title: [Non-invasive analysis of proteins in living cells using NMR spectroscopy].
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, (19)F-NMR to characterize protein-ligand interactions in cells is presented. Because (19)F nuclei rarely exist in natural cells, when (19)F-labeled proteins are delivered into cells and (19)F-NMR signals are observed, one can safely ascertain that these signals originate from the delivered proteins and not other molecules.
authors.affiliation: Department of Molecular Engineering, Graduate School of Engineering, Kyoto University.
authors.forename: Hidehito
authors.initials: H
authors.lastname: Tochio
authors.forename: Shuhei
authors.initials: S
authors.lastname: Murayama
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Inomata
authors.forename: Daichi
authors.initials: D
authors.lastname: Morimoto
authors.forename: Ayako
authors.initials: A
authors.lastname: Ohno
authors.forename: Masahiro
authors.initials: M
authors.lastname: Shirakawa
mesh_terms: D002470:Cell Survival; D006801:Humans; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000378:metabolism
chemical_list: D008024:Ligands; D011506:Proteins