Dihydrofolate reductase: Sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution

Journal Article · Tue Jun 25 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00239a035· OSTI ID:5014049

Carr, M D; Birdsall, B; Jimenez-Barbero, J; Polshakov, V I; McCormick, J E; Feeney, J; Frenkiel, T A; Bauer, C J ^[1]; Roberts, G C.K. ^[2]

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1. National Inst. for Medical Research, London (England)
2. Univ. of Leicester (England)

Three-dimensional (3D) heteronuclear NMR techniques have been used to make sequential {sup 1}H and {sup 15}H resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18,300 Da. A uniformly {sup 15}N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D {sup 15}N/{sup 1}H nuclear Overhauserheteronuclear multiple quantum coherence (NOESY-HMQC), Harmann-Hahn-heteronuclear multiple quantum coherence (HOHAHA-HMQC), and HMQC-NOESY-HMQC experiments. These experiments overcame most of the spectral overlap problems caused by chemical shift degeneracies in 2D spectra and allowed the {sup 1}H-{sup 1}H through-space and through-bond connectivities to be identified unambiguously, leading to the resonance assignments. The novel HMQC-NOESY-HMQC experiment allows NOE cross peaks to be detected between NH protons even when their {sup 1}H chemical shifts are degenerate as long as the amide {sup 15}N chemical shifts are nondegenerate. The 3D experiments, in combination with conventional 2D NOESY, COSY, and HOHAHA experiments on unlabelled and selectively deuterated DHFR, provide backbone assignments for 146 of the 162 residues and side-chain assignments for 104 residues of the protein. Data from the NOE-based experiments and identification of the slowly exchanging amide protons provide detailed information about the secondary structure of the binary complex of the protein with methotrexate.

• OSTI ID: 5014049
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:25; 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
BACTERIA
BARYONS
CHEMICAL SHIFT
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
HADRONS
ISOTOPES
LACTOBACILLUS
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
MOLECULAR STRUCTURE
NITROGEN 15
NITROGEN ISOTOPES
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEONS
ODD-EVEN NUCLEI
OVERHAUSER EFFECT
OXIDOREDUCTASES
PROTONS
RESONANCE
STABLE ISOTOPES