SLIM: A short‐linked, highly redox‐stable trityl label for high sensitivity in cell EPR distance measurements

The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules, preferably acquired under native conditions. In this regard, pulsed dipolar EPR spectroscopy (PDS) in conjunction with site-directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However, the currently available spin labels have diverse deficiencies for in cell applications, e.g. low radical stability or long bioconjugation linkers. In this work, a synthesis strategy is introduced for the derivatization of trityl radicals with a maleimide functionalized methylene group. The resulting trityl spin label SLIM yields narrow distance distributions, enables highly sensitive distance measurements down to concentrations of 90 nM and shows high stability against reduction. Using this label, the GDI domain of YopO is shown to change its conformation within eukaryotic cells.