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Utilization of lysine 13C-methylation NMR for protein–protein interaction studies

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Abstract

Chemical modification is an easy way for stable isotope labeling of non-labeled proteins. The reductive 13C-methylation of the amino group of the lysine side-chain by 13C-formaldehyde is a post-modification and is applicable to most proteins since this chemical modification specifically and quickly proceeds under mild conditions such as 4 °C, pH 6.8, overnight. 13C-methylation has been used for NMR to study the interactions between the methylated proteins and various molecules, such as small ligands, nucleic acids and peptides. Here we applied lysine 13C-methylation NMR to monitor protein–protein interactions. The affinity and the intermolecular interaction sites of methylated ubiquitin with three ubiquitin-interacting proteins were successfully determined using chemical-shift perturbation experiments via the 1H–13C HSQC spectra of the 13C-methylated-lysine methyl groups. The lysine 13C-methylation NMR results also emphasized the importance of the usage of side-chain signals to monitor the intermolecular interaction sites, and was applicable to studying samples with concentrations in the low sub-micromolar range.

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Acknowledgments

We thank M. Yoneyama and H. Kinoshita for sample preparation; S. Isogai and D. Morimoto for the gift of the p62UBA sample; H. Kobayashi and A. Ohno for the gift of the Dsk2UBA expression vector; and J. Tsukamoto, Y. Hara and A. Nakagawa for mass spectroscopy. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) through the Target Proteins Research Program, the Global COE Program at NAIST, and Grants-in-Aid for Scientific Research (20370038, 22121515, 23370053, 20770086) to CK and IO.

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Authors and Affiliations

  1. Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Yoshikazu Hattori, Kyoko Furuita, Takahisa Ikegami, Toshimichi Fujiwara & Chojiro Kojima

  2. Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan

    Izuru Ohki & Chojiro Kojima

  3. Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka, 599-8531, Japan

    Harumi Fukada

  4. Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, 606-8501, Japan

    Masahiro Shirakawa

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  1. Yoshikazu Hattori
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  2. Kyoko Furuita
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  3. Izuru Ohki
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Correspondence to Izuru Ohki or Chojiro Kojima.

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Hattori, Y., Furuita, K., Ohki, I. et al. Utilization of lysine 13C-methylation NMR for protein–protein interaction studies. J Biomol NMR 55, 19–31 (2013). https://doi.org/10.1007/s10858-012-9675-9

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  • DOI: https://doi.org/10.1007/s10858-012-9675-9

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