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 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 coordination approach is vulnerable to low binding-affinity. Herein, dissociation constants (K ) are investigated directly from the modulation depths of relaxation-induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low- to sub-μm Cu K 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 -nitroxide systems using a commercial Q-band EPR instrument.