Assignments of backbone 1H, 13C, and 15N resonances and secondary structure of ribonuclease H from Escherichia coli by heteronuclear three-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 1H-15N HMQC experiments with samples in which specific amino acids were labeled with 15N. Second, the amide 1H-15N 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, 1H-15N-1H 3D NOESY-HMQC, 1H-15N-1H 3D TOCSY-HMQC, 13C-1H-1H 3D HMQC-NOESY, and 13C-1H-1H 3D HMQC-TOCSY, were applied to proteins uniformly labeled either with 15N or with 13C. 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.
Yamazaki
T
T
Biometrology Lab, JEOL Limited, Tokyo, Japan.
Yoshida
M
M
Kanaya
S
S
Nakamura
H
H
Nagayama
K
K
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D016914:Ribonuclease H
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D006859:Hydrogen; D004722:Endoribonucleases; D016914:Ribonuclease H