Non-invasive Analysis of Proteins in Living Cells Using NMR Spectroscopy

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  • Tochio Hidehito
    Department of Molecular Engineering, Graduate School of Engineering, Kyoto University
  • Murayama Shuhei
    Department of Molecular Engineering, Graduate School of Engineering, Kyoto University
  • Inomata Kohsuke
    RIKEN, QBiC
  • Morimoto Daichi
    Department of Molecular Engineering, Graduate School of Engineering, Kyoto University
  • Ohno Ayako
    Departments of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School
  • Shirakawa Masahiro
    Department of Molecular Engineering, Graduate School of Engineering, Kyoto University

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Other Title
  • 核磁気共鳴法を用いた細胞内タンパク質の無侵襲解析
  • Symposium Review 核磁気共鳴法を用いた細胞内タンパク質の無侵襲解析
  • Symposium Review カク ジキ キョウメイホウ オ モチイタ サイボウ ナイ タンパクシツ ノ ムシンシュウカイセキ

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Abstract

  NMR spectroscopy enables structural analyses of proteins and has been widely used in the structural biology field in recent decades. NMR spectroscopy can be applied to proteins inside living cells, allowing characterization of their structures and dynamics in intracellular environments. The simplest “in-cell NMR” approach employs bacterial cells; in this approach, live Escherichia coli cells overexpressing a specific protein are subjected to NMR. The cells are grown in an NMR active isotope-enriched medium to ensure that the overexpressed proteins are labeled with the stable isotopes. Thus the obtained NMR spectra, which are derived from labeled proteins, contain atomic-level information about the structure and dynamics of the proteins. Recent progress enables us to work with higher eukaryotic cells such as HeLa and HEK293 cells, for which a number of techniques have been developed to achieve isotope labeling of the specific target protein. In this review, we describe successful use of electroporation for in-cell NMR. In addition, 19F-NMR to characterize protein-ligand interactions in cells is presented. Because 19F nuclei rarely exist in natural cells, when 19F-labeled proteins are delivered into cells and 19F-NMR signals are observed, one can safely ascertain that these signals originate from the delivered proteins and not other molecules.<br>

Journal

  • YAKUGAKU ZASSHI

    YAKUGAKU ZASSHI 135 (3), 391-398, 2015-03-01

    The Pharmaceutical Society of Japan

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