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

Abstract

Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double‐histidine motifs with CuII spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol‐specific labelling. However, the non‐covalent CuII coordination approach is vulnerable to low binding‐affinity. Herein, dissociation constants (KD) are investigated directly from the modulation depths of relaxation‐induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low‐ to sub‐μm CuII KDs under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double‐histidine motif for EPR applications even at sub‐μm protein concentrations in orthogonally labelled CuII–nitroxide systems using a commercial Q‐band EPR instrument.

In-cell destabilization of a homo-dimeric protein complex detected by DEER spectroscopy

“…S18, SI). The determined KD value is smaller than that obtained by DEER, which likely reflected temperature effects between NMR (room temperature) and DEER experiments (freezing point of samples) (75). The presence of spin-labels and 20% glycerol in the latter may have further contributed to the observed differences.…”

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.…”

“…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 .…”

“…As the Cu 2 + /nitroxide spin pair fits perfectly to this case, it has been successfully used in several RIDME studies. [54,[73][74][75] 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.…”

“…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. [75] 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. [54,73] 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.…”