Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in ...integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole–dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron–electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.
We describe the synthesis, characterization, and application of an isotopologue of the trityl radical OX071, labeled with
C at the central carbon (
C
). This spin probe features large anisotropy of ...the hyperfine coupling with the
C
(
= 1/2), leading to an EPR spectrum highly sensitive to molecular tumbling. The high biocompatibility and lack of interaction with blood albumin allow for systemic delivery and
measurement of tissue microviscosity by EPR.
The detection and characterization of paramagnetic species by electron spin resonance (ESR) spectroscopy is widely used throughout chemistry, biology and materials science, from in vivo imaging to ...distance measurements in spin-labelled proteins. ESR relies on the inductive detection of microwave signals emitted by the spins into a coupled microwave resonator during their Larmor precession. However, such signals can be very small, prohibiting the application of ESR at the nanoscale (for example, at the single-cell level or on individual nanoparticles). Here, using a Josephson parametric microwave amplifier combined with high-quality-factor superconducting microresonators cooled at millikelvin temperatures, we improve the state-of-the-art sensitivity of inductive ESR detection by nearly four orders of magnitude. We demonstrate the detection of 1,700 bismuth donor spins in silicon within a single Hahn echo with unit signal-to-noise ratio, reduced to 150 spins by averaging a single Carr-Purcell-Meiboom-Gill sequence. This unprecedented sensitivity reaches the limit set by quantum fluctuations of the electromagnetic field instead of thermal or technical noise, which constitutes a novel regime for magnetic resonance. The detection volume of our resonator is ∼ 0.02 nl, and our approach can be readily scaled down further to improve sensitivity, providing a new versatile toolbox for ESR at the nanoscale.
Soluble stable radicals are used as spin probes and spin labels for in vitro and in vivo electron paramagnetic resonance (EPR) spectroscopy and imaging applications. We report the synthesis and ...characterization of a perchlorinated triarylmethyl radical enriched 99% at the central carbon,
. The anisotropy of the hyperfine splitting with the
C
(
= 26,
= 25,
= 199.5 MHz) and the
(
= 2.0015,
= 2.0015,
= 2.0040) are responsible for a strong effect of the radical tumbling rate on the EPR spectrum. The rotational correlation time can be determined by spectral simulation or via the line width or the apparent
after calibration, so the spin probe
can be used to measure media microviscosity with high sensitivity.
Fluorine electron-nuclear double resonance (
F ENDOR) has recently emerged as a valuable tool in structural biology for distance determination between F atoms and a paramagnetic center, either ...intrinsic or conjugated to a biomolecule via spin labeling. Such measurements allow access to distances too short to be measured by double electron-electron resonance (DEER). To further extend the accessible distance range, we exploit the high-spin properties of Gd(III) and focus on transitions other than the central transition (|-1/2⟩ ↔ |+1/2⟩), that become more populated at high magnetic fields and low temperatures. This increases the spectral resolution up to ca. 7 times, thus raising the long-distance limit of
F ENDOR almost 2-fold. We first demonstrate this on a model fluorine-containing Gd(III) complex with a well-resolved
F spectrum in conventional central transition measurements and show quantitative agreement between the experimental spectra and theoretical predictions. We then validate our approach on two proteins labeled with
F and Gd(III), in which the Gd-F distance is too long to produce a well-resolved
F ENDOR doublet when measured at the central transition. By focusing on the |-5/2⟩ ↔ |-3/2⟩ and |-7/2⟩ ↔ |-5/2⟩ EPR transitions, a resolution enhancement of 4.5- and 7-fold was obtained, respectively. We also present data analysis strategies to handle contributions of different electron spin manifolds to the ENDOR spectrum. Our new extended
F ENDOR approach may be applicable to Gd-F distances as large as 20 Å, widening the current ENDOR distance window.
DNA G-quadruplexes (GQs) are of great interest due to their involvement in crucial biological processes such as telomerase maintenance and gene expression. Furthermore, they are reported as ...catalytically active DNAzymes and building blocks in bio-nanotechnology. GQs exhibit remarkable structural diversity and conformational heterogeneity, necessitating precise and reliable tools to unravel their structure-function relationships. Here, we present insights into the structure and conformational flexibility of a unimolecular GQ with high spatial resolution via electron-nuclear double resonance (ENDOR) experiments combined with Cu(II) and fluorine labeling. These findings showcase the successful application of the
F-ENDOR methodology at 34 GHz, overcoming the limitations posed by the complexity and scarcity of higher-frequency spectrometers. Importantly, our approach retains both sensitivity and orientational resolution. This integrated study not only enhances our understanding of GQs but also expands the methodological toolbox for studying other macromolecules.
Triplet ground-state organic molecules are of interest with respect to several emerging technologies but usually show limited stability, especially as thin films. We report an organic diradical, ...consisting of two Blatter radicals, that possesses a triplet ground state with a singlet–triplet energy gap, ΔE ST ≈ 0.4–0.5 kcal mol–1 (2J/k ≈ 220–275 K). The diradical possesses robust thermal stability, with an onset of decomposition above 264 °C (TGA). In toluene/chloroform, glassy matrix, and fluid solution, an equilibrium between two conformations with ΔE ST ≈ 0.4 kcal mol–1 and ΔE ST ≈ −0.7 kcal mol–1 is observed, favoring the triplet ground state over the singlet ground-state conformation in the 110–330 K temperature range. The diradical with the triplet ground-state conformation is found exclusively in crystals and in a polystyrene matrix. The crystalline neutral diradical is a good electrical conductor with conductivity comparable to the thoroughly optimized bis(thiazolyl)-related monoradicals. This is surprising because the triplet ground state implies that the underlying π-system is cross-conjugated and thus is not compatible with either good conductance or electron delocalization. The diradical is evaporated under ultra-high vacuum to form thin films, which are stable in air for at least 18 h, as demonstrated by X-ray photoelectron and electron paramagnetic resonance (EPR) spectroscopies.
The interaction of {Cryptand(K+)}(C60 •–) with Fe3(CO)12 produced {Cryptand(K+)}2{Fe(CO)2-μ 2-η 2,η 2-C60}2 2–·2.5C6H4Cl2 (1) as the first negatively charged iron-bridged fullerene C60 dimer. The ...bridged iron atoms are coordinated to two 6–6 bonds of one C60 hexagon with short and long C(C60)–Fe bonds with average lengths of 2.042(3) and 2.088(3) Å. Fullerenes are close to each other in the dimer with a center-to-center interfullerene distance of 10.02 Å. Optical spectra support the localization of negative electron density on the Fe2(CO)4 units, which causes a 50 cm–1 shift of the CO vibration bands to smaller wavenumbers, and the C60 cages. Dimers are diamagnetic and electron paramagnetic resonance silent and have a singlet ground state resulting from the formation of an Fe–Fe bond in the dimer with a length of 2.978(4) Å. According to density functional theory calculations, the excited triplet state is higher than the ground state by 6.5 kcal/mol. Compound 1 shows a broad near-infrared band with a maximum at 970 nm, which is attributable to the charge transfer from the orbitals localized mainly on iron atoms to the C60 ligand.
A series of seven Cu/SSZ-13 catalysts with Si/Al = 6.7 are used to elucidate key rate-controlling factors during low-temperature standard ammonia-selective catalytic reduction (NH
-SCR), via a ...combination of SCR kinetics and
electron paramagnetic resonance (EPR) spectroscopy. Strong Cu-loading-dependent kinetics, with Cu atomic efficiency increasing nearly by an order of magnitude, is found when per chabazite cage occupancy for Cu ion increases from ∼0.04 to ∼0.3. This is due mainly to the release of intercage Cu transfer constraints that facilitates the redox chemistry, as evidenced from detailed Arrhenius analysis.
EPR spectroscopy studies reveal strong connectivity between Cu-ion dynamics and SCR kinetics, based on which it is concluded that under low-temperature steady-state SCR, kinetically most relevant Cu species are those with the highest intercage mobility. Transient binuclear Cu species are mechanistically relevant species, but their splitting and cohabitation are indispensable for low-temperature kinetics.
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