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

直接观察蛋白质在其自然细胞内环境中的功能是细胞生物学的中心目标。除了检测细胞内的定位过程外，阐明蛋白质的结构和构象动力学是当前研究的重点。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特征，例如测量单个经过战略选择的氨基酸之间的绝对距离分布。这使我们能够全面了解蛋白质和蛋白质复合体的分子结构，作为其细胞功能的基础。另一方面，到目前为止，直接在自然细胞内环境中对内源自旋标记蛋白质进行EPR研究是不可能的，因为这种蛋白质自旋标记还没有细胞兼容的方法。为了使这类研究成为可能，我们将首次合成自旋标记氨基酸，并通过定向进化开发用于其遗传编码的氨基酰-tRNA合成酶。我们将研究这些氨基酸的EPR波谱特性，并开发它们在大肠杆菌细胞内EPR波谱中的应用方法。我们将利用这些见解首次在体外和直接在细胞中检测非晶态转录激活器样效应蛋白(TALE)的基本DNA识别机制。TALE蛋白具有DNA结合域，具有可编程的序列特异性，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.