Binding Affinity Determines the Success of Endogenous Cu^II-NTA Spin Labeling for In-Cell Electron Paramagnetic...

In-cell electron paramagnetic resonance (EPR) spectroscopy requires robust spin-labeling strategies compatible with cellular conditions. Cu^II-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 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, whereas 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 labeling and the substantial sensitivity gain offered by RIDME experiments.