Abstract
Relaxation induced dipolar modulation enhancement (RIDME) is a valuable method for measuring nanometer-scale distances between electron spin centers. Such distances are widely used in structural biology to study biomolecular structures and track their conformational changes. Despite significant improvements of RIDME in recent years, the background analysis of primary RIDME signals remains to be challenging. In particular, it was recently shown that the five-pulse RIDME signals contain an artifact which can hinder the accurate extraction of distance distributions from RIDME time traces [as reported by Ritsch et al. (Phys Chem Chem Phys 21: 9810, 2019)]. Here, this artifact, as well as one additionally identified artifact, are systematically studied on several model compounds and the possible origins of both artifacts are discussed. In addition, a new six-pulse RIDME sequence is proposed that eliminates the artifact with the biggest impact on the extracted distance distributions. The efficiency of this pulse sequence is confirmed on several examples.
Highlights
Nanometer-scale distance measurements using electron paramagnetic resonance (EPR) spectroscopy provide important information for studying the structure and conformational states of biomacromolecules, such as proteins, nucleic acids, and their complexes [1]
double quantum coherence EPR (DQC) and single-frequency technique for refocusing dipolar couplings (SIFTER) outperform PELDOR in terms of modulation depth and sensitivity for spin labels with spectral widths comparable to microwave pulse bandwidths [14], such as trityl-based spin labels [11, 15,16,17]
Distance distributions were extracted from the relaxation induced dipolar modulation enhancement (RIDME) time traces of 2 using the Tikhonov regularization implemented in DeerAnalysis2019
Summary
Nanometer-scale distance measurements using electron paramagnetic resonance (EPR) spectroscopy provide important information for studying the structure and conformational states of biomacromolecules, such as proteins, nucleic acids, and their complexes [1]. The five-pulse RIDME (5p-RIDME, Fig. 1a) sequence has been shown to have advantages over PELDOR when applied to spin centers with spectral widths that significantly exceed the bandwidths of microwave pulses, such as Cu2+ [18,19,20,21,22,23,24], Gd3+ [24,25,26,27,28], Mn2+ [24, 29, 30], Co2+ [31], low-spin, and highspin Fe3+ [32,33,34,35] For all these metal ions, RIDME provides significantly larger modulation depth and better sensitivity compared to PELDOR [36, 37]. Both compounds were dissolved in deuterated THF and had a concentration of 200 μM
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