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

Journal Article · Tue Jun 18 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00238a030· OSTI ID:5014135

Yamazaki, Toshio; Nagayama, Kuniaki ^[1]; Yoshida, Mayumi ^[2]; Kanaya, Shigenori; Nakamura, Haruki ^[3]

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1. JEOL Ltd., Tokyo (Japan)
2. Kyowa Hakko Kogyo Co., Ltd., Tokyo (Japan)
3. 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), Journal Name: Biochemistry; (United States) Vol. 30:24; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
AMIDES
BACTERIA
BARYONS
CARBON 13
CARBON ISOTOPES
CHEMICAL SHIFT
ELEMENTARY PARTICLES
ENZYMES
ESCHERICHIA COLI
ESTERASES
EVEN-ODD NUCLEI
FERMIONS
HADRONS
HYDROLASES
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NITROGEN 15
NITROGEN ISOTOPES
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEONS
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OVERHAUSER EFFECT
PHOSPHODIESTERASES
PROTONS
RESONANCE
RNA-ASE
STABLE ISOTOPES