Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing Cu(II)‐labelling of Double‐Histidine Motifs with Lower Temperature

Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double‐histidine motifs with Cu(II) spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol‐specific labelling. However, the non‐covalent Cu(II) coordination approach is vulnerable to low binding‐affinity. Herein, dissociation constants (K (D)) are investigated directly from the modulation depths of relaxation‐induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low‐ to sub‐μm Cu(II) K (D)s under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double‐histidine motif for EPR applications even at sub‐μm protein concentrations in orthogonally labelled Cu(II)–nitroxide systems using a commercial Q‐band EPR instrument.