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 solution to this problem: a hybrid (4d,3d) reduced-dimensionality HN(CO)CA(CON)CA sequence. In this experiment, the Ca(i) resonance is modulated with the frequency of the Ca(iâ??1) resonance, which helps in resolving the ambiguity involved in connecting the Ca(i) and Ca(iâ??1) resonances for overlapping NH roots. The experiment has limited sensitivity, and is only suited for small or unfolded proteins. In a companion experiment, (4d,3d) reduced-dimensionality HNCO(N)CA, the Ca(i) resonance is modulated with the frequency of the CO(iâ??1) resonance, hence resolving the ambiguity existent in pairing up the Ca(i) and CO(iâ??1) resonances for overlapping NH roots.
Content Type Journal Article
DOI 10.1007/s10858-010-9465-1
Authors
Ireena Bagai, Department of Biological Chemistry, University of Michigan Medical School, MSRB III, 1150Â*W. Medical Center Drive, Ann Arbor, MI 48109-5605, USA
Stephen W. Ragsdale, Department of Biological Chemistry, University of Michigan Medical School, MSRB III, 1150Â*W. Medical Center Drive, Ann Arbor, MI 48109-5605, USA
Erik R. P. Zuiderweg, Department of Biological Chemistry, University of Michigan Medical School, 4220D MSRB III, 1150Â*W. Medical Center Drive, Ann Arbor, MI 48109-5605, USA
Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment
Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment
Abstract Obtaining NMR assignments for slowly tumbling molecules such as detergent-solubilized membrane proteins is often compromised by low sensitivity as well as spectral overlap. Both problems can be addressed by amino-acid specific isotope labeling in conjunction with 15Nâ??1H correlation experiments. In this work an extended combinatorial selective in vitro labeling scheme is proposed that seeks to reduce the number of samples required for assignment. Including three...
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01-21-2012 06:26 PM
AUTOBA: Automation of backbone assignment from HN(C)N suite of experiments
AUTOBA: Automation of backbone assignment from HN(C)N suite of experiments
Abstract Development of efficient strategies and automation represent important milestones of progress in rapid structure determination efforts in proteomics research. In this context, we present here an efficient algorithm named as AUTOBA (Automatic Backbone Assignment) designed to automate the assignment protocol based on HN(C)N suite of experiments. Depending upon the spectral dispersion, the user can record 2D or 3D versions of the experiments for assignment. The algorithm uses as inputs: (i) protein primary...
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06-06-2011 12:53 AM
5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion
5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion
Abstract Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of 13C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and minimize the experimental time. The pulse sequences were successfully tested on the sample of partially disordered delta...
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03-22-2011 07:32 PM
Sparsely-sampled High-resolution 4-D Experiments for Efficient Backbone Resonance Assignment of Disordered Proteins
Sparsely-sampled High-resolution 4-D Experiments for Efficient Backbone Resonance Assignment of Disordered Proteins
Publication year: 2011
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 4 January 2011</br>
Jie, Wen , Jihui, Wu , Pei, Zhou</br>
Intrinsically disordered proteins (IDPs) play important roles in many critical cellular processes. Due to their limited chemical shift dispersion, IDPs often require four pairs of resonance connectivities (H?, C?, C? and CO) for establishing sequential backbone assignment. Because most conventional 4-D...
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01-05-2011 11:03 AM
Corrigendum to “BEST-HNN and 2D (HN)NH experiments for rapid backbone assignment in p
Corrigendum to “BEST-HNN and 2D (HN)NH experiments for rapid backbone assignment in proteins”
Publication year: 2010
Source: Journal of Magnetic Resonance, In Press, Corrected Proof, Available online 15 September 2010</br>
Dinesh, Kumar , Subhradip, Paul , Ramakrishna V., Hosur</br>
Source: Journal of Magnetic Resonance
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[NMR paper] Double and triple resonance NMR methods for protein assignment.
Double and triple resonance NMR methods for protein assignment.
Related Articles Double and triple resonance NMR methods for protein assignment.
Methods Mol Biol. 1997;60:29-52
Authors: Whitehead B, Craven CJ, Waltho JP
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08-22-2010 03:31 PM
[NMR paper] Solid-state NMR triple-resonance backbone assignments in a protein.
Solid-state NMR triple-resonance backbone assignments in a protein.
Related Articles Solid-state NMR triple-resonance backbone assignments in a protein.
J Biomol NMR. 1999 Apr;13(4):337-42
Authors: Tan WM, Gu Z, Zeri AC, Opella SJ
Triple-resonance solid-state NMR spectroscopy is demonstrated to sequentially assign the 13C' and 15N amide backbone resonances of adjacent residues in an oriented protein sample. The observed 13C' chemical shift frequency provides an orientational constraint complementary to those measured from the 1H and 15N amide...
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08-21-2010 04:03 PM
HA-detected experiments for the backbone assignment of intrinsically disordered prote
Abstract We propose a new alpha proton detection based approach for the sequential assignment of natively unfolded proteins. The proposed protocol superimposes on following features: HA-detection (1) enables assignment of natively unfolded proteins at any pH, i.e., it is not sensitive to rapid chemical exchange undergoing in natively unfolded proteins even at moderately high pH. (2) It allows straightforward assignment of proline-rich polypeptides without additional proline-customized experiments. (3) It offers more streamlined and less ambiguous assignment based on solely intraresidual...
Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins
Linear Mode
