Hydration water molecules of nucleotide-free RNase T1 studied by NMR spectroscopy in solution.
The hydration of uncomplexed RNase T1 was investigated by NMR spectroscopy at pH 5.5 and 313 K. Two-dimensional heteronuclear NOE and ROE difference experiments were employed to determine the spatial proximity and the residence times of water molecules at distinct sites of the protein. Backbone carbonyl oxygens involved in intermolecular hydrogen bonds to water molecules were identified based on 1J(NC) coupling constants. These coupling constants were determined from 2D-H(CA)CO and 15N-HSQC experiments with selective decoupling of the 13C alpha nuclei during the t1 evolution time. Our results support the existence of a chain of water molecules with increased residence times in the interior of the protein which is observed in several crystal structures with different inhibitor molecules and serves as a space filler between the alpha-helix and the central beta-sheet. The analysis of 1J(NC) coupling constants demonstrates that some of the water molecules seen in crystal structures are not involved in hydrogen bonds to backbone carbonyls as suggested by crystal structures. This is especially true for a water molecule, which is probably hydrogen bonded by the protonated carboxylate group of D76 and the hydroxyl group of T93 in solution, and for a water molecule, which was reported to connect four different amino acid residues in the core of the protein by intermolecular hydrogen bonds.
Pfeiffer
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S
Institut für Biophysikalische Chemie, Johann Wolfgang Goethe-Universität, Frankfurt, Germany.
Spitzner
N
N
Löhr
F
F
Rüterjans
H
H
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011522:Protons; D006163:Ribonuclease T1 / Q000737:chemistry*; D012996:Solutions; D014867:Water / Q000737:chemistry
D011522:Protons; D012996:Solutions; D014867:Water; D006163:Ribonuclease T1