title: High-resolution heteronuclear multidimensional NMR of proteins in living insect cells using a baculovirus protein expression system.
abstract: Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. In order to complement the existing protocols, and to extend the range of possible applications, we introduce a novel approach for observing in-cell NMR spectra using the sf9 cell/baculovirus system. High-resolution 2D (1)H-(15)N correlation spectra were observed for four model proteins expressed in sf9 cells. Furthermore, 3D triple-resonance NMR spectra of the Streptococcus protein G B1 domain were observed in sf9 cells by using nonlinear sampling to overcome the short lifetime of the samples and the low abundance of the labeled protein. The data were processed with a quantitative maximum entropy algorithm. These were assigned ab initio, yielding approximately 80% of the expected backbone NMR resonances. Well-resolved NOE cross peaks could be identified in the 3D (15)N-separated NOESY spectrum, suggesting that structural analysis of this size of protein will be feasible in sf9 cells.
authors: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji-shi, Tokyo 192-0373, Japan.
authors: Jumpei
authors: J
authors: Hamatsu
authors: Daniel
authors: D
authors: O'Donovan
authors: Takashi
authors: T
authors: Tanaka
authors: Takahiro
authors: T
authors: Shirai
authors: Yuichiro
authors: Y
authors: Hourai
authors: Tsutomu
authors: T
authors: Mikawa
authors: Teppei
authors: T
authors: Ikeya
authors: Masaki
authors: M
authors: Mishima
authors: Wayne
authors: W
authors: Boucher
authors: Brian O
authors: BO
authors: Smith
authors: Ernest D
authors: ED
authors: Laue
authors: Masahiro
authors: M
authors: Shirakawa
authors: Yutaka
authors: Y
authors: Ito
mesh_terms: D000465:Algorithms; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D000079922:Copper Transport Proteins; D019277:Entropy; D006801:Humans; D056511:Metallochaperones / Q000737:chemistry* / Q000235:genetics; D018832:Molecular Chaperones; D019906:Nuclear Magnetic Resonance, Biomolecular; D011789:Quantum Theory; D051381:Rats; D061987:Sf9 Cells; D018411:Spodoptera / Q000821:virology*; D016963:Thermus thermophilus / Q000235:genetics
chemical_list: C474500:ATOX1 protein, human; D001426:Bacterial Proteins; D002147:Calmodulin; D000079922:Copper Transport Proteins; D056511:Metallochaperones; D018832:Molecular Chaperones