Selenko Philipp, Serber Zach, Gadea Bedrick, Ruderman Joan, Wagner Gerhard
Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2006 Aug 8;103(32):11904-9. doi: 10.1073/pnas.0604667103. Epub 2006 Jul 27.
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.
我们引入了一种真核细胞系统——非洲爪蟾卵母细胞，用于对生物分子进行高分辨率的细胞内核磁共振（NMR）分析，并阐述了在这种细胞类型中成功进行体内NMR测量的实验参考条件。这种方法能够在确定的外源蛋白质细胞内浓度下进行定量NMR实验，蛋白质G B1结构域（GB1）的细胞内NMR特性描述即为实例。在非洲爪蟾卵提取物和人工拥挤的体外溶液中进行的其他实验表明，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。对于在纯态下具有高度内部流动性的蛋白质区域，这些影响尤为明显。我们还评估了该方法的实验局限性，并讨论了其在真核细胞活动原位结构表征方面的潜在应用。
