhibitor

Abstract

Solvent exchange of 18O-labeled buried water in bovine pancreatic trypsin inhibitor (BPTI), trypsin, and trypsin-BPTI complex is measured by high-precision isotope ratio mass spectrometry. Buried water is labeled by equilibration of the protein in 18O-enriched water. Protein samples are then rapidly dialyzed against water of normal isotope composition by gel filtration and stored. The exchangeable 18O label eluting with the protein in 10-300 s is determined by an H2O-CO2 equilibration technique. Exchange of buried waters with solvent water is complete before 10-15 s in BPTI, trypsin, and BPTI-trypsin, as well as in lysozyme and carboxypeptidase measured as controls. When in-exchange dialysis and storage are carried out at pH greater than or equal to 2.5, trypsin-BPTI and trypsin, but not free BPTI, have the equivalent of one 18O atom that exchanges slowly (after 300 s and before several days). This oxygen is probably covalently bound to a specific site in trypsin. When in-exchange dialysis and storage are carried out at pH 1.1, the equivalent of three to seven 18O atoms per molecule is associated with the trypsin-BPTI complex, apparently due to nonspecific covalent 18O labeling of carboxyl groups at low pH. In addition to 18O exchange of buried waters, the hydrogen isotope exchange of buried NH groups H bonded to buried waters was also measured. Their base-catalyzed exchange rate constants are on the order of NH groups that in the crystal are exposed to solvent (static accessibility greater than 0) and hydrogen-bonded main chain O, and their pH min is similar to that for model compounds. The pH dependence of their exchange rate constants suggests that direct exchange with water may significantly contribute to their observed exchange rate.

Protein Hydration Dynamics in Aqueous Solution: A Comparison of Bovine Pancreatic Trypsin Inhibitor and Ubiquitin by Oxygen-17 Spin Relaxation Dispersion

“…This picture is consistent, not only with the crystal structures (Deisenhofer & Steigemann, 1975;Wlodawer et al, 1984Wlodawer et al, , 1987a, but also with the recent high-resolution 1 H NMR studies (Otting & Wü thrich, 1989;Otting et al, 1991a) and computer simulations (Brunne et al, 1993) of BPTI. Our upper bound of four microseconds for the residence time of the exchanging internal water molecules is a factor 5000 lower than (but consistent with) the result from paramagnetic shift experiments (Otting et al, 1991b) and a factor 4 × 10 6 lower than the result from 18 O gel filtration experiments (Tü chsen et al, 1987). Furthermore, we believe that our picture of protein hydration is consistent with previous relaxation dispersion data, although not with all conclusions drawn therefrom.…”

How amide hydrogens exchange in native proteins

“…The HX rate, k HX , for H exchange into D 2 O has been measured by NMR for each of the 53 amides in BPTI (7,(50)(51)(52)(53). For our analysis, we use experimental PFs (at 300 K) for a subset of 41 amides.…”

A statistical mechanical model for hydrogen exchange in globular proteins

“…Proton NMR studies in BPTI show that exchange of water molecules between the protein core and bulk solvent can occur on a subsecond time scale under physiological conditions (Tuchsen et al, 1987;Otting et al, 1991).…”