Complete assignments of magnetic resonances of ribonuclease H from Escherichia coli by double- and triple-resonance 2D and 3D NMR spectroscopies.
Assignments of 1H, 15N, and 13C magnetic resonances for ribonuclease H from Escherichia coli have been completed using double- and triple-resonance 2D and 3D NMR experiments. These assignments include all types of 1H, 15N, and 13C nuclei detectable by NMR. The enzyme used, which cleaves the RNA moiety of an RNA-DNA duplex, consists of 155 amino acid residues and has 1962 nuclei (227 nitrogen, 762 carbons, and 973 protons) observable independently by NMR. Among those, 1868 nuclei (95%) have been assigned. Two methods, 3D HCH and 13C-13C-1H heteroSQC/homoSQC, were newly devised to complete the side chain assignments. These methods were used to elucidate the -CH2- and -C-CH-substructures. Triple-resonance experiments to detect other types of substructures, (e.g., -N-CH- and -C-NH-) were also applied. In total, 10 kinds of 3D NMR experiments were used to complete the assignments. The chemical shifts obtained through the assignments were analyzed in terms of the tertiary structure of the protein molecule. Among the 13C chemical shifts, larger secondary shifts (deviations from shifts at the random coil state) were observed for the C alpha, C beta, and C' nuclei, which reflect the local structures on the backbone, that is, the alpha-helix, beta-sheet, and left-handed helix, respectively.
Yamazaki
T
T
Biometrology Lab, JEOL Ltd., Tokyo, Japan.
Yoshida
M
M
Nagayama
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids; D010582:Bacteriophage lambda / Q000235:genetics; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009587:Nitrogen Isotopes; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics
D000596:Amino Acids; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D016914:Ribonuclease H