Versatile Trityl Spin Labels for Nanometer Distance Measurements on Biomolecules In Vitro and within Cells

Abstract

Abstract Structure determination of biomacromolecules under in‐cell conditions is a relevant yet challenging task. Electron paramagnetic resonance (EPR) distance measurements in combination with site‐directed spin labeling (SDSL) are a valuable tool in this endeavor but the usually used nitroxide spin labels are not well‐suited for in‐cell measurements. In contrast, triarylmethyl (trityl) radicals are highly persistent, exhibit a long relaxation time and a narrow spectral width. Here, the synthesis of a versatile collection of trityl spin labels and their application in in vitro and in‐cell trityl–iron distance measurements on a cytochrome P450 protein are described. The trityl labels show similar labeling efficiencies and better signal‐to‐noise ratios (SNR) as compared to the popular methanethiosulfonate spin label (MTSSL) and enabled a successful in‐cell measurement.