In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes

被引:99
作者
Sakai, Tomomi
Tochio, Hidehito [1 ]
Tenno, Takeshi
Ito, Yutaka
Kokubo, Tetsuro
Hiroaki, Hidekazu
Shirakawa, Masahiro
机构
[1] Kyoto Univ, Dept Mol Engn, Grad Sch Engn, Nishikyo Ku, Kyoto 6158510, Japan
[2] Yokohama City Univ, Int Grad Sch Arts & Sci, Yokohama, Kanagawa 2300045, Japan
[3] Tokyo Metropolitan Univ, Grad Sch Sci, Hachioji, Tokyo 1920397, Japan
[4] CREST, JST, Kawaguchi, Saitama, Japan
[5] RIKEN, Genome Sci Ctr, Yokohama, Kanagawa 2300045, Japan
基金
日本科学技术振兴机构;
关键词
calmodulin; in-cell NMR; microinjection; ubiquitin; Xenopus laevis oocyte;
D O I
10.1007/s10858-006-9079-9
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. N-15-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional H-1-N-15 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.
引用
收藏
页码:179 / 188
页数:10
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