In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes
In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes
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DOI：
10.1007/s10858-006-9079-9
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发表时间：
2006-11-01
影响因子：
2.7
通讯作者：
Shirakawa, Masahiro
中科院分区：
文献类型：
--
作者：
Sakai, Tomomi;Tochio, Hidehito;Shirakawa, Masahiro
细胞内NMR是溶液NMR的一种应用，可以研究活细胞内的蛋白质构象。我们测量了非洲爪蟾卵母细胞的细胞内NMR光谱。将N-15标记的泛素及其衍生物和钙调素注射到非洲爪蟾卵母细胞中，获得了蛋白质的二维H-1-N-15相关光谱。虽然在卵母细胞中的野生型泛素的光谱有相当少的交叉峰相比，其在体外光谱，泛素衍生物，大概是无法结合到泛素相互作用蛋白给出了一个显着更大的交叉峰的数量。这一观察结果表明，泛素和泛素相互作用蛋白之间的蛋白质-蛋白质相互作用可能会导致NMR信号增宽，从而破坏细胞内HSQC光谱的质量。此外，我们还利用细胞内核磁共振技术观察了泛素前体衍生物在活卵母细胞中的成熟过程。这一过程被部分抑制预加入的泛素醛，一个特定的抑制剂泛素C-末端水解酶（UCH）。我们的工作表明，在细胞内NMR与爪蟾卵母细胞的蛋白质构象和功能的调查在细胞内环境条件下的潜在用途。
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.