Abstract
Methyl moieties are highly valuable probes for quantitative NMR studies of large proteins. Hence, their assignment is of the utmost interest to obtain information on both interactions and dynamics of proteins in solution. Here, we present the synthesis of a new precursor that allows connection of leucine and valine pro-S methyl moieties to backbone atoms by linear 13C-chains. This optimized 2H/13C-labelled acetolactate precursor can be combined with existing 13C/2H-alanine and isoleucine precursors in order to directly transfer backbone assignment to the corresponding methyl groups. Using this simple approach leucine and valine pro-S methyl groups can be assigned using a single sample without requiring correction of 1H/2H isotopic shifts on 13C resonances. The approach was demonstrated on the N-terminal domain of human HSP90, for which complete assignment of Ala-β, Ile-δ1, Leu-δ2, Met-ε, Thr-γ and Val-γ2 methyl groups was obtained.
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Data availability
The FIDs acquired for this study are available in the biological magnetic resonance databank (bmrbig12) and the assignment have been deposited under the BMRB ID: 50786.
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Acknowledgements
The authors thank Dr. R Awad and Mr. L. Imbert for advice and stimulating discussions. This work used the high field NMR and isotopic labelling facilities at the Grenoble Instruct-ERIC Center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB). Platform access was supported by FRISBI (ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). This work was supported by grants from CEA/NMR-Bio (research program C24990) and by the French National Research Agency in the framework of the “Investissements d’avenir” program (ANR-15‐IDEX‐02).
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Henot, F., Kerfah, R., Törner, R. et al. Optimized precursor to simplify assignment transfer between backbone resonances and stereospecifically labelled valine and leucine methyl groups: application to human Hsp90 N-terminal domain. J Biomol NMR 75, 221–232 (2021). https://doi.org/10.1007/s10858-021-00370-0
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DOI: https://doi.org/10.1007/s10858-021-00370-0