A novel method of preparing totally α‐deuterated amino acids for selective incorporation into proteins

Feeney, J.; Birdsall, B.; Ostler, G.; Carr, Mark D.; Kairi, M.

doi:10.1016/0014-5793(90)80483-y

Cited by 11 publications

(8 citation statements)

References 14 publications

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“…The spin systems of valine residues in the DHFR-MTX complex proved to be more difficult to identify by this approach, and it was possible to identify only 11 complete valine spin systems and partially assign one other from the 2D and 3D spectra. However, after analysis of DQF-COSY and HOHAHA spectra recorded from specifically labeled DHFR samples, where either the methyl or the a protons of valine residues are deuterated, 15 of the 16 valine spin systems could be fully characterized (Feeney et al, 1990). In two cases there were no detectable aCH-0CH COSY cross peaks, or relay peaks involving the NH resonances and the backbone and side-chain protons could only be associated with each other by using NOEs from the NH protons to the ß, y, and y' protons.…”

Section: Methodsmentioning

confidence: 99%

Dihydrofolate reductase; sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution

Carr¹,

Birdsall²,

Frenkiel³

et al. 1991

Biochemistry

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Three-dimensional (3D) heteronuclear NMR techniques have been used to make sequential 1H and 15N resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18,300 Da. A uniformly 15N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D 15N/1H nuclear Overhauser-heteronuclear multiple quantum coherence (NOESY-HMQC), Hartmann-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 1H-1H 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 1H chemical shifts are degenerate as long as the amide 15N 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. Sequential NHi-NHi+1 NOEs define four regions with helical structure. Two of these regions, residues 44-49 and 79-89, correspond to within one amino acid to helices C and E in the crystal structure of the DHFR.methotrexate.NADPH complex [Bolin et al. (1982) J. Biol. Chem. 257, 13650-13662], while the NMR-determined helix formed by residues 26-35 is about one turn shorter at the N-terminus than helix B in the crystal structure, which spans residues 23-34. Similarly, the NMR-determined helical region comprising residues 102-110 is somewhat offset from the crystal structure's helix F, which encompasses residues 97-107. Regions of beta-sheet structure were characterized in the binary complex by strong alpha CHi-NHi+1 NOEs and by slowly exchanging amide protons. In addition, several long-range NOEs were identified linking together these stretches to form a beta-sheet. These elements align perfectly with corresponding elements in the crystal structure of the DHFR.methotrexate.NADPH complex, which contains an eight-stranded beta-sheet, indicating that the main body of the beta-sheet is preserved in the binary complex in solution.

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Section: Methodsmentioning

confidence: 99%

Dihydrofolate reductase; sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution

Carr¹,

Birdsall²,

Frenkiel³

et al. 1991

Biochemistry

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“…[77±82] By examining 2D COSY spectra from DHFRcontaining g-Me-deuterated valine [78,79] and a-deuterated valine, [80] it was possible to assign the signals for the protons normally at the deuterated sites. We have prepared L. casei DHFR containing (2S,4R)- [5,5, H 3 ]leucine [81] and have made stereospecific assignments for Leu methyl groups by examining the 1 H COSY spectra of the deuterated and nondeuterated samples (see Figure 3).…”

Section: Assignment Of the Nmr Signals Of The Proteinmentioning

confidence: 99%

NMR Studies of Ligand Binding to Dihydrofolate Reductase

Feeney¹

2000

Angewandte Chemie International Edition

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Unique capabilities are offered by NMR spectroscopy for probing specific interactions of enzymes with substrates and substrate analogues, for characterizing the multiple conformations of the complexes, and for measuring rates of a wide range of dynamic processes. The most extensive studies of this type have been directed at complexes formed by dihydrofolate reductase with antifolate drugs and are described in this review.

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“…Viele selektiv deuterierte L.-casei-DHFRs wurden durch biosynthetischen Einbau entsprechender deuterierter Aminosäuren hergestellt; durch Vergleich der Spektren der nicht deuterierten und der selektiv deuterierten Proben war es möglich, diejenigen 1 H-Signale zu entdecken und zuzuordnen, die nur in den Spektren der nicht deuterierten Proben vorkamen. [77±82] Durch Interpretation der 2D-COSY-Spektren von DHFR, die g-Me-deuteriertes Valin [78,79] und a-deuteriertes Valin [80] enthielt, war es möglich, die Signale der Protonen zuzuordnen, die sich normalerweise an den deuterierten Positionen befinden. Wir stellten L.-casei-DHFR her, die (2S,4R)-[5,5,5-2 H 3 ]Leucin [81] enthielt, und erstellten stereospezifische Zuordnungen für die Leu-Methylgruppen, indem wir die 1 H-COSY-Spektren der deuterierten mit denen nicht deuterierter Proben verglichen (Abbildung 3).…”

Section: Zuordnungen Der Nmr-signale Des Proteinsunclassified

NMR-Untersuchungen zur Ligandenbindung an Dihydrofolat-Reduktase

Feeney¹

2000

Angewandte Chemie

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Einzigartige Möglichkeiten bietet die NMR‐Spektroskopie zur Untersuchung spezifischer Wechselwirkungen von Enzymen mit Substraten und Substrat‐analoga, zur Charakterisierung multipler Konformationen der Komplexe und zur Messung der Geschwindigkeiten einer Vielzahl von dynamischen Prozessen. Die umfangreichsten Studien dieser Art befassten sich mit Komplexen aus Dihydrofolat‐Reduktase und Folsäure‐Antagonisten und werden im vorliegenden Aufsatz beschrieben.

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A novel method of preparing totally α‐deuterated amino acids for selective incorporation into proteins

Cited by 11 publications

References 14 publications

Dihydrofolate reductase; sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution

Dihydrofolate reductase; sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution

NMR Studies of Ligand Binding to Dihydrofolate Reductase

NMR-Untersuchungen zur Ligandenbindung an Dihydrofolat-Reduktase

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