Site selective and efficient spin labeling of proteins with a maleimide-functionalized trityl radical for pulsed dipolar EPR spectroscopy

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) in combination with site-directed spin labeling (SDSL) of proteins and oligonucleotides is a powerful tool in structural biology. Instead of using the commonly employed gem-dimethyl-nitroxide labels, triarylmethyl (trityl) spin labels enable such studies at room temperature, within the cells and with single-frequency electron paramagnetic resonance (EPR) experiments. However, it has been repeatedly reported that labeling of proteins with trityl radicals led to low labeling efficiencies, unspecific labeling and label aggregation. Therefore, this work introduces the synthesis and characterization of a maleimide-functionalized trityl spin label and its corresponding labeling protocol for cysteine residues in proteins. The label is highly cysteine-selective, provides high labeling efficiencies and outperforms the previously employed methanethiosulfonate-functionalized trityl label. Finally, the new label is successfully tested in PDS measurements on a set of doubly labeled Yersinia outer protein O (YopO) mutants.