Assignments of backbone sup 1 H, sup 13 C, and sup 15 N resonances and secondary structure of ribonuclease H from Escherichia coli by heteronuclear three-dimensional NMR spectroscopy
- JEOL Ltd., Tokyo (Japan)
- Kyowa Hakko Kogyo Co., Ltd., Tokyo (Japan)
- Protein Engineering Research Inst., Osaka (Japan)
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 {sup 1}H-{sup 15}N HMQC experiments with samples in which specific amino acids were labeled with {sup 15}N. Second, the amide {sup 1H}-{sup 15}N 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, {sup 1}H-{sup 15}N-{sup 1}H 3D NOESY-HMQC, {sup 1}H-{sup 15}N-{sup 1}H 3D TOCSY-HMQC, {sup 13}C-{sup 1}H-{sup 1}H 3D HMQC-NOESY, and {sup 13}C-{sup 1}H-{sup 1}H 3D HMQC-TOCSY, were applied to proteins uniformly labeled either with {sup 15}N or with {sup 13}C. This method gave a systematic way to assign backbone nuclei (N, NH, C{sup a}H, and C{sup a}) 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.
- OSTI ID:
- 5014135
- Journal Information:
- Biochemistry; (United States), Vol. 30:24; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
RNA-ASE
NUCLEAR MAGNETIC RESONANCE
AMIDES
CARBON 13
CHEMICAL SHIFT
ESCHERICHIA COLI
NITROGEN 15
OVERHAUSER EFFECT
PROTONS
BACTERIA
BARYONS
CARBON ISOTOPES
ELEMENTARY PARTICLES
ENZYMES
ESTERASES
EVEN-ODD NUCLEI
FERMIONS
HADRONS
HYDROLASES
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NITROGEN ISOTOPES
NUCLEI
NUCLEONS
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHODIESTERASES
RESONANCE
STABLE ISOTOPES
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