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

在Schwerpunktprogramm EPR光谱灵敏度的新前沿：从生物细胞到纳米材料中，这项研究的重点是优化正交自旋标记方法和脉冲偶极EPR光谱的最新设置，以提取细胞器和细胞中蛋白质的结构信息。我们将进行脉冲偶极EPR光谱的多频市售光谱仪配备高功率放大器和任意波形发生器（AWG），通过自旋间距检测凋亡Bcl-2蛋白的构象变化，在细胞凋亡的发病以及蛋白质-蛋白质相互作用的网络分离的线粒体和细胞。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开发：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）将镧系元素螯合剂连接到蛋白质上，以通过EPR获得结构信息，并通过基于发光的成像技术在细胞中定位蛋白质; ii）用于活细胞中His标记蛋白质的基于trisNTA的Gd标记。这项工作将有助于建立EPR作为一种有价值的技术，以检索高分辨率的生理条件下的特定位点的结构信息。

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.