NMR protein structure determination in living E. coli cells using nonlinear sampling
NMR protein structure determination in living E. coli cells using nonlinear sampling
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DOI：
10.1038/nprot.2010.69
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发表时间：
2010-01-01
期刊：
影响因子：
14.8
通讯作者：
Ito, Yutaka
中科院分区：
文献类型：
--
作者：
Ikeya, Teppei;Sasaki, Atsuko;Ito, Yutaka
细胞是一个拥挤的环境，在这个环境中，蛋白质与其他蛋白质、核酸、辅因子和配体特定地相互作用。对蛋白质在这种环境中的功能进行原子分辨结构解释是生物化学研究的主要目标。最近对核磁共振硬件和方法的改进使得能够测量活细胞中大分子的高分辨率异核多维核磁共振谱(细胞内核磁共振)。在这项研究中，我们描述了一种稳定同位素(C-13，N-15和H-2)标记和确定在大肠杆菌细胞中过度表达的蛋白质的结构的方法，该方法完全基于在活细胞中获得的信息。该方案结合了在大肠杆菌细胞中制备蛋白质、通过对间接获得的尺寸进行非线性采样来快速测量三维(3D)核磁共振光谱、结构计算和结构优化。在有利的条件下，这种细胞内核磁共振方法可以提供生活环境中蛋白质的高分辨率3D结构。该方案已被用于解决活细胞中的蛋白质的第一个三维结构，该蛋白质可能是来自嗜热嗜热杆菌HB8的重金属结合蛋白TTHA1718，在大肠杆菌中过表达。由于不需要蛋白质的提纯，因此可以在2-3d内获得用于细胞内核磁共振测量的样品，利用非线性采样方案，每次3D实验的持续时间可以缩短到2-3h，一旦完成了化学位移指定和NOESY峰表，用CYANA程序进行结构计算和能量精炼可以在功能强大的计算机系统上在不到1h的时间内完成。
The cell is a crowded environment in which proteins interact specifically with other proteins, nucleic acids, cofactors and ligands. atomic resolution structural explanation of proteins functioning in this environment is a main goal of biochemical research. recent improvements to nuclear magnetic resonance (NMR) hardware and methodology allow the measurement of high-resolution heteronuclear multidimensional NMR spectra of macromolecules in living cells (in-cell NMR). In this study, we describe a protocol for the stable isotope (C-13, N-15 and H-2) labeling and structure determination of proteins overexpressed in Escherichia coli cells exclusively on the basis of information obtained in living cells. the protocol combines the preparation of the protein in E. coli cells, the rapid measurement of the three-dimensional (3D) NMR spectra by nonlinear sampling of the indirectly acquired dimensions, structure calculation and structure refinement. under favorable circumstances, this in-cell NMR approach can provide high-resolution 3D structures of proteins in living environments. the protocol has been used to solve the first 3D structure of a protein in living cells for the putative heavy metal-binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in E. coli cells. as no protein purification is necessary, a sample for in-cell NMR measurements can be obtained within 2-3 d. With the nonlinear sampling scheme, the duration of each 3D experiment can be reduced to 2-3 h. once chemical shift assignments and NOESY peak lists have been prepared, structure calculation with the program CYANA and energy refinement can be completed in less than 1 h on a powerful computer system.