Enhanced sensitivity for pulse dipolar EPR spectroscopy using variable-time RIDME

Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Distance Measurements at Low Nanomolar Concentrations: The CuII-Trityl Case

“…For samples with a protein concentration of <100 nM, recording the reference traces became unfeasible due to the required extended averaging times and was therefore done only for concentrations of ≥100 nM. As observed previously, 13,35 modulation depths were slightly higher for the vtRIDME than for the ctRIDME. Deconvolution reduced the observed modulation depths for both ct-and vtRIDME measurements as expected.…”

“…In the next step, we investigated whether the recently reported variable-time RIDME experiment (vtRIDME) would enable a further sensitivity increase particularly valuable at these low concentrations. To ensure reproducibility and minimize errors from sample positioning and experiment optimization, four experiments were recorded on the samples of the dilution series: a ctRIDME similar to the measurement described above, a vtRIDME with otherwise identical parameters, and corresponding reference traces for both the ctRIDME and the vtRIDME to enable deconvolution of the data (division by the reference trace) .…”

“…Recent developments in electron paramagnetic resonance (EPR) spectroscopy have firmly established PDS in the toolbox for structural biology, due to its accuracy, reproducibility, and concentration sensitivity, and by providing access to full conformational ensembles of biomolecules. − PDS methods such as pulsed electron–electron double resonance (PELDOR or DEER), − relaxation-induced dipolar modulation enhancement (RIDME), − and double quantum coherence (DQC) ,, provide distance distributions in the nanometer range (from ∼2 to ∼16 nm) − and have become important tools for studying complex biomolecular systems. , …”

Spectroscopically Orthogonal Labelling to Disentangle Site-Specific Nitroxide Label Distributions

“…RIDME has the further advantage that orientation-dependent excitation of the broad copper(II) spectrum can be avoided as all orientations will contribute, simplifying analysis and interpretation [ 56 ]. Here, we compare the 5-pulse variable-time (vt) RIDME, recently introduced by our group [ 57 ], with the standard 5-pulse constant-time (ct) RIDME. Testing the vtRIDME sequence further will allow evaluating its performance compared to the constant-time sequence, to gain a deeper understanding of its potential and limitations.…”

Double Quantum Coherence ESR at Q-Band Enhances the Sensitivity of Distance Measurements at Submicromolar Concentrations

“…These achievements are paving the way for a broader applicability of ESR in the measurement of biological distance constraints, for instance, at physiological concentrations and in more complex systems. Recent work has shown the capability of pulsed dipolar ESR to reach submicromolar protein concentration measurements in vitro ,− and in cell . Current techniques appear to work with protein concentrations as low as 45–200 nM ,, depending on the spin label, but these measurements often require a few days of collection time at these low concentrations.…”