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Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids

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Abstract

The 1H–13C HMQC signals of the 13CH3 moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ1-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically 13CH3-labeled [U–2H;15N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G.

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Acknowledgments

This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science (MEXT), and also in part by the Targeted Protein Research Program (MEXT). Y. M. was supported by JSPS KAKENHI Grants-in-Aid for Young Scientists (B) (23770111 and 25840021), and M. T. was supported by a JSPS KAKENHI Grant-in-Aid for Young Scientists (B) (23770109), a Grant-in-Aid for Scientific Research (C) (25440018), and a grant from the Kurata Memorial Hitachi Science and Technology Foundation. We would like to thank Dr. Tugarinov, of the University of Maryland, for providing the MSG gene and his advice regarding MSG protein production. We also thank Dr. Jérôme Boisbouvier, of Institute de Biologie Structurale Jean-Pierre Ebel, for valuable discussions.

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Authors and Affiliations

  1. Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan

    Yohei Miyanoiri, Mitsuhiro Takeda & Masatsune Kainosho

  2. Center for Priority Areas, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, 192-0397, Japan

    Kosuke Okuma, Akira M. Ono, Tsutomu Terauchi & Masatsune Kainosho

  3. SAIL Technologies Inc., 1-40 Suehiro-cho 1-chome, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan

    Kosuke Okuma, Akira M. Ono & Tsutomu Terauchi

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  1. Yohei Miyanoiri
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  2. Mitsuhiro Takeda
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  3. Kosuke Okuma
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Correspondence to Masatsune Kainosho.

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Miyanoiri, Y., Takeda, M., Okuma, K. et al. Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids. J Biomol NMR 57, 237–249 (2013). https://doi.org/10.1007/s10858-013-9784-0

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