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 subunit from RNA polymerase from Bacillus subtilis. The unstructured part of this 20 kDa protein consists of 81 amino acids with frequent sequential repeats. A collection of 0.0003% of the data needed for a conventional experiment with linear sampling was sufficient to perform an unambiguous assignment of the disordered part of the protein from a single 5D spectrum.
Content Type Journal Article
Pages 1-11
DOI 10.1007/s10858-011-9496-2
Authors
JiĹ?Ă* NováÄ?ek, Faculty of Science, NCBR, and CEITEC, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
Anna Zawadzka-Kazimierczuk, Faculty of Chemistry, University of Warsaw, Pasteura 1, 02 093 Warsaw, Poland
Veronika Papoušková, Faculty of Science, NCBR, and CEITEC, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
Lukáš Ĺ˝Ă*dek, Faculty of Science, NCBR, and CEITEC, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
Hana Ĺ*anderová, Laboratory of Molecular Genetics of Bacteria and Department of Bacteriology, Institute of Microbiology, Academy of Sciences of the Czech Republic, VĂ*deĹ?ská 1083, 142 20 Prague, Czech Republic
Libor KrásnĂ˝, Laboratory of Molecular Genetics of Bacteria and Department of Bacteriology, Institute of Microbiology, Academy of Sciences of the Czech Republic, VĂ*deĹ?ská 1083, 142 20 Prague, Czech Republic
Wiktor KoĹşmiĹ?ski, Faculty of Chemistry, University of Warsaw, Pasteura 1, 02 093 Warsaw, Poland
VladimĂ*r SklenáĹ?, Faculty of Science, NCBR, and CEITEC, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
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
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
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
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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09-16-2010 04:29 PM
Nitrogen-detected CAN and CON experiments as alternative experiments for main chain N
Abstract Heteronuclear direct-detection experiments, which utilize the slower relaxation properties of low γ nuclei, such as 13C have recently been proposed for sequence-specific assignment and structural analyses of large, unstructured, and/or paramagnetic proteins. Here we present two novel 15N direct-detection experiments. The CAN experiment sequentially connects amide 15N resonances using 13Cα chemical shift matching, and the CON experiment connects the preceding 13C� nuclei. When starting from the same carbon polarization, the intensities of nitrogen signals detected in the CAN or...
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08-14-2010 04:19 AM
13C-direct detected NMR experiments for the sequential J-based resonance assignment o
Abstract We present here a set of 13C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose 13C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4â?˛ nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1â?˛,H1â?˛ ribose signals. The experiments were applied to two RNA hairpin structures....
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08-14-2010 04:19 AM
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...
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08-14-2010 04:19 AM
Suite of Six NMR Relaxation Dispersion Experiments to Study Multiple-Site Exchange in Proteins
http://pubs.acs.org/isubscribe/journals/jacsat/127/i44/figures/ja054550en00001.gif
Multiple-Site Exchange in Proteins Studied with a Suite of Six NMR Relaxation Dispersion Experiments: An Application to the Folding of a Fyn SH3 Domain Mutant
Dmitry M. Korzhnev, Philipp Neudecker, Anthony Mittermaier, Vladislav Yu. Orekhov, and Lewis E. Kay*
Contribution from the Departments of Medical Genetics, Biochemistry, and Chemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada, and Swedish NMR Center at Göteborg University, Box 465, 405 30 Göteborg, Sweden
J. Am. Chem....
5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion
Linear Mode
