Abstract
Specific isotopic labeling of methyl groups in proteins has greatly extended the applicability of solution NMR spectroscopy. Simultaneous labeling of the methyl groups of several different amino acid types can offer a larger number of useful probes that can be used for structural characterisations of challenging proteins. Herein, we propose an improved AILV methyl-labeling protocol in which L and V are stereo-specifically labeled. We show that 2-ketobutyrate cannot be combined with Ala and 2-acetolactate (for the stereo-specific labeling of L and V) as this results in co-incorporation incompatibility and isotopic scrambling. Thus, we developed a robust and cost-effective enzymatic synthesis of the isoleucine precursor, 2-hydroxy-2-(1′-[2H2], 2′-[13C])ethyl-3-keto-4-[2H3]butanoic acid, as well as an incorporation protocol that eliminates metabolic leakage. We show that application of this labeling scheme to a large 82 kDa protein permits the detection of long-range 1H–1H NOE cross-peaks between methyl probes separated by up to 10 Å.
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Acknowledgments
We would like to thank Dr. S. J. Remington for providing MSG plasmid, Dr. D. Chipman for providing AHAS II plasmid and Mrs I. Ayala as well as Dr. R. Sounier for stimulating discussions. 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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Kerfah, R., Plevin, M.J., Pessey, O. et al. Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-proS and valine-proS methyl groups for the detection of long range NOEs. J Biomol NMR 61, 73–82 (2015). https://doi.org/10.1007/s10858-014-9887-2
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DOI: https://doi.org/10.1007/s10858-014-9887-2