PROTEIN HYDRATION DYNAMICS IN AQUEOUS-SOLUTION - A COMPARISON OF BOVINE PANCREATIC TRYPSIN-INHIBITOR AND UBIQUITIN BY O-17 SPIN RELAXATION DISPERSION

Water oxygen-17 spin relaxation was used to study hydration and dynamics of the globular proteins bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin in aqueous solution. The frequency dispersion of the longitudinal and transverse relaxation rates was measured over the Larmor frequency range 2.6 to 49 MHz in the pD range 2 to 11 at 27 degrees 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.