ASSIGNMENTS OF BACKBONE H-1, C-13, AND N-15 RESONANCES AND SECONDARY STRUCTURE OF RIBONUCLEASE-H FROM ESCHERICHIA-COLI BY HETERONUCLEAR 3-DIMENSIONAL NMR-SPECTROSCOPY

The assignments of individual magnetic resonances of backbone nuclei of a larger protein, ribonuclease H from Escherichia coli, which consists of 155 amino acid residues and has a molecular mass of 17.6 kDa are presented. To remove the problem of degenerate chemical shifts, which is inevitable in proteins of this size, three-dimensional NMR was applied. The strategy for the sequential assignment was, first, resonance peaks of amides were classified into 15 amino acid types by H-1-N-15 HMQC experiments with samples in which specific amino acids were labeled with N-15. Second, the amide H-1-N-15 peaks were connected along the amino acid sequence by tracing intraresidue and sequential NOE cross peaks. In order to obtain unambiguous NOE connectivities, four types of heteronuclear 3D NMR techniques, H-1-N-15-H-1 3D NOESY-HMQC, H-1-N-15-H-1 3D TOCSY-HMQC, C-13-H-1-H-1 3D HMQC-NOESY, and C-13-H-1-H-1 3D HMQC-TOCSY, were applied to proteins uniformly labeled either with N-15 or with C-13. This method gave a systematic way to assign backbone nuclei (N, NH, C-alpha-H, and C-alpha) of larger proteins. Results of the sequential assignments and identification of secondary structure elements that were revealed by NOE cross peaks among backbone protons are reported.