Publication year: 2011 Source: Journal of Magnetic Resonance, Volume 213, Issue 2, December 2011, Pages 423-441
Lewis E.*Kay, Mitsuhiko*Ikura, Rolf*Tschudin, Ad*Bax
Four new and complementary three-dimensional triple-resonance experiments are described for obtaining complete backboneH,C, andN resonance assignments of proteins uniformly enriched withC andN. The new methods all rely onH detection and use multiple magnetization transfers through well-resolved one-bondJcouplings. Therefore, the 3D experiments are sensitive and permit relatively rapid recording of 3D spectra (l–2*days) for protein concentrations on the order of 1*mM. One experiment (HNCO) correlates the amideH andN shifts with theC shift of the carbonyl resonance of the preceding amino acid. A second experiment (HNCA) correlates the intraresidue amideH andN shifts with the C?chemical shift. This experiment often also provides a weak correlation between the amide NH andN resonances of one amino acid and the Ca resonance of the preceding amino acid. A third experiment (HCACO) correlates the H?and C?shifts with the intraresidue carbonyl shift. Finally, a 3D relay experiment, HCA(CO)N, correlates Ha and Cal resonances of one residue with theN frequency of the succeeding residue. The principles of these experiments are described in terms of the operator formalism. To optimize spectral resolution, special attention is paid to removal of undesiredJsplittings in the 3D spectra. Technical details regarding the implementation of these triple-resonance experiments on a commercial spectrometer are also provided. The experiments are demonstrated for the protein calmodulin (16.7*kDa).
Triple resonance three-dimensional protein NMR: Before it became a black box.
Triple resonance three-dimensional protein NMR: Before it became a black box.
Triple resonance three-dimensional protein NMR: Before it became a black box.
J Magn Reson. 2011 Aug 30;
Authors: Bax A
Abstract
Three-dimensional triple resonance experiments have become an integral part of virtually every solution NMR study of proteins. The approach relies on uniform isotopic enrichment of proteins with (13)C and (15)N, and establishes the scalar connectivity pathway between nuclei through the large (1)J(NH), (1)J(CH)(, 1)J(CC), and (1)J(CN)...
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Triple resonance three-dimensional protein NMR: Before it became a black box
Triple resonance three-dimensional protein NMR: Before it became a black box
Publication year: 2011
Source: Journal of Magnetic Resonance, In Press, Corrected Proof, Available online 31 August 2011</br>
Ad, Bax</br>
Three-dimensional triple resonance experiments have become an integral part of virtually every solution NMR study of proteins. The approach relies on uniform isotopic enrichment of proteins with 13C and 15N, and establishes the scalar connectivity pathway between nuclei through the large 1JNH, 1JCH, 1JCC, and 1JCN couplings. The magnetization transfer process takes place...
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08-31-2011 07:12 PM
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy.
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy.
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy.
J Biomol NMR. 2011 Jun 12;
Authors: Sastry M, Xu L, Georgiev IS, Bewley CA, Nabel GJ, Kwong PD
NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in (15)N, (13)C, or (2)H. The bacterial expression systems currently in use to...
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06-15-2011 01:15 PM
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy
Abstract NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in 15N, 13C, or 2H. The bacterial expression systems currently in use to obtain isotopic enrichment, however, cannot produce a number of eukaryotic proteins, especially those that require post-translational modifications such as N-linked glycosylation for proper folding or activity. Here, we report the use of an...
Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins
Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins
Abstract The solution NMR resonance assignment of the protein backbone is most commonly carried out using triple resonance experiments that involve 15N and 1HN resonances. The assignment becomes problematic when there is resonance overlap of 15Nâ??1HN cross peaks. For such residues, one cannot unambiguously link the â??leftâ?? side of the NH root to the â??rightâ?? side, and the residues associated with such overlapping HN resonances remain often unassigned. Here we present a...
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12-31-2010 08:38 PM
[NMR paper] Multidimensional triple resonance NMR spectroscopy of isotopically uniformly enriched
Multidimensional triple resonance NMR spectroscopy of isotopically uniformly enriched proteins: a powerful new strategy for structure determination.
Related Articles Multidimensional triple resonance NMR spectroscopy of isotopically uniformly enriched proteins: a powerful new strategy for structure determination.
Ciba Found Symp. 1991;161:108-19; discussion 119-35
Authors: Bax A, Ikura M, Kay LE, Barbato G, Spera S
A procedure is described that affords complete 1H, 13C and 15N resonance assignment in proteins of up to about 25 kDa. The new...
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08-21-2010 11:16 PM
[NMR paper] Four-dimensional heteronuclear triple-resonance NMR spectroscopy of interleukin-1 bet
Four-dimensional heteronuclear triple-resonance NMR spectroscopy of interleukin-1 beta in solution.
Related Articles Four-dimensional heteronuclear triple-resonance NMR spectroscopy of interleukin-1 beta in solution.
Science. 1990 Jul 27;249(4967):411-4
Authors: Kay LE, Clore GM, Bax A, Gronenborn AM
A method is presented that dramatically improves the resolution of protein nuclear magnetic resonance (NMR) spectra by increasing their dimensionality to four. The power of this technique is demonstrated by the application of four-dimensional...
Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins
Linear Mode
