Binding Affinity Determines the Success of Endogenous CuII–NTA Spin Labelling for In-Cell EPR Distance Measurements

In-cell electron paramagnetic resonance (EPR) spectroscopy requires robust spin-labelling strategies compatible with cellular conditions. Cu II -NTA coordination to genetically engineered double-histidine (dHis) motifs has shown promise for endogenous labelling in E. coli . Here, we evaluated the effect of varying Cu II -NTA affinity on the success of these experiments. Relaxation induced dipolar modulation enhancement (RIDME)-based titrations revealed dissociation constants ( K d ) in the 10 -6 and 10 -8 range for two different β-sheet i and i+2 dHis sites. In-cell EPR spectra demonstrated that the persistence of the Cu II EPR signal correlates with these binding affinities. The success of in-cell pulsed electron-electron double resonance (PELDOR) involving both β-sheet sites and a high-affinity α-helical site depended on the site; the higher-affinity site yielded analyzable results, the lower-affinity site did not. These results highlight the critical importance of low K d binding sites for reliable in-cell distance measurements with endogenous Cu II -NTA labelling and the substantial sensitivity gain offered by RIDME experiments.