Differentiating between Label and Protein Conformers in Pulsed Dipolar EPR Spectroscopy with the dHis-Cu²⁺(NTA) Motif

Pulsed dipolar EPR spectroscopy (PDS) in combination with site-directed spin labeling is a powerful tool in structural biology. However, the commonly used spin labels are conjugated to biomolecules via rather long and flexible linkers, which hampers the translation of distance distributions into biomolecular conformations. In contrast, the spin label copper(II)-nitrilotriacetic acid [Cu²⁺(NTA)] bound to two histidines (dHis) is rigid and yields narrow distance distributions, which can be more easily translated into biomolecular conformations. Here, we use this label on the 71 kDa Yersinia outer protein O (YopO) to decipher whether a previously experimentally observed bimodal distance distribution is due to two conformations of the biomolecule or of the flexible spin labels. Two different PDS experiments, that is, pulsed electron–electron double resonance (PELDOR aka DEER) and relaxation-induced dipolar modulation enhancement (RIDME), yield unimodal distance distribution with the dHis-Cu²⁺(NTA) motif; this result suggests that the α-helical backbone of YopO adopts a single conformation in frozen solution. In addition, we show that the Cu²⁺(NTA) label preferentially binds to the target double histidine (dHis) sites even in the presence of 22 competing native histidine residues. Our results therefore suggest that the generation of a His-null background is not required for this spin labeling methodology. Together these results highlight the value of the dHis-Cu²⁺(NTA) motif in PDS experiments.