In-organello and in-cell EPR on orthogonally spin-labeled proteins

在schwerpunktprogram中，EPR光谱灵敏度的新前沿：从生物细胞到纳米材料，本研究侧重于正交自旋标记方法的优化和脉冲偶极EPR光谱的最先进设置，以提取细胞器和细胞中蛋白质的结构信息。我们将在配备高功率放大器和任意波形发生器（AWG）的多频商用光谱仪上执行脉冲偶极EPR光谱，通过旋间距离检测凋亡时凋亡的Bcl-2蛋白的构象变化，以及分离线粒体和细胞中蛋白质-蛋白质相互作用的网络。对于细胞内EPR，选择的方法是将自旋标记的纯化蛋白内化到哺乳动物细胞中。与该项目相关的是这些标记策略的发展：i)用于同时检测同一样品上的三种蛋白质的三自旋正交标记方案；ii)将镧系螯合物附着在蛋白质上，通过EPR获得结构信息，并通过基于发光的成像技术获得蛋白质在细胞中的定位；ii)活细胞中his标记蛋白的基于trisnta的gd标记。这项工作将有助于建立EPR作为一种在生理条件下检索高分辨率位点特异性结构信息的有价值的技术。

Within the Schwerpunktprogramm New Frontiers in Sensitivity for EPR Spectroscopy: From Biological Cells to Nano Materials, this research focuses on the optimization of orthogonal spin labeling methodologies and state-of-the-art setups for pulsed dipolar EPR spectroscopy to extract structural information on proteins in organelles and in cells. We will perform pulsed dipolar EPR spectroscopy on multi-frequency commercially available spectrometers equipped with high power amplifiers and arbitrary waveform generators (AWG) to detect via interspin distances the conformational changes of apoptotic Bcl-2 proteins at the onset of apoptosis as well as the networking of protein-protein interaction in isolated mitochondria and in cells. For in-cell EPR, the method of choice is the internalization of spin-labeled purified proteins into mammalian cells. Relevant for the project is the development of these labeling strategies: i) a three-spin orthogonal labeling scheme for simultaneous detection of three proteins on the same sample; ii) lanthanide chelators to be attached to proteins to get both structural information by EPR and protein localization in cells via imaging techniques based on luminescence; ii) a trisNTA-based Gd-label for His-tagged proteins in living cells. This work will help establishing EPR as a valuable technique to retrieve high resolution site-specific structural information under physiological conditions.