title: Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes.
abstract: We introduce a eukaryotic cellular system, the Xenopus laevis oocyte, for in-cell NMR analyses of biomolecules at high resolution and delineate the experimental reference conditions for successful implementations of in vivo NMR measurements in this cell type. This approach enables quantitative NMR experiments at defined intracellular concentrations of exogenous proteins, which is exemplified by the description of in-cell NMR properties of the protein G B1 domain (GB1). Additional experiments in Xenopus egg extracts and artificially crowded in vitro solutions suggest that for this biologically inert protein domain, intracellular viscosity and macromolecular crowding dictate its in vivo behavior. These contributions appear particularly pronounced for protein regions with high degrees of internal mobility in the pure state. We also evaluate the experimental limitations of this method and discuss potential applications toward the in situ structural characterization of eukaryotic cellular activities.
authors.affiliation: Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Philipp
authors.initials: P
authors.lastname: Selenko
authors.forename: Zach
authors.initials: Z
authors.lastname: Serber
authors.forename: Bedrick
authors.initials: B
authors.lastname: Gadea
authors.forename: Joan
authors.initials: J
authors.lastname: Ruderman
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wagner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001671:Biochemistry / Q000379:methods; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D014982:Xenopus laevis / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus