Binding Affinity Determines the Success of Endogenous Cu II -NTA Spin Labeling for In-Cell Electron Paramagnetic Resonance Distance Measurements

In-cell electron paramagnetic resonance (EPR) spectroscopy requires robust spin-labeling strategies compatible with cellular conditions. CuII-NTA coordination to genetically engineered double-histidine (dHis) motifs has shown promise for endogenous labeling in Escherichia coli. Here, we evaluated the effect of varying CuII-NTA affinity on the success of these experiments. Relaxation induced dipolar modulation enhancement (RIDME)-based titrations revealed dissociation constants (Kd) 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 CuII 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, whereas the lower-affinity site did not. These results highlight the critical importance of low Kd binding sites for reliable in-cell distance measurements with endogenous CuII-NTA labeling and the substantial sensitivity gain offered by RIDME experiments.