Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes
Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes
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DOI：
10.1073/pnas.0604667103
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发表时间：
2006-08-08
影响因子：
11.1
通讯作者：
Wagner, Gerhard
中科院分区：
文献类型：
--
作者：
Selenko, Philipp;Serber, Zach;Wagner, Gerhard
我们介绍了一种真核细胞系统，非洲爪蟾卵母细胞，在细胞内的NMR分析的生物分子在高分辨率和描绘的实验参考条件成功实现在这种细胞类型的体内NMR测量。这种方法能够在限定的外源蛋白质的细胞内浓度下进行定量NMR实验，这通过蛋白质G B1结构域（GB1）的细胞内NMR性质的描述来举例说明。在非洲爪蟾卵提取物和人工拥挤的体外溶液中的其他实验表明，对于这种生物惰性蛋白质结构域，细胞内粘度和大分子拥挤决定了其体内行为。这些贡献似乎特别明显的蛋白质区域具有高度的内部流动性，在纯状态。我们还评估了这种方法的实验局限性，并讨论了对真核细胞活动的原位结构表征的潜在应用。
We introduce a eukaryotic cellular system, the Xenopus laevis oocyte, for in-cell NMR analyses of biomolecules at high resolution and delineate the experimental reference conditions for successful implementations of in vivo NMR measurements in this cell type. This approach enables quantitative NMR experiments at defined intracellular concentrations of exogenous proteins, which is exemplified by the description of in-cell NMR properties of the protein G B1 domain (GB1). Additional experiments in Xenopus egg extracts and artificially crowded in vitro solutions suggest that for this biologically inert protein domain, intracellular viscosity and macromolecular crowding dictate its in vivo behavior. These contributions appear particularly pronounced for protein regions with high degrees of internal mobility in the pure state. We also evaluate the experimental limitations of this method and discuss potential applications toward the in situ structural characterization of eukaryotic cellular activities.