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A Photo-Crosslinking Approach to Monitoring the Assembly of an LptD Intermediate with LptE in a Living Cell

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Lipopolysaccharide Transport

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2548))

Abstract

Elucidating the dynamic behavior of proteins in living cells is extremely important for understanding the physiological roles of biological processes. The site-specific in vivo photo-crosslinking approach using a photoreactive unnatural amino acid enables the analysis of protein interactions with high spatial resolution in vivo. Recently, by improving the photo-crosslinking technique, we developed the “PiXie” method for the analysis of dynamic interactions of newly synthesized proteins. Here, we describe the detailed protocols of the “PiXie” method and its application to the analysis of the assembly processes of the lipopolysaccharide translocon components, a β-barrel outer membrane protein, LptD, and a lipoprotein, LptE.

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Acknowledgments

This work was supported by JSPS KAKENHI and MEXT KAKENHI Grants 15J05262, 18H06047, 19K21179, and 20K15715 (to R.M.); 17K07334, 17H05666, 17H05879, and 20K06556 (to H. M.); and 15H01532 and 18H023404 (to Y.A.), as well as research grants from the Institute for Fermentation, Osaka Y-2020-02-027 (to R.M.) and the Nagase Science and Technology Foundation (to Y.A.).

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Authors and Affiliations

  1. Nara Institute of Science and Technology, Ikoma, Nara, Japan

    Ryoji Miyazaki

  2. Institute for Life and Medical Sciences, Kyoto University, Kyoto, Japan

    Hiroyuki Mori & Yoshinori Akiyama

Authors
  1. Ryoji Miyazaki
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  2. Hiroyuki Mori
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  3. Yoshinori Akiyama
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Corresponding author

Correspondence to Yoshinori Akiyama .

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Editors and Affiliations

  1. Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy

    Paola Sperandeo

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Miyazaki, R., Mori, H., Akiyama, Y. (2022). A Photo-Crosslinking Approach to Monitoring the Assembly of an LptD Intermediate with LptE in a Living Cell. In: Sperandeo, P. (eds) Lipopolysaccharide Transport. Methods in Molecular Biology, vol 2548. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2581-1_7

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  • DOI: https://doi.org/10.1007/978-1-0716-2581-1_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2580-4

  • Online ISBN: 978-1-0716-2581-1

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