Measuring Cu2+-Nitroxide Distances Using Double Electron-Electron Resonance and Saturation Recovery

Distance measurements were obtained between a bound Cu2+ and a spin label on two polypeptides of differing length using the double electron-electron resonance (DEER) and saturation recovery experiments. Distance distributions obtained from the DEER results resolved differences between the average distance and distribution of distances for each peptide. An average distance was also obtained for each peptide using the relaxation-based saturation recovery experiment. Predicted average distances for the relaxation-based method, < r(ESR)>, were calculated using the distance distributions from the DEER experiment. The predicted < r(ESR)> values were similar to those measured by saturation recovery; both were biased to shorter values compared with the DEER results. The breadth of the distance distributions had a significant effect on the average distance measured by saturation recovery. This work highlights the advantage of using DEER to measure metal-nitroxide distances in that the average distances measured are less biased than in relaxation-based techniques.