In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes |
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Authors: | Tomomi Sakai Hidehito Tochio Takeshi Tenno Yutaka Ito Tetsuro Kokubo Hidekazu Hiroaki Masahiro Shirakawa |
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Institution: | (1) International Graduate School of Arts and Sciences, Yokohama City University, Yokohama 230-0045, Japan;(2) Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-Ku Kyoto, 615-8510, Japan;(3) Graduate Schools of Science, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji Tokyo, 192-0397, Japan;(4) CREST, JST, 4-1-8 Honcho Kawaguchi, Saitama, Japan;(5) Genome Science Center, RIKEN, Yokohama 230-0045, Japan |
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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. 15N-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional 1H–15N 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.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at . |
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Keywords: | calmodulin In-cell NMR microinjection ubiquitin Xenopus laevis oocyte |
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