Trityl Radicals: New Spin Labels for Nanometer Distance Measurements with higher Sensitivity, at Room Temperature and within Cells

结构生物学的重点正在转向研究在非常复杂的环境中的越来越大的大分子，例如在膜或活细胞内。EPR光谱提供了方法，允许在这种条件下测量自旋中心之间的偶极耦合，从而研究纳米范围内的大分子结构及其构象变化。这种方法的实例是cw-EPR、脉冲电子-电子双共振（PELDOR）和双量子相干（DQC）实验。由于大多数大分子是抗磁性的，它们必须首先用自旋标记物标记，最常见的是氮氧化合物。然而，氮氧化物的化学和EPR光谱特性导致了一些限制，包括必须在冷冻样品上进行测量。在上一个资助期间，我们能够证明，这些限制中的许多可以通过使用三苯甲基自旋标记来规避或减轻。我们已经合成了两个三苯甲基标签的纳米材料和一个三苯甲基标签的选择性标记的半胱氨酸蛋白质。我们已经表明，根据EPR方法，距离测量的灵敏度可以增加2至30倍，当使用三苯甲基标签，而不是氮氧化物标签。使用bistrityl模型化合物，我们能够证明，所得到的距离分布的宽度是可比的，其氮氧当量。在新的资助期内，我们现在希望使用三苯甲基标记和基于EPR的距离测量来研究两个完整的膜或膜相关分子机器的结构和构象变化：FeoB和YopO。我们将在低温和室温下以及体外和细胞中进行实验。对于细胞内实验，我们将合成三苯甲基标记物，其通过稳定的硫醚键而不是常规的二硫键与蛋白质YopO偶联。对于FeoB，我们将合成新的三苯甲基标记，其可以与非天然氨基酸偶联。我们将使用各种膜系统和天然的细胞骨架蛋白网络（F-肌动蛋白），在其自然环境中的蛋白质，并以这种方式对这些条件下的室温距离测量工作。

The focus of structural biology is shifting towards investigations of ever-larger macromolecules in very complex environments, for example in membranes or inside living cells. EPR spectroscopy offers methods, which allow measuring the dipolar coupling between spin centers under such conditions and therefore to study macromolecular structures and their conformational changes in the nanometer range. Examples of such methods are cw-EPR, Pulsed Electron-Electron Double Resonance (PELDOR) and Double Quantum Coherence (DQC) experiments. Since most macromolecules are diamagnetic, they must first be labelled with spin labels, most commonly nitroxide compounds. However, the chemical and EPR spectroscopic properties of nitroxides lead to several limitations, including that the measurements must be performed on frozen samples. In the previous funding period, we were able to show that many of these limitations can be circumvented or alleviated by the use of trityl spin labels. We have synthesized two trityl labels for nanomaterials and one trityl label for the selective labelling of cysteines in proteins. We have shown that, depending on the EPR method, the sensitivity of distance measurements can be increased by a factor of 2 to 30 when trityl labels are used instead of nitroxide labels. Using bistrityl model compounds, we were able to demonstrate that the widths of the resulting distance distributions are comparable to those of their nitroxide equivalents. Furthermore, it turned out that the trityl label is much more stable under in-cell conditions than the nitroxide group.In the new funding period, we now want to use trityl labels and EPR-based distance measurements to investigate the structure and conformational changes of two integral membrane or membrane-associated molecular machines: FeoB and YopO. We will perform our experiments both at low temperature and room temperature, as well as in vitro and in cells. For the in-cell experiments, we will synthesize trityl labels, which are coupled to the protein YopO via stable thioether linkages instead of the conventional disulfide bridges. For FeoB we will synthesize new trityl labels, which can be coupled to unnatural amino acids. We will use various membrane systems and native cytoskeletal protein networks (F-actin) to immobilize the proteins in their natural environment and in this way work towards room temperature distance measurements under these conditions.