Studies of Protein Hydration by Direct NMR Observation of Individual Protein-Bound Water Molecules

Abstract

A novel, improved scheme for two-dimensional NMR experiments with proteins in H2O solution was used to observe individual hydration water molecules. In these experiments the location of the hydration waters is determined from their nuclear Overhauser effects to distinct amino acid residues. In basic pancreatic trypsin inhibitor (BPTI) four internal water molecules which had been reported in three different crystal forms (Deisenhofer and Steigeman, 1975; Wlodawer et al., 1984, 1987) were found to be in the same locations also in the solution structure, with life times with respect to exchange of the water protons in excess of 0.30 ns. Additional NOE’s with polypeptide protons located on the protein surface may involve either hydration water molecules or hydroxyl protons of amino acid side chains. Their total number is small compared to the number of NOE’s expected from the hydration water molecules identified in the crystal structures of BPTI.