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 minimal scrambling within the ribose ring. To demonstrate the utility of this labeling approach, the new site-specific labeled and the uniformly labeled nucleotides were used to synthesize a 36-nt RNA containing the catalytically essential domain 5 (D5) of the brown algae group II intron self-splicing ribozyme. The D5 RNA was used in binding and relaxation studies probed by NMR spectroscopy. Key nucleotides in the D5 RNA that are implicated in binding Mg2+ ions are well resolved. As a result, spectra obtained using selectively labeled nucleotides have higher signal-to-noise ratio compared to those obtained using uniformly labeled nucleotides. Thus, compared to the uniformly 13C/15N-labeled nucleotides, these specifically labeled nucleotides eliminate the extensive 13Câ??13C coupling within the nitrogenous base and ribose ring, give rise to less crowded and more resolved NMR spectra, and accurate relaxation rates without the need for constant-time or band-selective decoupled NMR experiments. These position selective labeled nucleotides should, therefore, find wide use in NMR analysis of biologically interesting RNA molecules.
Content Type Journal Article
Category Article
Pages 1-12
DOI 10.1007/s10858-011-9586-1
Authors
Chandar S. Thakur, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), College Park, MD 20742-3360, USA
Yiling Luo, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and 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 and Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), College Park, MD 20742-3360, USA
Nadukkudy V. Eldho, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and 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 and Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), College Park, MD 20742-3360, USA
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
Abstract An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract,...
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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 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle
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...
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11-09-2010 03:17 PM
[NMR paper] Tumor suppressor p16INK4A: structural characterization of wild-type and mutant protei
Tumor suppressor p16INK4A: structural characterization of wild-type and mutant proteins by NMR and circular dichroism.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles Tumor suppressor p16INK4A: structural characterization of wild-type and mutant proteins by NMR and circular dichroism.
Biochemistry. 1996 Jul 23;35(29):9475-87
Authors: Tevelev A, Byeon IJ, Selby T, Ericson K, Kim HJ, Kraynov V, Tsai MD
The tumor suppressor p16INK4A with eight N-terminal amino acids deleted (p16/delta 1-8)...
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08-22-2010 02:20 PM
[NMR paper] Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www3.interscience.wiley.com-aboutus-images-wiley_interscience_pubmed_logo_FREE_120x27.gif Related Articles Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
Eur J Biochem. 1991 Aug 15;200(1):139-48
Authors: Folkers PJ, Stassen AP, van Duynhoven JP, Harmsen BJ, Konings RN, Hilbers CW
Recording of good quality NMR spectra of the single-stranded DNA binding protein gene V of the...
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08-21-2010 11:12 PM
[NMR paper] Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www3.interscience.wiley.com-aboutus-images-wiley_interscience_pubmed_logo_FREE_120x27.gif Related Articles Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
Eur J Biochem. 1991 Aug 15;200(1):139-48
Authors: Folkers PJ, Stassen AP, van Duynhoven JP, Harmsen BJ, Konings RN, Hilbers CW
Recording of good quality NMR spectra of the single-stranded DNA binding protein gene V of the...
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08-21-2010 11:12 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
High Resolution 1H Detected 1H,13C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective 1H,2H Isotopic Labeling of Methyl Groups
High Resolution <SUP>1</SUP>H Detected <SUP>1</SUP>H,<SUP>13</SUP>C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective <SUP>1</SUP>H,<SUP>2</SUP>H Isotopic Labeling of Methyl Groups
Vipin Agarwal, Anne Diehl, Nikolai Skrynnikov, and Bernd Reif
J. Am. Chem. Soc.; 2006; 128(39) pp 12620 - 12621;
Abstract:
MAS solid-state NMR experiments applied to biological solids are still hampered by low sensitivity and resolution. In this work, we employ a deuteration scheme in which individual methyl groups are selectively protonated. This labeling scheme...
Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR
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