In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes

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 ooc...

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Bibliographic Details
Published in:Journal of biomolecular NMR 2006-11, Vol.36 (3), p.179-188
Main Authors: Sakai, Tomomi, Tochio, Hidehito, Tenno, Takeshi, Ito, Yutaka, Kokubo, Tetsuro, Hiroaki, Hidekazu, Shirakawa, Masahiro
Format: Article
Language:English
Subjects:
Animals
Anura
calmodulin
Calmodulin - chemistry
Cells - chemistry
Cytological Techniques
Environmental conditions
In-cell NMR
microinjection
Nuclear Magnetic Resonance, Biomolecular - methods
Oocytes - chemistry
Protein Binding
Protein Conformation
Proteins
Spectrum analysis
ubiquitin
Ubiquitin - chemistry
Xenopus laevis
Xenopus laevis oocyte
Xenopus Proteins - chemistry
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Summary: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.
ISSN:0925-2738
1573-5001
DOI:10.1007/s10858-006-9079-9