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.C ombining double-histidine motifs with Cu II spin labels can further increase the precision of distance measurements.Itisalso useful for proteins containing essential cysteines that can interfere with thiol-specific labelling.However,the non-covalent Cu II coordination approachis vulnerable to lowb inding-affinity.H erein, dissociation constants (K D )a re investigated directly from the modulation depths of relaxation-induced dipolar modulation enhancement (RIDME) EPR experiments.This reveals low-to sub-mm Cu II K D sunder EPR distance measurement conditions at cryogenic temperatures.W eshowthe feasibility of exploiting the doublehistidine motif for EPR applications even at sub-mm protein concentrations in orthogonally labelled Cu II -nitroxide systems using acommercial Q-band EPR instrument.

Site-directed attachment of photoexcitable spin labels for light-induced pulsed dipolar spectroscopy

“…As the applications of pulsed dipolar spectroscopy (PDS) broaden, the repertory of spin labels now comprises not only the routinely used nitroxides 7 but also other paramagnetic species. [8][9][10][11] To accommodate the spectral and relaxational differences between these labels, other techniques than the established double electronelectron resonance (DEER) experiment, 12 such as relaxationinduced dipolar modulation enhancement (RIDME) 13,14 and double-quantum coherence (DQC), 15 are stepping into the spotlight. Several examples have also illustrated the advantages of combining spectroscopically orthogonal spin labels, e.g., Cu(II) and nitroxides.…”

EPR Distance Measurements as a Tool to Characterize Protein‐DNA Interactions

“…Moreover, the disulfide bond generated by the protein‐MTSL conjugates is also unstable in reducing environments. Over the last decade, to overcome the instability of nitroxide radicals, researchers have begun to explore the use of alternative spin labels, based on paramagnetic metal ions, including Cu(II), Mn(II) and Gd(III) . For Mn(II) and Gd(III) spin labels, high‐field (>30 GHz) EPR experiments are preferable, owing to the presence of zero‐field splitting at low fields.…”

“…In our own experiments, we have observed that for MTSL spin labels, it is possible to use labeled‐protein concentrations that are as low as 5–10 μM, whereas for Cu(II) spin labels it is necessary to use about 50 μM labeled protein. Nonetheless, recently it was reported of using sub‐micromolar concentrations with Cu(II)‐NTA spin labels . For low protein concentrations, the time‐domain DEER trace is relatively short and is up to 2 μs for nitroxide spin‐labels; therefore distances up to 5.0 nm can be evaluated.…”

Accessing distributions of exchange and dipolar couplings in stiff molecular rulers with Cu(ii) centres

“…[24][25][26][27] Significant progress in performing and analysing Cu(II)-based PDS experiments has been achieved over the past few years. [28][29][30][31][32][33] Exchange interactions are often observed for Cu(II) ions that are bound to conjugated p-systems, [34][35][36][37][38][39] or in strongly coupled systems, such as molecular magnets. 7,40,41 The exchange coupling may give additional information on the system of interest, yet, the dipolar and exchange couplings can become difficult to disentangle.…”