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 C4ā?² 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 C1ā?²,H1ā?² ribose signals. The experiments were applied to two RNA hairpin structures. The current set of 13C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, 13C-direct detected NMR methods constitute useful complements to the conventional 1H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.
Content Type Journal Article
DOI 10.1007/s10858-010-9429-5
Authors
Christian Richter, Johann Wolfgang Goethe-University Frankfurt Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance Max-von-Laue-Str. 7 60438 Frankfurt/M Germany
Helena Kovacs, Bruker BioSpin AG Industriestrasse 26 8117 FƤllanden Switzerland
Janina Buck, Johann Wolfgang Goethe-University Frankfurt Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance Max-von-Laue-Str. 7 60438 Frankfurt/M Germany
Anna Wacker, Johann Wolfgang Goethe-University Frankfurt Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance Max-von-Laue-Str. 7 60438 Frankfurt/M Germany
Boris FĆ¼rtig, Johann Wolfgang Goethe-University Frankfurt Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance Max-von-Laue-Str. 7 60438 Frankfurt/M Germany
Wolfgang Bermel, Bruker BioSpin GmbH Silberstreifen 76287 Rheinstetten Germany
Harald Schwalbe, Johann Wolfgang Goethe-University Frankfurt Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance Max-von-Laue-Str. 7 60438 Frankfurt/M Germany
Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers
Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers
Abstract Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring 1H-15N dipolar couplings (DC) and 15N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal...
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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
Rapid acquisition of (1) H and (19) F NMR experiments for direct and competition ligand-based screening.
Rapid acquisition of (1) H and (19) F NMR experiments for direct and competition ligand-based screening.
Rapid acquisition of (1) H and (19) F NMR experiments for direct and competition ligand-based screening.
Magn Reson Chem. 2011 Mar 9;
Authors: Dalvit C, Gossert AD, Coutant J, Piotto M
Direct and competition ligand-based NMR experiments are often used in the screening of chemical fragment libraries against a protein target due to the high relative sensitivity of NMR for protein-binding events. A plethora of NMR methods has been proposed...
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03-10-2011 03:51 PM
Extension of the HA-detection based approach: (HCA)CON(CA)H and (HCA)NCO(CA)H experiments for the main-chain assignment of intrinsically disordered proteins
Extension of the HA-detection based approach: (HCA)CON(CA)H and (HCA)NCO(CA)H experiments for the main-chain assignment of intrinsically disordered proteins
Abstract Extensive resonance overlap exacerbates assignment of intrinsically disordered proteins (IDPs). This issue can be circumvented by utilizing 15N, 13Cā?² and 1HN spins, where the chemical shift dispersion is mainly dictated by the characteristics of consecutive amino acid residues. Especially 15N and 13Cā?² spins offer superior chemical shift dispersion in comparison to 13CĪ± and 13CĪ² spins. However, HN-detected experiments...
[NMR paper] Sequential NMR resonance assignment and structure determination of the Kunitz-type in
Sequential NMR resonance assignment and structure determination of the Kunitz-type inhibitor domain of the Alzheimer's beta-amyloid precursor protein.
Related Articles Sequential NMR resonance assignment and structure determination of the Kunitz-type inhibitor domain of the Alzheimer's beta-amyloid precursor protein.
Biochemistry. 1991 Oct 29;30(43):10467-78
Authors: Heald SL, Tilton RF, Hammond LJ, Lee A, Bayney RM, Kamarck ME, Ramabhadran TV, Dreyer RN, Davis G, Unterbeck A
Certain precursor proteins (APP751 and APP770) of the amyloid...
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08-21-2010 11:12 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 Ī³ 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 13CĪ± chemical shift matching, and the CON experiment connects the preceding 13Cā?² 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
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...
13C-direct detected NMR experiments for the sequential J-based resonance assignment o
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