Water oxygen-17 and deuteron spin relaxation rates, measured as a function of resonance frequency, have been used to study the dynamics of protein hydration in aqueous solutions of ribonuclease A, lysozyme, myoglobin, trypsin and serum albumin. The relaxation data conform to the picture of protein hydration dynamics, proposed on the basis of previous studies of smaller proteins, where the long-lived water molecules responsible for the relaxation dispersion are identified with a small number of integral water molecules seen in the crystal structures. These integral water molecules, with residence times in the range 10-9-10-3 s, are either buried in internal cavities, trapped in narrow clefts or coordinated to metal ions. For the water molecules in the traditional hydration layer at the protein surface, the relaxation data suggest an average residence time in the range 10-50 ps, consistent with high-resolution 'H spectroscopy and computer simulations. The relaxation data also reveal some more specific features of protein hydration, relating to hydration of cavities that appear empty by crystallography, entrapment of water between structural domains of large proteins and subnanosecond 180" flips in buried water clusters.

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Water oxygen-17 spin relaxation was used to study hydration and dynamics Condensed Matter Magnetic Resonance Group, Chemical of the globular proteins bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin in aqueous solution. The frequency dispersion of the longitudinal Center, Lund University P. O. Box 124, S-22100 Lund and transverse relaxation rates was measured over the Larmor frequency Sweden range 2.6 to 49 MHz in the pD range 2 to 11 at 27°C. While the protein-induced relaxation enhancement was similar for the two proteins at high frequencies, it was an order of magnitude smaller for ubiquitin than for BPTI at low frequencies. This difference was ascribed to the abscence, in ubiquitin, of highly ordered internal water molecules, which are known to be present in BPTI and in most other globular proteins. These observations demonstrate that the water relaxation dispersion in protein solutions is essentially due to a few structural water molecules buried within the protein matrix, but exchanging rapidly with the external water. The relaxation data indicate that the internal water molecules of BPTI exchange with bulk water on the time-scale 10 −8 to 10 −6 second thus lowering the recently reported upper bound on the residence time of these internal water molecules by four orders of magnitude, and implying that local unfolding occurs on the submicrosecond time-scale. The water molecules residing at the surface of the two proteins were found to be highly mobile, with an average rotational correlation time of approximately 20 picoseconds. For both proteins, the oxygen-17 relaxation depended only very weakly on pD, showing that ionic residues do not perturb hydration water dynamics more than other surface residues. We believe that the present results resolve the long-standing controversy regarding the mechanism behind the spin relaxation dispersion of water nuclei in protein solutions, thus establishing oxygen-17 relaxation as a powerful tool for studies of structurally and functionally important water molecules in proteins and other biomolecules.

Buried water molecules constitute a highly conserved, integral part of nearly all known protein structures. Such water molecules exchange with external solvent as a result of protein conformational fluctuations. We report here the results of water (17)O and (2)H magnetic relaxation dispersion measurements on wild-type and mutant bovine pancreatic trypsin inhibitor in aqueous solution at 4-80 degrees C. These data lead to the first determination of the exchange rate of a water molecule buried in a protein. The strong temperature dependence of this rate is ascribed to large-scale conformational fluctuations in an energy landscape with a statistical ruggedness of approximately 10 kJ mol(-1).

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