Two-dimensional 1H NMR investigation of ribonuclease A and ribonuclease-A--pyrimidine-nucleotide complexes.
Ribonuclease A was studied by two-dimensional 1H NMR spectroscopy. 10 out of 12 alanine and 9 out of 10 threonine spin systems as well as all valine [9], leucine [2] and isoleucine [3] spin systems were identified from the correlated spectroscopy (COSY) and relayed coherence transfer spectroscopy (RCT). Sequence-specific assignments were obtained from nuclear Overhauser effect spectra for proton resonances of 21 amino acid moieties. 2' and 3'-pyrimidine-nucleotide-RNase-A complexes were also investigated by two-dimensional NMR. We were able to monitor structural changes in the active center, the vicinity of the active center and in regions far from the catalytic region. Chemical shift changes of resonances of protons near Thr-45 reflected the binding of the same moiety. This in turn is also dependent on the position of the nucleotide phosphate group. Binding of 2' nucleotides led to characteristic changes in protein regions not affected by the binding of 3' nucleotides. These results are interpreted in terms of structural differences between the 2' and 3'-nucleotide-RNase-A complexes; the structure of the complex of the native 3' nucleotide inhibitor being more closely related to that of the free protein.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons; D011742:Pyrimidine Nucleotides / Q000032:analysis*; D012259:Ribonuclease, Pancreatic / Q000032:analysis*
D000596:Amino Acids; D011522:Protons; D011742:Pyrimidine Nucleotides; D012259:Ribonuclease, Pancreatic