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CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample

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Abstract

A new strategy for the NMR assignment of aliphatic side-chains in large perdeuterated proteins is proposed. It involves an alternative isotopic labeling protocol, the use of an out-and-back 13C–13C TOCSY experiment ((H)C-TOCSY-C-TOCSY-(C)H) and an optimized non-uniform sampling protocol. It has long been known that the non-linearity of an aliphatic spin-system (for example Ile, Val, or Leu) substantially compromises the efficiency of the TOCSY transfers. To permit the use of this efficient pulse scheme, a series of optimized precursors were designed to yield linear 13C perdeuterated side-chains with a single protonated CH3 group in these three residues. These precursors were added to the culture medium for incorporation into expressed proteins. For Val and Leu residues, the topologically different spin-systems introduced for the pro-R and pro-S methyl groups enable stereospecific assignment. All CH3 can be simultaneously assigned on a single sample using a TOCSY experiment. It only requires the tuning of a mixing delay and is thus more versatile than the relayed COSY experiment. Enhanced resolution and sensi-tivity can be achieved by non-uniform sampling combined with the removal of the large JCC coupling by deconvolution prior to the processing by iterative soft thresholding. This strategy has been used on malate synthase G where a large percentage of the CH3 groups could be correlated directly up to the backbone Ca. It is anticipated that this robust combined strategy can be routinely applied to large proteins.

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Acknowledgments

We would like to thank Dr. S.J. Remington for providing the MSG plasmid, Dr. D. Chipman for providing the ALSII plasmid, Pr. Vladislav Yu. Orekhov for providing MDDNMR software, Mrs I. Ayala, Drs. P. Gans and M. Plevin for stimulating discussions and Drs Laura Lerner and Michael Caffrey for manuscript editing. This work used the high-field NMR and the isotopic labeling facilities at the Grenoble Instruct Centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The research leading to these results has received funding from the European Research Council under the European Community’s Seventh Framework Program FP7/2007-2013 Grant Agreement No. 260887.

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Authors and Affiliations

  1. Université Grenoble Alpes, IBS, 38044, Grenoble, France

    Rime Kerfah, Jérôme Boisbouvier & Dominique Marion

  2. CNRS, IBS, 38044, Grenoble, France

    Rime Kerfah, Jérôme Boisbouvier & Dominique Marion

  3. CEA, IBS, 38044, Grenoble, France

    Rime Kerfah, Jérôme Boisbouvier & Dominique Marion

  4. Chemistry and Biology of Metals Laboratory, University Grenoble Alpes, 38027, Grenoble, France

    Olivier Hamelin

  5. Biomolecular NMR Spectroscopy Group, Institut de Biologie Structurale, 71 Avenue des Martyrs, CS 10090, 38044, Grenoble Cedex 9, France

    Dominique Marion

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  1. Rime Kerfah
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  2. Olivier Hamelin
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  3. Jérôme Boisbouvier
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  4. Dominique Marion
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Correspondence to Dominique Marion.

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Kerfah, R., Hamelin, O., Boisbouvier, J. et al. CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample. J Biomol NMR 63, 389–402 (2015). https://doi.org/10.1007/s10858-015-9998-4

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