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

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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.