RIDME distance measurements using Gd(iii) tags with a narrow central transition

Methods based on pulse electron paramagnetic resonance allow measurement of the electron-electron dipolar coupling between two spin labels. Here we compare the most popular technique, Double Electron-Electron Resonance (DEER or PELDOR), with the dead-time free 5-pulse Relaxation-Induced Dipolar Modulation Enhancement (RIDME) method for Gd(iii)-Gd(iii) distance measurements at W-band (94.9 GHz, ≈3.5 T) using Gd(iii) tags with a small zero field splitting (ZFS). Such tags are important because of their high EPR sensitivity arising from their narrow central transition. Two systems were investigated: (i) a rigid model compound with an inter-spin distance of 2.35 nm, and (ii) two mutants of a homodimeric protein, both labeled with a DOTA-based Gd(iii) chelate and characterized by an inter-spin distance of around 6 nm, one having a narrow distance distribution and the other a broad distribution. Measurements on the model compound show that RIDME is less sensitive to the complications arising from the failure of the weak coupling approximation which affect DEER measurements on systems characterized by short inter-spin distances between Gd(iii) tags having a narrow central transition. Measurements on the protein samples, which are characterized by a long inter-spin distance, emphasize the complications due to the appearance of harmonics of the dipolar interaction frequency in the RIDME traces for S > 1/2 spin systems, as well as enhanced uncertainties in the background subtraction. In both cases the sensitivity of RIDME was found to be significantly better than DEER. The effects of the experimental parameters on the RIDME trace are discussed.This research was supported by the Israeli Science Foundation (grant 334/14) and was made possible in part by the historic generosity of the Harold Perlman Family (D. G.). We also acknowledge the Australian Research Council for a Discovery grant to G. O. and B. G. (DP150100383) and a Future Fellowship to B. G. (FT130100838). D. G. holds the Erich Klieger professorial chair in Chemical Physics

Efficient determination of the accessible conformation space of multi-domain complexes based on EPR PELDOR data

Many proteins can adopt multiple conformations which are important for their function. This is also true for proteins and domains that are covalently linked to each other. One important example is ubiquitin, which can form chains of different conformations depending on which of its lysine side chains is used to form an isopeptide bond with the C-terminus of another ubiquitin molecule. Similarly, ubiquitin gets covalently attached to active-site residues of E2 ubiquitin-conjugating enzymes. Due to weak interactions between ubiquitin and its interaction partners, these covalent complexes adopt multiple conformations. Understanding the function of these complexes requires the characterization of the entire accessible conformation space and its modulation by interaction partners. Long-range (1.8-10 nm) distance restraints obtained by EPR spectroscopy in the form of probability distributions are ideally suited for this task as not only the mean distance but also information about the conformation dynamics is encoded in the experimental data. Here we describe a computational method that we have developed based on well-established structure determination software using NMR restraints to calculate the accessible conformation space using PELDOR/DEER data

How do cardiologists select patients for dual antiplatelet therapy continuation beyond 1 year after a myocardial infarction? Insights from the EYESHOT Post-MI Study

Background: Current guidelines suggest to consider dual antiplatelet therapy (DAPT) continuation for longer than 12 months in selected patients with myocardial infarction (MI). Hypothesis: We sought to assess the criteria used by cardiologists in daily practice to select patients with a history of MI eligible for DAPT continuation beyond 1 year. Methods: We analyzed data from the EYESHOT Post-MI, a prospective, observational, nationwide study aimed to evaluate the management of patients presenting to cardiologists 1 to 3 years from the last MI event. Results: Out of the 1633 post-MI patients enrolled in the study between March and December 2017, 557 (34.1%) were on DAPT at the time of enrolment, and 450 (27.6%) were prescribed DAPT after cardiologist assessment. At multivariate analyses, a percutaneous coronary intervention (PCI) with multiple stents and the presence of peripheral artery disease (PAD) resulted as independent predictors of DAPT continuation, while atrial fibrillation was the only independent predictor of DAPT interruption for patients both at the second and the third year from MI at enrolment and the time of discharge/end of the visit. Conclusions: Risk scores recommended by current guidelines for guiding decisions on DAPT duration are underused and misused in clinical practice. A PCI with multiple stents and a history of PAD resulted as the clinical variables more frequently associated with DAPT continuation beyond 1 year from the index MI

Allosteric activation of an ion channel triggered by modification of mechanosensitive nano-pockets

Lipid availability within transmembrane nano-pockets of ion channels is linked with mechanosensation. However, the effect of hindering lipid-chain penetration into nano-pockets on channel structure has not been demonstrated. Here we identify nano-pockets on the large conductance mechanosensitive channel MscL, the high-pressure threshold channel. We restrict lipid-chain access to the nano-pockets by mutagenesis and sulfhydryl modification, and monitor channel conformation by PELDOR/DEER spectroscopy. For a single site located at the entrance of the nano-pockets and distal to the channel pore we generate an allosteric response in the absence of tension. Single-channel recordings reveal a significant decrease in the pressure activation threshold of the modified channel and a sub-conducting state in the absence of applied tension. Threshold is restored to wild-type levels upon reduction of the sulfhydryl modification. The modification associated with the conformational change restricts lipid access to the nano-pocket, interrupting the contact between the membrane and the channel that mediates mechanosensitivity

Gd(III) complexes as paramagnetic tags: Evaluation of the spin delocalization over the nuclei of the ligand

Collauto A, Feintuch A, Qi M, Godt A, Meade T, Goldfarb D. Gd(III) complexes as paramagnetic tags: Evaluation of the spin delocalization over the nuclei of the ligand. Journal of Magnetic Resonance. 2016;263:156-163

Graphite anodes for li-ion batteries: an electron paramagnetic resonance investigation

Graphite is the most commercially successful anode material for lithium (Li)-ion batteries: its low cost, low toxicity, and high abundance make it ideally suited for use in batteries for electronic devices, electrified transportation, and grid-based storage. The physical and electrochemical properties of graphite anodes have been thoroughly characterized. However, questions remain regarding their electronic structures and whether the electrons occupy localized states on Li, delocalized states on C, or an admixture of both. In this regard, electron paramagnetic resonance (EPR) spectroscopy is an invaluable tool for characterizing the electronic states generated during electrochemical cycling as it measures the properties of the unpaired electrons in lithiated graphites. In this work, ex situ variable-temperature (10–300 K), variable-frequency (9–441 GHz) EPR was carried out to extract the g tensors and line widths and understand the effect of metallicity on the observed EPR spectra of electrochemically lithiated graphites at four different states of lithiation. We show that the increased resolution offered by EPR at high frequencies (>300 GHz) enables up to three different electron environments of axial symmetry to be observed, revealing heterogeneity within the graphite particles and the presence of hyperfine coupling to Li nuclei. Importantly, our work demonstrates the power of EPR spectroscopy to investigate the local electronic structure of graphite at different lithiation stages, paving the way for this technique as a tool for screening and investigating novel materials for use in Li-ion batteries

Effects of slow-release verapamil and nitrendipine on office and 24-hour ambulatory blood pressure in hypertensive patients.

The aim of the present study was to compare the effects of slow-release verapamil (V), 240 mg and nitrendipine (N), 20 mg, administered once daily, on office (OBP) and 24-h ambulatory blood pressure (ABP) in patients with mild-to-moderate hypertension. Twenty patients were entered into this open, randomized, two-group (V, N) parallel study. The study groups had similar age and sex distribution. The OBP (V, 155/103 +/- 19/8 mm Hg; N, 141/98 +/- 13/4 mm Hg), heart rate (HR) (V, 74 +/- 7 beats/min; N, 77 +/- 10 beats/min), daytime systolic ABP (V, 149 +/- 14 mm Hg; N, 147 +/- 13 mm Hg), and nighttime ABP of the two groups were not statistically different after a 2-week washout period. The daytime diastolic ABP (V, 99 +/- 6 mm Hg; N, 93 +/- 6 mm Hg) was slightly lower (p less than 0.05) in group N. Both the OBP (V, 136/90 +/- 19/9 mm Hg; N, 135/85 +/- 10/4 mm Hg) and daytime ABP (V, 132/85 +/- 14/4 mm Hg; N, 136/87 +/- 13/8 mm Hg) dropped in the two groups after 8 weeks of treatment. Nonparametric analysis did not show statistical differences between the groups in OBP and ABP percentage drop. There was no significant change in nighttime ABP, HR (V, 73 +/- 10 beats/min; N, 74 +/- 12 beats/min), ECG, and laboratory exams. We conclude that both verapamil SR and nitrendipine are effective in reducing blood pressure in hypertensive patients without altering the HR

Chemisorption of nitronyl-nitroxide radicals on gold surface: An assessment of morphology, exchange interaction and decoherence time

A combined Tof-SIMS, XPS and STM characterization has been performed to study the deposition of a sulphur-functionalized nitronyl nitroxide radical on Au(111) clearly demonstrating the chemisorption of intact molecules. Continuous -wave EPR characterization showed that the radical molecules maintain their paramagnetic character. Pulsed EPR measurements allowed to determine the decoherence time of the nanostructure at 80 K, which turned out to be comparable to the one measured in frozen solution and longer than previously reported for many radicals and other paramagnetic molecules at much lower temperatures. Furthermore, we conducted a state-of-the-art ab initio molecular dynamics study, suggesting different possible scenarios for chemisorption geometries and predicting the energetically favoured geometry. Calculation of the magnetic properties indicates a partial non-innocent role of the gold surface in determining the magnetic interactions between radicals in packed structures. This suggests that the observed EPR spectrum is to be attributed to low-density domains of disordered radicals interacting via dipolar interactions

Compaction of RNA duplexes in the cell

The structure and flexibility of RNA depend sensitively on the microenvironment. Using pulsed electron-electron double-resonance (PELDOR) spectroscopy combined with advanced labeling techniques, we show that the structure of double-stranded RNA (dsRNA) changes upon internalization into Xenopus laevis oocytes. Compared to dilute solution, the dsRNA A-helix is more compact in cells. We recapitulate this compaction in a densely crowded protein solution. Atomic-resolution molecular dynamics simulations of dsRNA capture semi-quantitatively the compaction, and identify non-specific electrostatic interactions between proteins and dsRNA as a possible driver of this effect