High-Resolution Heteronuclear Multidimensional NMR of Proteins in Living Insect Cells Using a Baculovirus Protein Expression System
High-Resolution Heteronuclear Multidimensional NMR of Proteins in Living Insect Cells Using a Baculovirus Protein Expression System
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DOI：
10.1021/ja310928u
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发表时间：
2013-02-06
影响因子：
15
通讯作者：
Ito, Yutaka
中科院分区：
文献类型：
--
作者：
Hamatsu, Jumpei;O'Donovan, Daniel;Ito, Yutaka
细胞内NMR技术的最新发展使我们能够详细研究活真核细胞内的蛋白质。为了补充现有的协议，并扩大可能的应用范围，我们介绍了一种新的方法，观察在细胞内的NMR光谱使用sf 9细胞/杆状病毒系统。高分辨率的二维H-1-N-15相关光谱观察到的四个模型蛋白在sf 9细胞中表达。此外，在sf 9细胞中观察到的链球菌蛋白G B1结构域的三维三重共振NMR光谱，通过使用非线性采样，以克服样品的短寿命和标记的蛋白质的低丰度。用定量最大熵算法处理数据。这些被分配从头算，产生约80%的预期骨架NMR共振。在3D N-15分离的NOESY光谱中可以鉴定出分辨良好的NOE交叉峰，这表明在sf 9细胞中对这种大小的蛋白质进行结构分析是可行的。
Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. In order to complement the existing protocols, and to extend the range of possible applications, we introduce a novel approach for observing in-cell NMR spectra using the sf9 cell/baculovirus system. High-resolution 2D H-1-N-15 correlation spectra were observed for four model proteins expressed in sf9 cells. Furthermore, 3D triple-resonance NMR spectra of the Streptococcus protein G B1 domain were observed in sf9 cells by using nonlinear sampling to overcome the short lifetime of the samples and the low abundance of the labeled protein. The data were processed with a quantitative maximum entropy algorithm. These were assigned ab initio, yielding approximately 80% of the expected backbone NMR resonances. Well-resolved NOE cross peaks could be identified in the 3D N-15-separated NOESY spectrum, suggesting that structural analysis of this size of protein will be feasible in sf9 cells.