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An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated 15N, 13C-labeled proteins

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Summary

A biosynthetic strategy has recently been developed for the production of 15N, 13C, 2H-labeled proteins using 1H3C-pyruvate as the sole carbon source and D2O as the solvent. The methyl groups of Ala, Val, Leu and Ile (γ2 only) remain highly protonated, while the remaining positions in the molecule are largely deuterated. An (H)C(CO)NH-TOCSY experiment is presented for the sequential assignment of the protonated methyl groups. A high-sensitivity spectrum is recorded on a 15N, 13C, 2H, 1H3C-labeled SH2 domain at 3°C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30–40 kDa molecular weight range.

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Author notes

  1. Robert Konrat

    Present address: The Institute of Organic Chemistry, University of Innsbruck, Innrain 52A, A-6060, Innsbruck, Austria

  2. Michael K. Rosen

    Present address: Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, 10021, New York, NY, USA

Authors and Affiliations

  1. The Protein Engineering Centers of Excellence, University of Toronto, M5S 1A8, Toronto, ON, Canada

    Kevin H. Gardner, Robert Konrat, Michael K. Rosen & Lewis E. Kay

  2. Department of Medical Genetics, University of Toronto, M5S 1A8, Toronto, ON, Canada

    Kevin H. Gardner, Robert Konrat, Michael K. Rosen & Lewis E. Kay

  3. Department of Biochemistry, University of Toronto, M5S 1A8, Toronto, ON, Canada

    Kevin H. Gardner, Robert Konrat, Michael K. Rosen & Lewis E. Kay

  4. Department of Chemistry, University of Toronto, M5S 1A8, Toronto, ON, Canada

    Kevin H. Gardner, Robert Konrat, Michael K. Rosen & Lewis E. Kay

Authors
  1. Kevin H. Gardner
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  2. Robert Konrat
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  4. Lewis E. Kay
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Gardner, K.H., Konrat, R., Rosen, M.K. et al. An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated 15N, 13C-labeled proteins. J Biomol NMR 8, 351–356 (1996). https://doi.org/10.1007/BF00410333

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  • DOI: https://doi.org/10.1007/BF00410333

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