title: A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells.
abstract: Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N-H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji, Tokyo, 192-0397, Japan.|Synthetic Organic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Hikone
authors.affiliation: Synthetic Organic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.|AMED-CREST, Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan.
authors.forename: Go
authors.initials: G
authors.lastname: Hirai
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji, Tokyo, 192-0397, Japan.|CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Mishima
authors.affiliation: Quantitative Biology Center, RIKEN, 1-7-22 Suehirocho, Tsurumi-ku, Yokohama, 230-0045, Japan.|PRESTO/Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan.
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Inomata
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji, Tokyo, 192-0397, Japan.|CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan.
authors.forename: Teppei
authors.initials: T
authors.lastname: Ikeya
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji, Tokyo, 192-0397, Japan.
authors.forename: Souichiro
authors.initials: S
authors.lastname: Arai
authors.affiliation: AMED-CREST, Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan.|Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto, 615-8510, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Shirakawa
authors.affiliation: Synthetic Organic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.|AMED-CREST, Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan.
authors.forename: Mikiko
authors.initials: M
authors.lastname: Sodeoka
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji, Tokyo, 192-0397, Japan. ito-yutaka@tmu.ac.jp.|CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan. ito-yutaka@tmu.ac.jp.
authors.forename: Yutaka
authors.identifier: 0000-0002-1030-4660
authors.initials: Y
authors.lastname: Ito
mesh_terms: D002614:Chelating Agents / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D009154:Mutation; D011487:Protein Conformation; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics
chemical_list: D002614:Chelating Agents; D028581:Lanthanoid Series Elements; D011506:Proteins; D025801:Ubiquitin
keywords: Cultured human cells; Electroporation; In-cell NMR; Lanthanoid-chelating tag; Paramagnetic NMR