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Deuteration and selective labeling of alanine methyl groups of β2-adrenergic receptor expressed in a baculovirus-insect cell expression system

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Abstract

G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl-13C1H3-labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of β2-adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.

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Acknowledgements

This work was supported by the development of core technologies for innovative drug development based upon IT, from the Japan Agency for Medical Research and Development, AMED, and by the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP16H01353, JP16H01531, JP17H04999, JP17H06097, JP17J11420. Y.S. and M.N. are JSPS fellows.

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Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-0033, Japan

    Yutaka Kofuku, Tomoki Yokomizo, Shunsuke Imai, Yutaro Shiraishi, Mei Natsume, Hiroaki Itoh, Masayuki Inoue, Takumi Ueda & Ichio Shimada

  2. Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, 210-8681, Japan

    Kunio Nakata, Shunsuke Igarashi, Hideyuki Yamaguchi, Toshimi Mizukoshi & Ei-ichiro Suzuki

  3. Japan Biological Informatics Consortium (JBiC), Koto-ku, Tokyo, 135-8073, Japan

    Ei-ichiro Suzuki

  4. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Chiyoda-ku, Tokyo, 102-0075, Japan

    Takumi Ueda

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  1. Yutaka Kofuku
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  2. Tomoki Yokomizo
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  3. Shunsuke Imai
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  12. Ei-ichiro Suzuki
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  13. Takumi Ueda
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  14. Ichio Shimada
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Correspondence to Ichio Shimada.

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Kofuku, Y., Yokomizo, T., Imai, S. et al. Deuteration and selective labeling of alanine methyl groups of β2-adrenergic receptor expressed in a baculovirus-insect cell expression system. J Biomol NMR 71, 185–192 (2018). https://doi.org/10.1007/s10858-018-0174-5

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