Related ArticlesAromatic 19F-13C TROSY - [19F, 13C] pyrimidine labeling for NMR spectroscopy of RNA.
Angew Chem Int Ed Engl. 2020 Jun 18;:
Authors: Kreutz C, Nußbaumer F, Plangger R, Roeck M
Abstract
We present the access to [5-19F, 5-13C] uridine and -cytidine phosphoramidites for the production of site-specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5-19F, 5-13C]-pyrimidine labels into five RNAs - the 30nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61nt human hepatitis B virus ? (hHBV ?) RNA, the 49nt SAM VI riboswitch aptamer domain from B. angulatum, the 29nt apical stem loop of the pre-microRNA (miR) 21 and the 59nt full length pre-miR 21. The main stimulus to introduce the aromatic 19F-13C-spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole-dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic 13C-19F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5-19F, 5-13C]/[5-19F] pyrimidine labeling. For the 61nt hHBV ? we find a beneficial 19F-13C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [19F, 13C]-labeling of the SAM VI aptamer domain and the pre-miR 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting.
PMID: 32558232 [PubMed - as supplied by publisher]
[NMR paper] Erratum to: Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
Erratum to: Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
Related Articles Erratum to: Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
J Biomol NMR. 2016 Feb 19;
Authors: Clark L, Zahm JA, Ali R, Kukula M, Bian L, Patrie SM, Gardner KH, Rosen MK, Rosenbaum DM
PMID: 26894386
[NMR paper] Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
Related Articles Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
J Biomol NMR. 2015 May 30;
Authors: Clark L, Zahm JA, Ali R, Kukula M, Bian L, Patrie SM, Gardner KH, Rosen MK, Rosenbaum DM
Abstract
(13)C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific...
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05-31-2015 01:57 PM
Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris
Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris
Abstract
13C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of...
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05-29-2015 01:24 PM
Increased resolution of aromatic cross peaks using alternate 13 C labeling and TROSY
Increased resolution of aromatic cross peaks using alternate 13 C labeling and TROSY
Abstract
For typical globular proteins, contacts involving aromatic side chains would constitute the largest number of distance constraints that could be used to define the structure of proteins and protein complexes based on NOE contacts. However, the 1H NMR signals of aromatic side chains are often heavily overlapped, which hampers extensive use of aromatic NOE cross peaks. Some of this overlap can be overcome by recording 13C-dispersed NOESY spectra. However, the...
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05-10-2015 07:49 PM
Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection
Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection
Abstract
Protein dynamics on the microsecondâ??millisecond time scales often play a critical role in biological function. NMR relaxation dispersion experiments are powerful approaches for investigating biologically relevant dynamics with site-specific resolution, as shown by a growing number of publications on enzyme catalysis, protein folding, ligand binding, and allostery. To date, the majority of studies has probed the...
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06-19-2014 10:21 PM
Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion
Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion
Abstract Protein dynamics on the millisecond time scale commonly reflect conformational transitions between distinct functional states. NMR relaxation dispersion experiments have provided important insights into biologically relevant dynamics with site-specific resolution, primarily targeting the protein backbone and methyl-bearing side chains. Aromatic side chains represent attractive probes of protein dynamics because they are over-represented in protein binding...
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07-30-2012 07:42 AM
[NMR paper] Optimization of three-dimensional TROSY-type HCCH NMR correlation of aromatic (1)H-(1
Optimization of three-dimensional TROSY-type HCCH NMR correlation of aromatic (1)H-(13)C groups in proteins.
Related Articles Optimization of three-dimensional TROSY-type HCCH NMR correlation of aromatic (1)H-(13)C groups in proteins.
J Magn Reson. 1999 Aug;139(2):447-50
Authors: Meissner A, Sorensen OW
Improved methods for three-dimensional TROSY-Type HCCH correlation involving protons of negligible CSA are presented. The TROSY approach differs from the conventional approach of heteronuclear decoupling in evolution and detection periods by...
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Aromatic 19F-13C TROSY - [19F, 13C] pyrimidine labeling for NMR spectroscopy of RNA.
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