Abstract
A strategy for the introduction of (1H,13C-methyl)-alanine into perdeuterated proteins is described. Specific protonation of alanine methyl groups to a level of 95% can be achieved by overexpressing proteins in M9/D2O based bacterial growth medium supplemented with 800 mg/l of 2-[2H], 3-[13C] l-alanine. However, though simple, this approach results in undesired, non-specific background labeling due to isotope scrambling via different amino acid metabolic pathways. Following a careful analysis of known metabolic pathways we found that co-addition of perdeuterated forms of α-ketoisovalerate-d7, succinate-d4 and l-isoleucine-d10 with labeled l-alanine, reduces undesired background labeling to <1%. When combined with recently developed methyl TROSY experiments, this methyl-specific labeling protocol permits the acquisition of excellent quality correlation spectra of alanine methyl groups in high molecular weight proteins. Our cost effective strategy offers a significant enhancement in the level of incorporation of methyl-labeled alanine in overexpressed proteins over previously reported methods.
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Acknowledgements
This work was supported by the CEA, the CNRS, and the UJF. J.B acknowledges funding from HFSP (CDA #0029/2004) and CNRS (Interdisciplinary program interface between physics, biology and chemistry). The authors thank Drs. Kay and Remington for providing clones of Malate Synthase G, Drs. Amero and Marion for stimulating discussion, and Dr. Plevin for suggestions and careful reading of this manuscript.
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Ayala, I., Sounier, R., Usé, N. et al. An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein. J Biomol NMR 43, 111–119 (2009). https://doi.org/10.1007/s10858-008-9294-7
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DOI: https://doi.org/10.1007/s10858-008-9294-7