A Suite of Triple Resonance NMR Experiments for the Backbone Assignment of 15N, 13C, 2H Labeled Proteins with High Sensitivity

Yamazaki, Toshio; Lee, Weontae; Arrowsmith, C.H.; Muhandiram, D. R.; Kay, Lewis E.

doi:10.1021/ja00105a005

Cited by 486 publications

(442 citation statements)

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“…Spectral assignments were obtained using the following 3D experiments: CT-HNCO, CT-HNCA, CT-HNCOCA, CBCACONH, HNCAHA, HN(COCA)HA, HACACO, (H)CCH-COSY, H(C)CH-COSY, H(C)CH-TOCSY, (H)CCH-TOCSY, C-CONH-TOCSY, 15 N edited 1 H TOCSY (Clore & Gronenborn, 1991a,b;Bax & Grzesiek, 1993). The following 3D experiments were acquired for the perdeuterated sample: CT-HNCA, CT-HNCOCA, HNCOCACB, HNCACB (Yamazaki et al, 1994).…”

Section: Data Collection and Assignmentmentioning

confidence: 99%

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism

Barbato

Cicero

Nardi

et al. 1999

Journal of Molecular Biology

108

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The solution structure of the hepatitis C virus (BK strain) NS3 protein Nterminal domain (186 residues) has been solved by NMR spectroscopy. The protein is a serine protease with a chymotrypsin-type fold, and is involved in the maturation of the viral polyprotein. Despite the knowledge that its activity is enhanced by the action of a viral protein cofactor, NS4A, the mechanism of activation is not yet clear. The analysis of the folding in solution and the differences from the crystallographic structures allow the formulation of a model in which, in addition to the NS4A cofactor, the substrate plays an important role in the activation of the catalytic mechanism. A unique structural feature is the presence of a zincbinding site exposed on the surface, subject to a slow conformational exchange process.

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Section: Data Collection and Assignmentmentioning

confidence: 99%

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism

Barbato

Cicero

Nardi

et al. 1999

Journal of Molecular Biology

108

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“…Here we report the results of deuterium-proton (D-H) exchange experiments [22][23][24] on WT-TTR at pH 5.75 (nonamyloidogenic tetramer) and 4.5 (amyloidogenic monomer), monitored by two-dimensional NMR spectroscopy, that provide site specific information complementing previous mass spectroscopic studies 18 . The differences in the backbone amide D-H exchange rates under these two conditions reveal that one 15 N resonances of 118 out of 120 nonproline residues in tetrameric WT-TTR were assigned by NMR using uniformly 2 H, 13 C and 15 N labeled samples 25 . In D-H exchange experiments, protected amide hydrogens are generally involved in hydrogen bonds.…”

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confidence: 98%

Untitled

Cho²,

Lashuel

et al. 2000

Nat. Struct Biol.

125

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Studies have indicated that partially unfolded states occur under conditions that favor amyloid formation by transthyretin (TTR), as well as other amyloidogenic proteins. In this study, we used hydrogen exchange measurements to show that there is selective destabilization of one half of the β-sandwich structure of TTR under such conditions. This provides more direct information about conformational fluctuations than previously available, and will facilitate design of future experiments to probe the intermediates critical to amyloid formation.

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“…However, the extension of these experiments to proteins that have higher molecular weights and 7, 2 10 ns is difficult, mainly due to the short transverse relaxation times of the C a nuclei. In these situations, the 13Ca line widths approach the 'H-13C coupling constants, resulting in poor magnetization transfer efficiencies and low signal to noise ratios in some key experiments Yamazaki et al, 1994). Nevertheless, the solution structure of the p53 homotetramer (20 kDa, 42 residues/subunit) with a correlation time (14.8 ns) comparable to that of 4-OT (14.5 ns) has been solved (Clore et al, 1994(Clore et al, , 1995Clubb et al, 1995).…”

mentioning

confidence: 99%

“…Nevertheless, the solution structure of the p53 homotetramer (20 kDa, 42 residues/subunit) with a correlation time (14.8 ns) comparable to that of 4-OT (14.5 ns) has been solved (Clore et al, 1994(Clore et al, , 1995Clubb et al, 1995). Recently, it has been shown that fractional substitution of deuterons for protons attached to I3C nuclei in uniformly "N-and 13C-labeled proteins can significantly narrow the I3C line widths and improve the magnetization transfer efficiencies (Yamazaki et al, 1994).…”

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confidence: 99%

4‐Oxalocrotonate tautomerase, a 41‐kDa homohexamer: Backbone and side‐chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy

Stivers¹,

Abeygunawardana²,

Whitman³

et al. 1996

Protein Science

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Abstract4-Oxalocrotonate tautomerase (4-OT), a homohexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated a-keto acids using Pro-1 as a general base (Stivers et al., 1996a(Stivers et al., , 1996b. We report the backbone and side-chain 'H, "N, and I3C NMR assignments and the solution secondary structure for 4-OT using 2D and 3D homonuclear and heteronuclear NMR methods. The subunit secondary structure consists of an a-helix (residues 13-30), two 0-strands (PI, residues 2-8; &, residues 39-45), a @-hairpin (residues 50-57), two loops (I, residues 9-12; 11, 34-38), and two turns (I, residues 30-33; II,47-50). The remaining residues form coils. The PI strand is parallel to the pz strand of the same subunit on the basis of cross strand NHi-NHj NOEs in a 2D "N-edited 'H-NOESY spectrum of hexameric 4-OT containing two I5N-labeled subunits/hexamer. The 0' strand is also antiparallel to another PI strand from an adjacent subunit forming a subunit interface. Because only three such pairwise interactions are possible, the hexamer is a trimer of dimers. The diffusion constant, determined by dynamic light scattering, and the rotational correlation time (14.5 ns) estimated from I5N T l / T , measurements, are consistent with the hexameric molecular weight of 41 kDa. Residue Phe-50 is in the active site on the basis of transferred NOEs to the bound partial substrate 2-0~0-1,6-hexanedioate. Modification of the general base, Pro-1, with the active site-directed irreversible inhibitor, 3-bromopyruvate, significantly alters the amide I5N and NH chemical shifts of residues in the 0-hairpin and in loop 11, providing evidence that these regions change conformation when the active site is occupied.

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A Suite of Triple Resonance NMR Experiments for the Backbone Assignment of 15N, 13C, 2H Labeled Proteins with High Sensitivity

Cited by 486 publications

References 1 publication

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism

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4‐Oxalocrotonate tautomerase, a 41‐kDa homohexamer: Backbone and side‐chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy

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