Distance measurements in the nanometer range by In-Cell Electron Paramagnetic Resonance Spectroscopy

直接观察蛋白质在其天然细胞内环境中的功能是细胞生物学的中心目标。除了检测细胞内定位过程的蛋白质结构和构象动力学的阐明是当前研究的重点。自旋标记蛋白质的细胞内电子顺磁共振（In-cell EPR）光谱为这些目标提供了独特的功能，例如测量个体之间的绝对距离分布，战略选择的氨基酸。这允许全面深入了解蛋白质和蛋白质复合物的分子结构作为其细胞功能的基础。另一方面，到目前为止，直接在天然细胞内环境中对内源性自旋标记蛋白质进行EPR研究还不可能，因为不存在与细胞兼容的用于此类蛋白质自旋标记的方法。为了实现此类研究，我们将首次合成自旋标记的氨基酸并开发氨酰-tRNA合成酶，通过定向进化进行遗传编码。我们将研究这些氨基酸的EPR光谱特性，并开发用于大肠杆菌细胞内EPR光谱的方法。我们将使用这些见解来研究基本的DNA识别机制的转录激活因子样效应（TALE）蛋白在非结晶状态的第一次，在体外和直接在细胞中。TALE蛋白具有可编程序列特异性的DNA结合结构域，是修饰和分析基因组功能的关键工具。

The direct observation of the function of proteins in their natural intracellular environment is a central goal of cell biology. In addition to the detection of intracellular localization processes the elucidation of protein structures and conformational dynamics is in the focus of current research.Intracellular electron paramagnetic resonance (In-cell EPR) spectroscopy of spin-labeled proteins offers unique features for these objectives, such as the measurement of absolute distance distributions between individual, strategically selected amino acids. This allows a comprehensive insight into the molecular architecture of proteins and protein complexes as the basis of their cellular function. On the other hand, EPR investigations of endogenous spin-labeled proteins directly in their natural intracellular environment have not been possible so far, because no cell-compatible approaches for this kind of protein spin labeling exists.To enable such studies for the first time, we will synthesize spin-labeled amino acids and develop aminoacyl-tRNA synthetases for their genetic encoding by directed evolution. We will examine the EPR spectroscopic properties of these amino acids and develop methods for their use in intracellular EPR spectroscopy in E. coli.We will use these insights to examine basic DNA recognition mechanisms of transcription activator-like effector (TALE) proteins in the non-crystalline state for the first time, both in vitro and directly in cells. TALE proteins have DNA-binding domains with a programmable sequence specificity and are key tools for the modification and analysis of genome functions.