High-resolution multi-dimensional NMR spectroscopy of proteins in human cells
High-resolution multi-dimensional NMR spectroscopy of proteins in human cells
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DOI：
10.1038/nature07839
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发表时间：
2009-03-05
期刊：
影响因子：
64.8
通讯作者：
Shirakawa, Masahiro
中科院分区：
文献类型：
--
作者：
Inomata, Kohsuke;Ohno, Ayako;Shirakawa, Masahiro
细胞内NMR是一种同位素辅助的多维NMR技术，可以在原子水平上观察活细胞中蛋白质的构象和功能（1）。该方法已成功应用于细菌中过表达的蛋白质，提供了蛋白质-配体相互作用（2）和构象（3，4）的信息。然而，细胞内NMR对真核细胞的应用仅限于非洲爪蟾卵母细胞（5-7）。同位素标记的蛋白质进入真核生物体细胞的效率低下，阻碍了该技术的广泛应用。在这里，我们描述了一种方法，以获得高分辨率的二维（2D）杂波NMR光谱的蛋白质内活的人类细胞。通过芘丁酸酯介导的与蛋白质共价连接的细胞穿透肽（8）的作用，蛋白质被递送至细胞溶质。随后通过内源性酶活性或通过自主还原裂解从细胞穿透肽释放蛋白质。人类细胞内三种不同蛋白质的异源二维光谱证明了这种技术在研究相互作用和蛋白质加工方面的广泛应用。FKBP 12（也称为FKBP 1A）的细胞内NMR光谱显示蛋白质和细胞外施用的免疫抑制剂之间形成特异性复合物，证明了该技术在药物筛选程序中的实用性。此外，细胞内NMR光谱表明，泛素在细胞内环境中具有更高的氢交换速率，可能是由于与内源性蛋白质的多种相互作用。
In-cell NMR is an isotope-aided multi-dimensional NMR technique that enables observations of conformations and functions of proteins in living cells at the atomic level(1). This method has been successfully applied to proteins overexpressed in bacteria, providing information on protein-ligand interactions(2) and conformations(3,4). However, the application of in-cell NMR to eukaryotic cells has been limited to Xenopus laevis oocytes(5-7). Wider application of the technique is hampered by inefficient delivery of isotope-labelled proteins into eukaryote somatic cells. Here we describe a method to obtain high-resolution two-dimensional (2D) heteronuclear NMR spectra of proteins inside living human cells. Proteins were delivered to the cytosol by the pyrenebutyrate-mediated action of cell-penetrating peptides(8) linked covalently to the proteins. The proteins were subsequently released from cell-penetrating peptides by endogenous enzymatic activity or by autonomous reductive cleavage. The heteronuclear 2D spectra of three different proteins inside human cells demonstrate the broad application of this technique to studying interactions and protein processing. The in-cell NMR spectra of FKBP12 (also known as FKBP1A) show the formation of specific complexes between the protein and extracellularly administered immunosuppressants, demonstrating the utility of this technique in drug screening programs. Moreover, in-cell NMRspectroscopy demonstrates that ubiquitin has much higher hydrogen exchange rates in the intracellular environment, possibly due to multiple interactions with endogenous proteins.