2-DIMENSIONAL H-1, N-15-NMR INVESTIGATION OF UNIFORMLY 15N-LABELED RIBONUCLEASE-T1 COMPLETE ASSIGNMENT OF N-15 RESONANCES

Uniformly N-15-enriched ribonuclease T1 (RNase T1) was obtained from Escherichia coli by recombinant techniques. Heteronuclear H-1, N-15-shift correlation spectra were recorded utilizing proton detection. Direct H-1, N-15 connectivities were established applying the heteronuclear multiple-quantum coherence technique. Additional H-1, H-1-TOCSY or H-1, H-1-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 (NH)-N-15 and (NH2)-N-15 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 H-1, N-15 NMR investigations were performed on the RNase-T1-3'GMP inhibitor complex. Results were interpreted with respect to nucleotide binding.