Protein structure determination in living cells by in-cell NMR spectroscopy
Protein structure determination in living cells by in-cell NMR spectroscopy
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DOI：
10.1038/nature07814
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发表时间：
2009-03-05
期刊：
影响因子：
64.8
通讯作者：
Ito, Yutaka
中科院分区：
文献类型：
--
作者：
Sakakibara, Daisuke;Sasaki, Atsuko;Ito, Yutaka
以原子分辨率研究在生活环境中“工作”的蛋白质是分子生物学的主要目标，尽管用于单晶或溶液中纯化蛋白质的三维（3D）结构测定的方法被广泛使用，但该目标尚未实现。NMR硬件和方法学的最新发展使得能够测量活细胞中大分子的高分辨率异相多维NMR光谱（细胞内NMR）（1-5）。各种细胞内的事件，如构象变化，动力学和结合事件已被这种方法进行了研究。然而，低灵敏度和短寿命的样品，到目前为止，阻止了收购足够的结构信息，以确定蛋白质结构的细胞内NMR。在这里，我们展示了第一个，据我们所知，完全基于活细胞中获得的信息计算的3D蛋白质结构。通过细胞内NMR解析了在大肠杆菌细胞中过表达的嗜热栖热菌HB 8的推定重金属结合蛋白TTHA 1718的结构。通过对间接获得的三维数据进行非线性采样来快速测量三维核磁共振谱，以克服活体E.大肠杆菌样本。几乎所有预期的骨架NMR共振和大多数侧链NMR共振都被观察到并分配，使得能够计算出与独立测定的TTHA 1718的体外结构非常相似的高质量（0.96埃骨架均方根偏差）结构。因此，细胞内NMR方法可以提供生活环境中蛋白质的精确高分辨率结构。
Investigating proteins 'at work' in a living environment at atomic resolution is a major goal of molecular biology, which has not been achieved even though methods for the three-dimensional (3D) structure determination of purified proteins in single crystals or in solution are widely used. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR)(1-5). Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and the short lifetime of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure calculated exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions was used to overcome problems caused by the instability and low sensitivity of living E. coli samples. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned, enabling high quality (0.96 angstrom backbone root mean squared deviation) structures to be calculated that are very similar to the in vitro structure of TTHA1718 determined independently. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments.