Two-dimensional 1H, 15N-NMR investigation of uniformly 15N-labeled ribonuclease T1. Complete assignment of 15N resonances.
Uniformly 15N-enriched ribonuclease T1 (RNase T1) was obtained from Escherichia coli by recombinant techniques. Heteronuclear 1H, 15N-shift correlation spectra were recorded utilizing proton detection. Direct 1H, 15N connectivities were established applying the heteronuclear multiple-quantum coherence technique. Additional 1H, 1H-TOCSY or 1H, 1H-NOESY transfer steps allowed for sequential assignments. Nitrogen atoms without directly bonded protons were detected by means of the heteronuclear multiple-bond correlation experiment. Signals emerging from 15NH and 15NH2 groups were distinguished by heteronuclear triple-quantum filtering methods. 119 nitrogen resonances out of the expected 127 were assigned unambiguously; in addition, previously obtained proton assignments were extended. Preliminary 1H, 15N NMR investigation were performed on the RNase-T1-3'GMP inhibitor complex. Results were interpreted with respect to nucleotide binding.
Schmidt
J M
JM
Institut für Biophysikalische Chemie, Johann-Wolfgang-Goethe-Universität Frankfurt, Federal Republic of Germany.
Thüring
H
H
Werner
A
A
Rüterjans
H
H
Quaas
R
R
Hahn
U
U
D009682:Magnetic Resonance Spectroscopy; D006163:Ribonuclease T1 / Q000737:chemistry*
D006163:Ribonuclease T1