Site-specific labeling of nucleotides for making RNA for high resolution NMR studies
Abstract Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to 1JC5C6 scalar coupling. However, the C2/C8 sites within purine rings and the C1â?²/C3â?²/C5â?² positions within the ribose rings have reduced labeling. Addition of 13C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1â?²/C3â?²/C5â?² positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and 15N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs.
Content Type Journal Article
DOI 10.1007/s10858-010-9405-0
Authors
T. Kwaku Dayie, University of Maryland Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization 1115 Biomolecular Sciences Bldg (#296) College Park MD 20742-3360 USA
Chandar S. Thakur, University of Maryland Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization 1115 Biomolecular Sciences Bldg (#296) College Park MD 20742-3360 USA
Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR
Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR
Abstract Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at different carbon sites is shown to facilitate cost-effective and large scale production of useful nucleotides. These nucleotides are site specifically labeled in C1â?² and C5â?² with...
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11-30-2011 10:45 PM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Abstract Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives,...
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Proteins
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01-22-2011 03:46 AM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
J Biomol NMR. 2011 Jan 19;
Authors: Fan Y, Shi L, Ladizhansky V, Brown LS
Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties...
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01-21-2011 01:22 AM
Site-specific labeling of proteins with NMR-active unnatural amino acids
Site-specific labeling of proteins with NMR-active unnatural amino acids
Abstract A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling...
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01-09-2011 12:46 PM
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Biosynthetic site-specific (13) C labeling of the light-harvesting 2 protein complex: a model for solid state NMR structure determination of transmembrane proteins.
Related Articles Biosynthetic site-specific (13) C labeling of the light-harvesting 2 protein complex: a model for solid state NMR structure determination of transmembrane proteins.
J Biomol NMR. 2004 Nov;30(3):267-74
Authors: van Gammeren AJ, Hulsbergen FB, Hollander JG, de Groot HJ
Partly biosynthetic site-directed isotopically (13)C enriched photosynthetic light-harvesting...
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11-24-2010 10:03 PM
[NMR paper] Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and ir
Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.
Related Articles Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.
J Biomol NMR. 2000 Aug;17(4):311-22
Authors: Sorkin DL, Miller AF
We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide...
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11-19-2010 08:29 PM
[NMR paper] Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-N
Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of Trp 62-lysozyme.
Related Articles Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of Trp 62-lysozyme.
J Biochem. 1991 Aug;110(2):295-300
Authors: Nakazawa T, Sakiyama F
The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed...
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08-21-2010 11:12 PM
[NMR paper] Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-N
Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of Trp 62-lysozyme.
Related Articles Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of Trp 62-lysozyme.
J Biochem. 1991 Aug;110(2):295-300
Authors: Nakazawa T, Sakiyama F
The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed...
Site-specific labeling of nucleotides for making RNA for high resolution NMR studies
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