Improving the accuracy of Cu(ii)–nitroxide RIDME in the presence of orientation correlation in water-soluble Cu(ii)–nitroxide rulers

Abstract

Detailed analysis of artefacts in the Cu(ii)–nitroxide RIDME experiments, related to orientation averaging, echo-crossing, ESEEM and background-correction is presented.

Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII‐labelling of Double‐Histidine Motifs with Lower Temperature

“…Similarly,R itsch et al have shown that Cu II -nitroxide RIDME outperforms PELDOR by asignificant margin, even in as tate-of-the-art ultra-wide band- . [22] Importantly,RIDME is also less prone to orientation selection than PELDOR, facilitating distance extraction. c) Background-corrected 5-pulse RIDME traces of 500 nm I6R1/K28H/ Q32H GB1 measured as apseudo-titration with 100-8100 nm Cu II -NTA.…”

Pulsed Dipolar EPR Spectroscopy and Metal Ions: Methodology and Biological Applications

“…For such spin pairs, the RIDME time trace is acquired on the slow relaxing organic radical, whereas the fast‐relaxing metal center is flipped by spontaneous relaxation. As the Cu 2+ /nitroxide spin pair fits perfectly to this case, it has been successfully used in several RIDME studies . Due to the infinite bandwidth of the relaxation‐driven Cu 2+ spin flips, the reported RIDME time traces showed good modulation depths of 30–45 % and no orientation selectivity for the Cu 2+ spins.…”

“…Overall, the sensitivity of Q‐band RIDME experiments was estimated to be ∼100 times higher than the sensitivity of the corresponding PELDOR experiments with rectangular pulses, enabling PDS measurements even at sub‐micromolar concentrations . The RIDME time traces of rigid model compounds were still orientation selective because of the selective excitation of the nitroxide spin centers by rectangular detection pulses . In order to reduce orientation selectivity, Meyer et al .…”

“…recorded several RIDME time traces at different positions across the nitroxide spectrum and then averaged them . Recently, the substitution of the rectangular detection pulses by shaped ones enabled the full excitation of the nitroxide spectrum and, consequently, provided the possibility to detect the entire set of nitroxide orientations in a single RIDME experiment . When applied to the Cu 2+ /Cu 2+ spin pair, RIDME measurements were shown to be less efficient due to an unavoidable trade‐off between sensitivity and modulation depth .…”

“…Despite the excellent modulation depth, the Gd 3+ /Gd 3+ RIDME experiments revealed several challenges. One of them are unwanted ESEEM contributions, which can be suppressed by introducing the strategies mentioned above . Another complication arises from the fact that the RIDME time traces have a steeper background decay as compared to PELDOR time traces .…”