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.

Ribonucleic acid function is governed by its structure, dynamics, and interaction with other biomolecules and influenced by the local environment. Thus, methods are needed that enable one to study RNA under conditions as natural as possible, possibly within cells. Site-directed spin-labeling of RNA with nitroxides in combination with, for example, pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy has been shown to provide such information. However, for in-cell measurements, the usually used gem-dimethyl nitroxides are less suited, because they are quickly reduced under in-cell conditions. In contrast, gem-diethyl nitroxides turned out to be more stable, but labeling protocols for binding these to RNA have been sparsely reported. Therefore, we describe here the bioconjugation of an azide functionalized gem-diethyl isoindoline nitroxide to RNA using a copper (I)-catalyzed azide–alkyne cycloaddition (“click”-chemistry). The labeling protocol provides high yields and site selectivity. The analysis of the orientation selective PELDOR data show that the gem-diethyl and gem-dimethyl labels adopt similar conformations. Interestingly, in deuterated buffer, both labels attached to RNA yield T_M relaxation times that are considerably longer than observed for the same type of label attached to proteins, enabling PELDOR time windows of up to 20 microseconds. Together with the increased stability in reducing environments, this label is very promising for in-cell Electron Paramagnetic Resonance (EPR) studies.