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

Proton nuclear magnetic resonance was used to study individual molecules of hydration water bound to the protein basic pancreatic trypsin inhibitor (BPTI) in aqueous solution. The experimental observations are nuclear Overhauser effects (NOE) between protons of individual amino acid residues of the protein and those of sufficiently tightly bound water molecules. These NOEs were recorded by two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) in the laboratory frame, and by the corresponding experiment in the rotating frame (ROESY). The detection of NOEs with water protons was enabled by a solvent suppression technique which provides a uniform excitation profile in NOESY and ROESY, except at the ω₂frequency of the water signal. From NOESY and ROESY spectra recorded at 5, 36, 50, and 68 °C, intermolecular ¹H-¹H NOEs between the protein and four water molecules buried in its interior were individually assigned, and additional NOEs between surface residues of the protein and labile protons with the chemical shift of the bulk water were identified. For the hydration waters that can be observed by NOEs at 36 °C, an upper limit for the proton-exchange rate with the bulk water is estimated to be 3 × 10⁹ s⁻¹. These NOE-observable water molecules account only for a small percentage of the hydration waters seen in the crystal structure of BPTI. This observation supports the independently established picture of increased disorder near the molecular surface in protein structures in solution.