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

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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.