Abstract Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3â?² and C5â?² carbon positions. Consequently the C1â?², C2â?² and C4â?² positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4â?² carbon position, such that the C2â?² and C3â?² positions suffer from multiplet splitting but the C5â?² position remains singlet and the C1â?² position shows a small amount of residual C1â?²â??C2â?² coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5â?² position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4â?² carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems.
Content Type Journal Article
DOI 10.1007/s10858-010-9454-4
Authors
Chandar S. Thakur, Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), College Park, MD 20742-3360, USA
Jacob N. Sama, Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), College Park, MD 20742-3360, USA
Melantha E. Jackson, Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), College Park, MD 20742-3360, USA
Bin Chen, Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), College Park, MD 20742-3360, USA
T. Kwaku Dayie, Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 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
Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR.
Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR.
Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR.
J Magn Reson. 2011 Mar 17;
Authors: Traaseth NJ, Veglia G
We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR...
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04-13-2011 11:57 PM
Frequency-Selective Heteronuclear Dephasing and Selective Carbonyl Labeling to Deconvolute Crowded Spectra of Membrane Proteins By Magic Angle Spinning NMR
Frequency-Selective Heteronuclear Dephasing and Selective Carbonyl Labeling to Deconvolute Crowded Spectra of Membrane Proteins By Magic Angle Spinning NMR
Publication year: 2011
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 17 March 2011</br>
Nathaniel J., Traaseth , Gianluigi, Veglia</br>
We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR spectra of membrane proteins in fluid lipid membranes with broad lines and...
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03-18-2011 06:43 AM
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle.
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle.
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle.
J Biomol NMR. 2010 Dec;48(4):179-92
Authors: Thakur CS, Sama JN, Jackson ME, Chen B, Dayie TK
Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon...
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03-01-2011 12:14 PM
Selective 1H-13C NMR spectroscopy of methyl groups in residually protonated samples of large proteins
Selective 1H-13C NMR spectroscopy of methyl groups in residually protonated samples of large proteins
Abstract Methyl 13CHD2 isotopomers of all methyl-containing amino-acids can be observed in residually protonated samples of large proteins obtained from -glucose/D2O-based bacterial media, with sensitivity sufficient for a number of NMR applications. Selective detection of some subsets of methyl groups (Alaβ, Thrγ2) is possible using simple â??out-and-backâ?? NMR methodology. Such selective methyl-detected â??out-and-backâ?? NMR experiments allow complete assignments of threonine γ2...
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01-09-2011 12:46 PM
[NMR paper] Chain-selective isotopic labeling for NMR studies of large multimeric proteins: appli
Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.
Related Articles Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.
Biophys J. 2000 Aug;79(2):1146-54
Authors: Simplaceanu V, Lukin JA, Fang TY, Zou M, Ho NT, Ho C
Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of...
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11-19-2010 08:29 PM
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...
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08-14-2010 04:19 AM
A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
Kit I. Tong, Masayuki Yamamoto and Toshiyuki Tanaka
Journal of Biomolecular NMR; 2008; 42(1); pp 59-67
Abstract:
A simple and user-friendly method of labeling protein selectively with amino acids in vivo is introduced. This technique does not require the use of transaminase-deficient or auxotrophic strains. By manipulating the product feedback inhibitory loops of the E. coli amino acid metabolic pathways and, if necessary, by using enzyme inhibitors, proteins were labeled efficiently in vivo...
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli
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