HYDRATION WATER-MOLECULES OF NUCLEOTIDE-FREE RNASE T-1 STUDIED BY NMR-SPECTROSCOPY IN SOLUTION

The hydration of uncomplexed RNase T-1 was investigated by NMR spectro scopy at pH 5.5 and 313 K. Two-dimensional heteronuclear NOE and ROE d ifference experiments were employed to determine the spatial proximity and the residence times of water molecules at distinct sites of the p rotein. Backbone carbonyl oxygens involved in intermolecular hydrogen bonds to water molecules were identified based on (1)J(NC') coupling c onstants. These coupling constants were determined from 2D-H(CA)CO and N-15-HSQC experiments with selective decoupling of the C-13(alpha) du ring the t(1) evolution time. Our results support the existence of a c hain of water molecules with increased residence times in the interior of the protein which is observed in several crystal structures with d ifferent inhibitor molecules and serves as a space filler between the alpha-helix and the central beta-sheet. The analysis of (1)J(NC') coup ling constants demonstrates that some of the water molecules seen in c rystal structures are not involved in hydrogen bonds to backbone carbo nyls as suggested by crystal structures. This is especially true for a water molecule, which is probably hydrogen bonded by the protonated c arboxylate group of D76 and the hydroxyl group of T93 in solution, and for a water molecule, which was reported to connect four different am ino acid residues in the core of the protein by intermolecular hydroge n bonds.