Monitoring Complex Formation by Relaxation‐Induced Pulse Electron Paramagnetic Resonance Distance Measurements

Abstract

Biomolecular complexes are often multimers fueling the demand for methods that allow unraveling their composition and geometric arrangement. Pulse electron paramagnetic resonance (EPR) spectroscopy is increasingly applied for retrieving geometric information on the nanometer scale. The emerging RIDME (relaxation‐induced dipolar modulation enhancement) technique offers improved sensitivity in distance experiments involving metal centers (e.g. on metalloproteins or proteins labelled with metal ions). Here, a mixture of a spin labelled ligand with increasing amounts of paramagnetic CuII ions allowed accurate quantification of ligand‐metal binding in the model complex formed. The distance measurement was highly accurate and critical aspects for identifying multimerization could be identified. The potential to quantify binding in addition to the high‐precision distance measurement will further increase the scope of EPR applications.

Intermolecular background decay in RIDME experiments

“…Over the last four years, several studies have reported on RIDME with pairs of paramagnetic metal centers, [27][28][29][30][31] or pairs of an organic radical and a paramagnetic metal center. [32][33][34][35][36][37] These studies demonstrated the overall good performance of the RIDME technique for such systems. To appropriately analyze the RIDME data in practically important cases, it is necessary to correctly discriminate between the so-called intramolecular form factor contribution and the intermolecular background contribution.…”

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

“…In recent work it was demonstrated that such measurements reveal otherwise inaccessible information on the electronic structure of the systems. 16,25 Secondly, complexes of Cu(II) ions are attracting attention in bio-EPR spectroscopy, [26][27][28][29] both as native metal centres in proteins, 23,26,[30][31][32][33][34][35][36][37][38] and as spin labels for orthogonal pulse dipolar spectroscopy. 39,40 The potential of the latter approach has been recognised previously and is reflected in a growing interest in Cu(II)-based spin labelling strategies.…”

“…39,40 The potential of the latter approach has been recognised previously and is reflected in a growing interest in Cu(II)-based spin labelling strategies. 17,26,29,40,41 Preparation of orthogonally spin labelled samples often requires some additional experimental effort, which is why orthogonal spin labelling methods may continue to play a minor role in structure determination of monomeric biomolecules. Their strength, however, lies in the possibility of spectroscopic selection in systems of several interacting molecules, and they thus may well become a method of choice in the studies of intermolecular biomolecule interactions.…”

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.…”

“…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.…”