title: In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes.
abstract: In-cell NMR is an application of solution NMR that enables the investigation of protein conformations inside living cells. We have measured in-cell NMR spectra in oocytes from the African clawed frog Xenopus laevis. (15)N-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional (1)H-(15)N correlation spectra of the proteins were obtained. While the spectrum of wild-type ubiquitin in oocytes had rather fewer cross-peaks compared to its in vitro spectrum, ubiquitin derivatives that are presumably unable to bind to ubiquitin-interacting proteins gave a markedly larger number of cross-peaks. This observation suggests that protein-protein interactions between ubiquitin and ubiquitin-interacting proteins may cause NMR signal broadening, and hence spoil the quality of the in-cell HSQC spectra. In addition, we observed the maturation of ubiquitin precursor derivative in living oocytes using the in-cell NMR technique. This process was partly inhibited by pre-addition of ubiquitin aldehyde, a specific inhibitor for ubiquitin C-terminal hydrolase (UCH). Our work demonstrates the potential usefulness of in-cell NMR with Xenopus oocytes for the investigation of protein conformations and functions under intracellular environmental conditions.
authors.affiliation: International Graduate School of Arts and Sciences, Yokohama City University, Yokohama, 230-0045, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Sakai
authors.forename: Hidehito
authors.initials: H
authors.lastname: Tochio
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tenno
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Ito
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Kokubo
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Hiroaki
authors.forename: Masahiro
authors.initials: M
authors.lastname: Shirakawa
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000737:chemistry; D002477:Cells / Q000737:chemistry; D003584:Cytological Techniques; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009865:Oocytes / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D025801:Ubiquitin / Q000737:chemistry; D029867:Xenopus Proteins / Q000737:chemistry*
chemical_list: D002147:Calmodulin; D025801:Ubiquitin; D029867:Xenopus Proteins