Biochemistry, Genetics and Molecular Biology › Biophysics

Electron Spin Resonance Studies

Works Count: 53723

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Description

This cluster of papers focuses on the application of Electron Spin Resonance (ESR) in studying biomolecular systems, including distance measurements using nitroxide compounds, protein dynamics, and the assessment of redox status and oxidative stress. The papers also cover techniques such as pulsed EPR spectroscopy, spin labeling, and the use of EPR in magnetic resonance imaging.

Keywords

Electron Spin Resonance; Distance Measurements; Nitroxide Compounds; Protein Dynamics; Oxidative Stress; Pulsed EPR Spectroscopy; Spin Labeling; Redox Status; Magnetic Resonance Imaging; Biomolecular Structure

Principles of Pulse Electron Paramagnetic Resonance

2001-05-31

Arthur Schweiger , Gunnar Jeschke

Abstract This book explains the foundations of pulse EPR, a field of spectroscopy which has now come of age and has found widespread application in investigations of structure, dynamics, and … Abstract This book explains the foundations of pulse EPR, a field of spectroscopy which has now come of age and has found widespread application in investigations of structure, dynamics, and function of biological systems and synthetic materials. For the first time a systematic overview of the whole field is given, including coverage of Fourier-transform EPR, relaxation measurements, electron spin echo envelope modulation (ESEEM), pulse electron- nuclear double resonance (ENDOR), pulse electron-electron double resonance (ELDOR), transient nutation, and a number of advanced techniques. Researchers approaching the field will find here the basic theory as well as a description and critical evaluation of the existing methods needed for selecting the proper experiment, conducting it, and analysing the results. The experienced researcher active in the field should find this book a useful reference and a guide to adapting EPR pulse sequences to new problems.

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Free Radical Mechanisms in Tissue Injury

1988-01-01

T. F. Slater

MOLECULAR MECHANISMS OF QUENCHING OF REACTIVE OXYGEN SPECIES BY PROLINE UNDER STRESS IN PLANTS

2002-01-01

Matysik J.Alia , B Bhalu , Partha Sarathi Mohanty

Less than 5% of the total pool of free amino acids in plants under stress-free conditions is provided by proline. In many plants under various forms of stress, the concentration … Less than 5% of the total pool of free amino acids in plants under stress-free conditions is provided by proline. In many plants under various forms of stress, the concentration increases up to 80% of the amino acid pool.This observation raises the question about the molecular mechanisms, making a high proline concentration favourable under stress conditions. Therefore, the literature about the chemical properties of proline is reviewed, linking it to the plant physiological observations. In addition to its role as an osmolyte and a reservoir of carbon and nitrogen, etc. proline has been shown to protect plants against free radical-induced damage. A recent concept that proline accumulation is linked with the quenching of singlet oxygen has inspired us to look at the molecular mechanism of singlet oxygen quenching by proline. In this review, the key properties and the chemical reactivity of proline with singlet oxygen and other reactive oxygen species are discussed.

Role of oxygen free radicals in carcinogenesis and brain ischemia

1990-06-01

Robert A. Floyd

Even though oxygen is necessary for aerobic life, it can also participate in potentially toxic reactions involving oxygen free radicals and transition metals such as Fe that damage membranes, proteins, … Even though oxygen is necessary for aerobic life, it can also participate in potentially toxic reactions involving oxygen free radicals and transition metals such as Fe that damage membranes, proteins, and nucleic acids. Oxygen free radical reactions and oxidative damage are in most cases held in check by antioxidant defense mechanisms, but where an excessive amount of oxygen free radicals are produced or defense mechanisms are impaired, oxidative damage may occur and this appears to be important in contributing to several pathological conditions including aging, carcinogenesis, and stroke. Several newer methods, such as in vivo spin‐trapping, have become available to monitor oxygen free radical flux and quantitate oxidative damage. Using a combination of these newer methods collectively focused on one model, recent results show that oxidative damage plays a key role in brain injury that occurs in stroke. Subtle changes, such as oxidative damage‐induced loss of glutamine synthetase activity, may be a key event in stroke‐induced brain injury. Oxygen free radicals may play a key role in carcinogenesis by mediating formation of base adducts, such as 8‐hydroxyguanine, which can now be quantitated to very low levels. Evidence is presented that a new class of free radical blocking agents, nitrone spin‐traps, may help not only to clarify if free radical events are involved, but may help prevent the development of injury in certain pathological conditions.—F loyd , R. A. Role of oxygen free radicals in carcinogenesis and brain ischemia. FASEB J. 4: 2587‐2597, 1990.

Identifying conformational changes with site-directed spin labeling.

2000-09-01

Wayne L. Hubbell , David S. Cafiso , Christian Altenbach

Three Distinct Mechanisms Generate Oxygen Free Radicals in Neurons and Contribute to Cell Death during Anoxia and Reoxygenation

2007-01-31

Andrey Y. Abramov , Antonella Scorziello , Michael R. Duchen

Ischemia is a major cause of brain damage, and patient management is complicated by the paradoxical injury that results from reoxygenation. We have now explored the generation of reactive oxygen … Ischemia is a major cause of brain damage, and patient management is complicated by the paradoxical injury that results from reoxygenation. We have now explored the generation of reactive oxygen species (ROS) in hippocampal and cortical neurons in culture in response to oxygen and glucose deprivation or metabolic inhibition and reoxygenation. Fluorescence microscopy was used to measure the rate of ROS generation using hydroethidine, dicarboxyfluorescein diacetate, or MitoSOX. ROS generation was correlated with changing mitochondrial potential (rhodamine 123), [Ca 2+ ] c (fluo-4, fura-2, or Indo-1), or ATP consumption, indicated by increased [Mg 2+ ] c . We found that three distinct mechanisms contribute to neuronal injury by generating ROS and oxidative stress, each operating at a different stage of ischemia and reperfusion. In response to hypoxia, mitochondria generate an initial burst of ROS, which is curtailed once mitochondria depolarize or prevented by previous depolarization with uncoupler. A second phase of ROS generation that followed after a delay was blocked by the xanthine oxidase (XO) inhibitor oxypurinol. This phase correlated with a rise in [Mg 2+ ] c , suggesting XO activation by accumulating products of ATP consumption. A third phase of ROS generation appeared at reoxygenation. This was blocked by NADPH oxidase inhibitors and was absent in cells from gp91 phox−/− knock-out mice. It was Ca 2+ dependent, suggesting activation by increased [Ca 2+ ] c during anoxia, itself partly attributable to glutamate release. Inhibition of either the NADPH oxidase or XO was significantly neuroprotective. Thus, oxidative stress contributes to cell death over and above the injury attributable to energy deprivation.

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Radiation Damage in Organic Crystals. II. Electron Spin Resonance of (CO2H)CH2CH(CO2H) in β-Succinic Acid

1960-05-01

C. Heller , Harden M. McConnell

An analysis of the electron spin resonance of x-irradiated single crystals of β-succinic acid shows that: (a) the principal long-lived paramagnetic species produced by the radiation damage is (CO2H)CH2–ĊH(CO2H); (b) … An analysis of the electron spin resonance of x-irradiated single crystals of β-succinic acid shows that: (a) the principal long-lived paramagnetic species produced by the radiation damage is (CO2H)CH2–ĊH(CO2H); (b) the radical is oriented in the crystal lattice in nearly the same way that the parent succinic acid molecule is oriented in the undamaged lattice; (c) the strongly anisotropic hyperfine interaction due to the σ proton is very nearly the same as that previously found for the σ proton in the malonic acid radical, (CO2H)ĊH(CO2H). In these molecules the σ proton is directly bonded to the carbon atom on which the odd electron is largely localized. The two methylene protons in the radical are not equivalent, and their hyperfine interactions are nearly isotropic, and in the range 80–100 Mc.

Electron Spin Resonance Absorption in Metals. I. Experimental

1955-04-15

G. Fehér , A. F. Kip

The electron spin resonance absorption in metals was investigated at 300 and 9000 Mc/sec, in the temperature range from 4\ifmmode^\circ\else\textdegree\fi{}K to 296\ifmmode^\circ\else\textdegree\fi{}K. The metals investigated were: Li, Na, K, Be, … The electron spin resonance absorption in metals was investigated at 300 and 9000 Mc/sec, in the temperature range from 4\ifmmode^\circ\else\textdegree\fi{}K to 296\ifmmode^\circ\else\textdegree\fi{}K. The metals investigated were: Li, Na, K, Be, Mg, Al, Pd, W. Resonances were observed in Li, Na, Be, for which accurate $g$ values and relaxation times were obtained and compared with existing theories. K gave a signal only at 4\ifmmode^\circ\else\textdegree\fi{}K and 300 Mc/sec, with too poor a signal-to-noise ratio to make accurate measurements. No resonances were observed in Mg, Al, Pd, W. The line shapes predicted by Dyson's theory for the resonance absorption in metals were checked experimentally for different diffusion times and relaxation times. A satisfactory agreement between the theory and experiment is obtained for both the normal and the anomalous skin effect region.

Biologically relevant metal ion‐dependent hydroxyl radical generation An update

1992-07-27

Barry Halliwell , John M.C. Gutteridge

Transition metal ions, especially iron, appear to be important mediators of oxidative damage in vivo. Iron(II) reacts with H 2 O 2 to give more‐reactive radicals. On the basis of … Transition metal ions, especially iron, appear to be important mediators of oxidative damage in vivo. Iron(II) reacts with H 2 O 2 to give more‐reactive radicals. On the basis of ESR spin‐trapping data with DMPO, supported by aromatic hydroxylation studies and patterns of DNA base modification, it is concluded that hydroxyl radical (OH • ) is likely to be the major damaging species formed in Fenton Systems under biologically‐relevant conditions (which include iron concentrations no higher than the micromolar range). Although reactive oxo‐iron species (such as ferryl and perferryl) may also be important, chemical evidence for their formation and identity in biologically relevant Fenton systems is currently lacking. Studies at alkaline pH values show that iron(IV) and iron(V) species are highly oxidizing under those reaction conditions, with a pattern of reactivity different from that of OH • .

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Spin trapping. Kinetics of the reaction of superoxide and hydroxyl radicals with nitrones

1980-07-01

Eli Finkelstein , Gerald M. Rosen , Elmer J. Rauckman

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSpin trapping. Kinetics of the reaction of superoxide and hydroxyl radicals with nitronesEli Finkelstein, Gerald M. Rosen, and Elmer J. RauckmanCite this: J. Am. Chem. Soc. 1980, … ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSpin trapping. Kinetics of the reaction of superoxide and hydroxyl radicals with nitronesEli Finkelstein, Gerald M. Rosen, and Elmer J. RauckmanCite this: J. Am. Chem. Soc. 1980, 102, 15, 4994–4999Publication Date (Print):July 1, 1980Publication History Published online1 May 2002Published inissue 1 July 1980https://pubs.acs.org/doi/10.1021/ja00535a029https://doi.org/10.1021/ja00535a029research-articleACS PublicationsRequest reuse permissionsArticle Views2790Altmetric-Citations535LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Distributed multipole analysis, or how to describe a molecular charge distribution

1981-10-01

Anthony J. Stone

Electron Spin Resonance Studies of Transient Alkyl Radicals

1963-11-01

Richard W. Fessenden , Robert H. Schüler

Electron spin resonance methods have been used to observe alkyl radicals in liquid hydrocarbon systems during irradiation with 2.8-MeV electrons. These investigations provide detailed structural, radiation chemical, and kinetic information … Electron spin resonance methods have been used to observe alkyl radicals in liquid hydrocarbon systems during irradiation with 2.8-MeV electrons. These investigations provide detailed structural, radiation chemical, and kinetic information about a large number of radicals. In general, in these studies the ESR lines are found to be narrow; considerable fine structure is observable, permitting positive assignment of the radical species. Accurate hyperfine constants are reported for 21 alkyl and cycloalkyl radicals (including several deuterated species), vinyl, 1-methylvinyl, 3-butenyl, allyl, and cyclohexadienyl radicals, and hydrogen and deuterium atoms. Except for cyclopropyl radical, all the alkyl and cycloalkyl radicals have α coupling constants in the range 21–23 G. The β coupling constants in cases where they have been rotationally averaged isotropically are found to decrease with increasing substitution of alkyl groups on the α carbon atom. In general the values for primary, secondary, and tertiary radicals appear to be represented by the splittings observed for the methyl protons in ethyl (26.87 G), isopropyl (24.68 G), and tert-butyl (22.72 G) radicals. A possible explanation for this trend is discussed. A number of examples showing a significant departure from the above isotropically averaged values have been found. In several cases this departure, and the resultant strong temperature dependence of the β coupling constants, is taken as evidence for a barrier hindering rotation about the α-carbon—β-carbon bond. Splittings caused by γ protons and which range from 0.4 to 1.1 G have been resolved in five cases. A pronounced angular dependence of this coupling constant has been demonstrated in the case of propyl radical. This angular dependence is important in considerations of the mechanism of the γ hyperfine interaction. Three radicals which do not have the usual π-electron configuration have been observed: vinyl, 1-methylvinyl, and cyclopropyl. The splittings by the α protons of vinyl and cyclopropyl radicals are 13.39 and 6.51 G. These small values indicate that as the orbital associated with the unpaired electron acquires s character the coupling constant increases from ∼−23 G. The coupling constants for the two conjugated radicals, allyl and cyclohexadienyl, support the theoretical prediction of negative spin density at the unstarred positions of these odd-alternant radicals. Spectra with relatively narrow lines are reported for transient species in a number of solid hydrocarbons. The radicals observed in various hydrocarbons are discussed in terms of the radiation chemical reactions expected in these systems. In most cases the radicals represent fragments which result from rupture of a single bond. In certain cases secondary reactions, such as the addition of hydrogen atoms to unsaturates, are also found to be important. The use of ESR methods in obtaining information concerning the rate of radical reactions is illustrated by two studies. In the first an activation energy of 3.3 kcal/mole is estimated for the addition of vinyl radicals to ethylene from measurements on the temperature dependence of the relative vinyl and 3-butenyl radical concentrations in liquid ethylene. In the second the kinetics of the disappearance of ethyl radicals in liquid ethane are examined in detail. The absolute second-order rate constant for this disappearance as obtained from absolute concentration and dose rate measurements is found to be 3×10 liters mole−1 sec−1 at −175°C. The activation energy for the reaction of ethyl radicals in liquid ethane is 780 cal/mole, or essentially that for the diffusion-controlled process.

The deoxyribose method: A simple “test-tube” assay for determination of rate constants for reactions of hydroxyl radicals

1987-08-01

Barry Halliwell , John M.C. Gutteridge , Okezie I. Aruoma

Spin Trapping: ESR parameters of spin adducts 1474 1528V

1987-01-01

Garry R. Buettner

Dead-Time Free Measurement of Dipole–Dipole Interactions between Electron Spins

2000-02-01

M. Pannier , Stephan Veit , Adelheid Godt +2 more

A four-pulse version of the pulse double electron-electron resonance (DEER) experiment is presented, which is designed for the determination of interradical distances on a nanoscopic length-scale. With the new pulse … A four-pulse version of the pulse double electron-electron resonance (DEER) experiment is presented, which is designed for the determination of interradical distances on a nanoscopic length-scale. With the new pulse sequence electron-electron couplings can be studied without dead-time artifacts, so that even broad distributions of electron-electron distances can be characterized. A version of the experiment that uses a pulse train in the detection period exhibits improved signal-to-noise ratio. Tests on two nitroxide biradicals with known length indicate that the accessible range of distances extends from about 1.5 to 8 nm. The four-pulse DEER spectra of an ionic spin probe in an ionomer exhibit features due to probe molecules situated both on the same and on different ion clusters. The former feature provides information on the cluster size and is inaccessible with previous methods.

Effect of librational motion on fluorescence depolarization and nuclear magnetic resonance relaxation in macromolecules and membranes

1980-06-01

Giovanni Lipari , Attila Szabó

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Iterative schemes for bilinear operators; application to spin decoupling

1988-04-01

A.J. Shaka , C.J Lee , Alexander Pines

Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ

1979-05-01

John A. Thomas , Robert Buchsbaum , Andrzej Zimniak +1 more

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIntracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situJohn A. Thomas, Robert N. Buchsbaum, Andrzej Zimniak, and Efraim RackerCite this: Biochemistry 1979, … ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIntracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situJohn A. Thomas, Robert N. Buchsbaum, Andrzej Zimniak, and Efraim RackerCite this: Biochemistry 1979, 18, 11, 2210–2218Publication Date (Print):May 1, 1979Publication History Published online1 May 2002Published inissue 1 May 1979https://pubs.acs.org/doi/10.1021/bi00578a012https://doi.org/10.1021/bi00578a012research-articleACS PublicationsRequest reuse permissionsArticle Views4053Altmetric-Citations1615LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Direct measurement of free radical generation following reperfusion of ischemic myocardium.

1987-03-01

Jay L. Zweíer , John T. Flaherty , Myron L. Weisfeldt

Electron paramagnetic resonance spectroscopy was used to directly measure free radical generation in perfused rabbit hearts. Hearts were freeze-clamped at 77 degrees K during control perfusion, after 10 min of … Electron paramagnetic resonance spectroscopy was used to directly measure free radical generation in perfused rabbit hearts. Hearts were freeze-clamped at 77 degrees K during control perfusion, after 10 min of normothermic global ischemia (no coronary flow), or following post-ischemic reperfusion with oxygenated perfusate. The spectra of these hearts exhibited three different signals with different power saturation and temperature stability: signal A was isotropic with g = 2.004; signal B was anisotropic with axial symmetry with g parallel = 2.033 and g perpendicular = 2.005; signal C was an isotropic triplet with g = 2.000 and hyperfine splitting an = 24 G (1 G = 0.1 mT). The g values, linewidth, power saturation, and temperature stability of signal A are identical to those of a carbon-centered semiquinone, whereas those of signal B are similar to alkyl peroxyl or superoxide oxygen-centered free radicals; signal C is most likely a nitrogen-centered free radical. In the control heart samples signal A predominated, whereas in ischemic hearts signal A decreased in intensity, and signals B and C became more intense; with reperfusion all three signals markedly increased. Free radical concentrations derived from the intensities of the B and C signals peaked 10 sec after initiation of reflow. At this time the oxygen-centered free radical concentration derived from the intensity of signal B was increased over six times the concentration measured in control hearts and over two times the concentration measured in ischemic hearts. Hypoxic reperfusion did not increase any of the free radical signals over the levels observed during ischemia. These experiments directly demonstrate that reactive oxygen-centered free radicals are generated in hearts during ischemia and that a burst of oxygen radical generation occurs within moments of reperfusion.

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Free Radicals in Biological Materials

1954-10-01

Barry Commoner , J. Townsend , G. E. Pake

Demonstration of free radical generation in "stunned" myocardium of intact dogs with the use of the spin trap alpha-phenyl N-tert-butyl nitrone.

1988-08-01

Roberto Bolli , Brijeshkumar S. Patel , Mohamed O. Jeroudi +2 more

Recent studies suggest that oxygen free radicals may mediate postischemic myocardial dysfunction ("stunning"), but all the evidence for this hypothesis is indirect. Thus, we used electron paramagnetic resonance (EPR) spectroscopy … Recent studies suggest that oxygen free radicals may mediate postischemic myocardial dysfunction ("stunning"), but all the evidence for this hypothesis is indirect. Thus, we used electron paramagnetic resonance (EPR) spectroscopy and the spin trap, alpha-phenyl N-tert-butyl nitrone (PBN), to directly investigate whether free radicals are produced after a 15-min coronary artery occlusion and subsequent reperfusion in 30 open-chest dogs. After intracoronary infusion of PBN, EPR signals characteristic of oxygen- and carbon-centered radical adducts were detected in the venous blood draining from the ischemic/reperfused vascular bed. The myocardial release of PBN adducts began during coronary occlusion but increased dramatically in the first few minutes after reperfusion. After this initial burst, the production of radicals abated but did not cease, persisting up to 3 h after reflow. The EPR spectra (aH beta = 2.67-2.79 G, aN = 14.75-15.00 G) were consistent with the trapping by PBN of secondary oxygen- and carbon-centered radicals, such as alkoxy and alkyl radicals, which could be formed by reactions of primary oxygen radicals with membrane lipids. There was a linear, direct relationship between the magnitude of PBN adduct production and the degree of ischemic flow reduction. Recovery of contractile function (measured as systolic wall thickening) after reperfusion was greater (P less than 0.05) in dogs given PBN than in controls. This study demonstrates that reversible regional myocardial ischemia in the intact animal is associated with prolonged free radical generation, and that the intensity of such generation is related to the severity of ischemia. The results provide direct evidence to support the hypothesis that reactive oxygen metabolites contribute to the persistent contractile dysfunction (myocardial stunning) observed after brief ischemia in vivo.

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Bemerkung zur Quantelung des harmonischen Oszillators im Magnetfeld

1928-05-01

V. Fock

Simulation of Multiple Isotropic Spin-Trap EPR Spectra

1994-06-01

David Duling

Molecular motion in spin-labeled phospholipids and membranes

1971-01-01

Harden M. McConnell , Wayne L. Hubbell

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMolecular motion in spin-labeled phospholipids and membranesHarden M. McConnell and Wayne L. HubbellCite this: J. Am. Chem. Soc. 1971, 93, 2, 314–326Publication Date (Print):January 1, 1971Publication History … ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMolecular motion in spin-labeled phospholipids and membranesHarden M. McConnell and Wayne L. HubbellCite this: J. Am. Chem. Soc. 1971, 93, 2, 314–326Publication Date (Print):January 1, 1971Publication History Published online1 May 2002Published inissue 1 January 1971https://pubs.acs.org/doi/10.1021/ja00731a005https://doi.org/10.1021/ja00731a005research-articleACS PublicationsRequest reuse permissionsArticle Views1210Altmetric-Citations1236LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Spin trapping of superoxide and hydroxyl radical: Practical aspects

1980-03-01

Eli Finkelstein , Gerald M. Rosen , Elmer J. Rauckman

Oxidative damage to brain proteins, loss of glutamine synthetase activity, and production of free radicals during ischemia/reperfusion-induced injury to gerbil brain.

1990-07-01

Constance Oliver , Pamela Starke‐Reed , Earl R. Stadtman +3 more

Free radical-mediated oxidative damage has been implicated in tissue injury resulting from ischemia/reperfusion events. Global cortical ischemia/reperfusion injury to Mongolian gerbil brains was produced by transient occlusion of both common … Free radical-mediated oxidative damage has been implicated in tissue injury resulting from ischemia/reperfusion events. Global cortical ischemia/reperfusion injury to Mongolian gerbil brains was produced by transient occlusion of both common carotid arteries. Protein oxidation, as measured by protein carbonyl content, increased significantly during the reperfusion phase that followed 10 min of ischemia. The activity of glutamine synthetase, an enzyme known to be inactivated by metal-catalyzed oxidation reactions, decreased to 65% of control levels after 2 hr of reperfusion that followed 10 min of ischemia. We also report that the free radical spin trap N-tert-butyl-alpha-phenylnitrone [300 mg/kg (body weight)] administered 60 min before ischemia/reperfusion is initiated, partially prevents protein oxidation and protects from loss of glutamine synthetase activity. In addition, we report a N-tert-butyl-alpha-phenylnitrone-dependent nitroxide radical obtained in the lipid fraction of the ischemia/reperfusion-lesioned brains, but there was very little radical present in the comparable sham-operated control brains. These data strengthen the previous observation utilizing in vivo-trapping methods, that free radical flux is increased during the reperfusion phase of the ischemia-lesioned gerbil brain. The loss of glutamine synthetase would be expected to increase the levels of brain L-glutamate. Thus, the oxidative inactivation of glutamine synthetase may be a critical factor in the neurotoxicity produced after cerebral ischemia/reperfusion injury.

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Ellman's reagent: 5,5′-dithiobis(2-nitrobenzoic acid)—a reexamination

1979-04-01

Peter W. Riddles , Robert L. Blakeley , Burt Zerner

Electron Paramagnetic Resonance: Elementary Theory and Practical Applications

1995-05-01

John A. Weil , James R. Bolton , John E. Wertz +1 more

Basic Principles of Electron Paramagnetic Resonance Magnetic Interactions Between Particles Isotropic Hyperfine Effects in EPR Spectra g-Anisotropy in Solids Hyperfine Anisotropy in Solids Systems with More than One Unpaired Electron … Basic Principles of Electron Paramagnetic Resonance Magnetic Interactions Between Particles Isotropic Hyperfine Effects in EPR Spectra g-Anisotropy in Solids Hyperfine Anisotropy in Solids Systems with More than One Unpaired Electron Paramagnetic Species in Gas Phase Transition-Group Ions The Interpretation of EPR Parameters Relaxation Times, Linewidths and Kinetic Phenomena Time-Dependent Excitation of Spins Double-Resonance Techniques Other Topics.

Indirect Hyperfine Interactions in the Paramagnetic Resonance Spectra of Aromatic Free Radicals

1956-04-01

Harden M. McConnell

It is shown that an electron spin polarization associated with an odd electron in a π-molecular orbital in an aromatic free radical can, through an atomic exchange coupling mechanism, produce … It is shown that an electron spin polarization associated with an odd electron in a π-molecular orbital in an aromatic free radical can, through an atomic exchange coupling mechanism, produce an appreciable resultant electron spin polarization in s-atomic orbitals at the aromatic protons. This resultant spin polarization gives rise to hyperfine interactions with aromatic protons which are of the same order of magnitude as those observed in the paramagnetic resonance spectra of aromatic free radicals. An equation is developed which relates (proton-spin)-(electron-spin) hyperfine coupling constants to the π distribution of the odd electron in the aromatic free radicals.

Study of Moderately Rapid Chemical Exchange Reactions by Means of Nuclear Magnetic Double Resonance

1963-12-01

Sture Forsén , Ragnar A. Hoffman

A nuclear magnetic double-resonance method for the determination of chemical exchange rates has been developed. The method is applicable to systems in which a nuclear spin is reversibly transferred between … A nuclear magnetic double-resonance method for the determination of chemical exchange rates has been developed. The method is applicable to systems in which a nuclear spin is reversibly transferred between two nonequivalent sites, A and B. The lifetime (τA) and spin—lattice relaxation time (T1A) in Site A are obtained through the study of the decay to a new equilibrium value of Signal A upon the sudden saturation of Signal B. The converse experiment permits the determination of τB and T1B. A number of data for cross checks are furthermore obtained through the study of the recovery of the signals upon the release of various combinations of saturating rf fields. A simple theory based on the Bloch equations as modified by McConnell to incorporate the effects of chemical exchange is given. Experimental results on the hydroxyl proton exchange in the system salicylaldehyde and 2-hydroxyacetophenone are well described by this simple theory. The present method, which can readily be extended to systems with several sites, offers a complement to the Gutowsky—Saika single-resonance method and is particularly suited to the study of exchange rates slower than those accessible by the single-resonance method.

A Molecular Beam Resonance Method with Separated Oscillating Fields

1950-06-15

Norman F. Ramsey

A new molecular beam resonance method using separated oscillating fields at the incident and emergent ends of the homogeneous field region is theoretically investigated in this paper. An expression is … A new molecular beam resonance method using separated oscillating fields at the incident and emergent ends of the homogeneous field region is theoretically investigated in this paper. An expression is obtained for the quantum mechanical transition probability of a system between two states when the system is subjected to such separated oscillating fields. This is numerically averaged over the molecular velocity distribution and provides the theoretical shape of the resonance curves. It is found that resonances with such a technique have a theoretical half-width only 0.6 as great as those by conventional molecular beam resonance methods. In addition to producing sharper resonance minima, the new method has its resonances much less broadened by inhomogeneities of the fixed field, it makes possible resonance experiments in regions into which an oscillating field cannot be introduced, and it is more convenient and effective with short wave-length radiation.

Time Saving in Measurement of NMR and EPR Relaxation Times

1970-02-01

D. C. Look , D. R. Locker

By producing a train of absorption or dispersion signals (continuous-wave magnetic resonance) or free induction decays (pulsed magnetic resonance) it is possible to save time in spin-lattice relaxation measurements due … By producing a train of absorption or dispersion signals (continuous-wave magnetic resonance) or free induction decays (pulsed magnetic resonance) it is possible to save time in spin-lattice relaxation measurements due to the fact that it is not necessary to wait for equilibrium magnetization before initiating the train. The relaxation time may be calculated from the train according to a simple rapidly converging iteration.

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Resonance Energy Transfer: Methods and Applications

1994-04-01

P.G. Wu , Ludwig Brand

Resonance energy transfer is widely used in studies of biomolecular structure and dynamics. It provides information about distances on the order of 10 to 100 Å and is thus suitable … Resonance energy transfer is widely used in studies of biomolecular structure and dynamics. It provides information about distances on the order of 10 to 100 Å and is thus suitable for investigating spatial relationships of interest in biochemistry. The information available from energy transfer studies has been enhanced by the advances in instrumental methods and procedures of data analysis related to fluorescence decay studies. Some practical aspects of the method are reviewed. These include sample preparation, Förster type distance determination, methods of detecting energy transfer and calculating transfer efficiency, time-resolved measurements, and data analysis. Applications of resonance energy transfer, including qualitative measurements as well as microscopy, average distance estimation, and distance distribution analysis, are surveyed.

DEER Distance Measurements on Proteins

2012-04-04

Gunnar Jeschke

Distance distributions between paramagnetic centers in the range of 1.8 to 6 nm in membrane proteins and up to 10 nm in deuterated soluble proteins can be measured by the … Distance distributions between paramagnetic centers in the range of 1.8 to 6 nm in membrane proteins and up to 10 nm in deuterated soluble proteins can be measured by the DEER technique. The number of paramagnetic centers and their relative orientation can be characterized. DEER does not require crystallization and is not limited with respect to the size of the protein or protein complex. Diamagnetic proteins are accessible by site-directed spin labeling. To characterize structure or structural changes, experimental protocols were optimized and techniques for artifact suppression were introduced. Data analysis programs were developed, and it was realized that interpretation of the distance distributions must take into account the conformational distribution of spin labels. First methods have appeared for deriving structural models from a small number of distance constraints. The present scope and limitations of the technique are illustrated.

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ωB97X-V: A 10-parameter, range-separated hybrid, generalized gradient approximation density functional with nonlocal correlation, designed by a survival-of-the-fittest strategy

2013-12-18

Narbe Mardirossian , Martin Head‐Gordon

A 10-parameter, range-separated hybrid (RSH), generalized gradient approximation (GGA) density functional with nonlocal correlation (VV10) is presented. Instead of truncating the B97-type power series inhomogeneity correction factors (ICF) for the … A 10-parameter, range-separated hybrid (RSH), generalized gradient approximation (GGA) density functional with nonlocal correlation (VV10) is presented. Instead of truncating the B97-type power series inhomogeneity correction factors (ICF) for the exchange, same-spin correlation, and opposite-spin correlation functionals uniformly, all 16,383 combinations of the linear parameters up to fourth order (m = 4) are considered. These functionals are individually fit to a training set and the resulting parameters are validated on a primary test set in order to identify the 3 optimal ICF expansions. Through this procedure, it is discovered that the functional that performs best on the training and primary test sets has 7 linear parameters, with 3 additional nonlinear parameters from range-separation and nonlocal correlation. The resulting density functional, ωB97X-V, is further assessed on a secondary test set, the parallel-displaced coronene dimer, as well as several geometry datasets. Furthermore, the basis set dependence and integration grid sensitivity of ωB97X-V are analyzed and documented in order to facilitate the use of the functional.

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DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data

2006-06-01

Gunnar Jeschke , Victor Chechik , Petre Ioniță +6 more

Mitochondrial Electron Transport Complex I Is a Potential Source of Oxygen Free Radicals in the Failing Myocardium

1999-08-20

Tomomi Ide , Hiroyuki Tsutsui , Shintaro Kinugawa +7 more

Abstract —Oxidative stress in the myocardium may play an important role in the pathogenesis of congestive heart failure (HF). However, the cellular sources and mechanisms for the enhanced generation of … Abstract —Oxidative stress in the myocardium may play an important role in the pathogenesis of congestive heart failure (HF). However, the cellular sources and mechanisms for the enhanced generation of reactive oxygen species (ROS) in the failing myocardium remain unknown. The amount of thiobarbituric acid reactive substances increased in the canine HF hearts subjected to rapid ventricular pacing for 4 weeks, and immunohistochemical staining of 4-hydroxy-2-nonenal ROS-induced lipid peroxides was detected in cardiac myocytes but not in interstitial cells of HF animals. The generation of superoxide anion was directly assessed in the submitochondrial fractions by use of electron spin resonance spectroscopy with spin trapping agent, 5,5′-dimethyl-1-pyrroline- N -oxide, in the presence of NADH and succinate as a substrate for NADH–ubiquinone oxidoreductase (complex I) and succinate–ubiquinone oxidoreductase (complex II), respectively. Superoxide production was increased 2.8-fold ( P <0.01) in HF, which was due to the functional block of electron transport at complex I. The enzymatic activity of complex I decreased in HF (274±13 versus 136±9 nmol · min −1 · mg −1 protein, P <0.01), which may thus have caused the functional uncoupling of the respiratory chain and the deleterious ROS production in HF mitochondria. The present study provided direct evidence for the involvement of ROS in the mitochondrial origin of HF myocytes, which might be responsible for both contractile dysfunction and structural damage to the myocardium.

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Oxygen radical mechanisms of brain injury following ischemia and reperfusion

1991-10-01

Richard J. Traystman , Jeffrey R. Kirsch , Raymond C. Koehler

This review addresses current understanding of oxygen radical mechanisms as they relate to the brain during ischemia and reperfusion. The mechanism for radical production remains speculative in large part because … This review addresses current understanding of oxygen radical mechanisms as they relate to the brain during ischemia and reperfusion. The mechanism for radical production remains speculative in large part because of the difficulty of measuring radical species in vivo. Breakdown of lipid membranes during ischemia leads to accumulation of free fatty acids. Decreased energy stores during ischemia result in the accumulation of adenine nucleotides. During reperfusion, metabolism of free fatty acids via the cyclooxygenase pathway and metabolism of adenine nucleotides via the xanthine oxidase pathway are the most likely sources of oxygen radicals. Although leukocytes have been found to accumulate in some models of ischemia and reperfusion, their mechanistic role remains in question. Therapeutic strategies aimed at decreasing brain injury have included administration of radical scavengers at the time of reperfusion. Efficacy of traditional oxygen radical scavengers such as superoxide dismutase and catalase may be limited by their inability to cross the blood-brain barrier. Lipid-soluble antioxidants appear more efficacious because of their ability to cross the blood-brain barrier and because of their presence in membrane structures where peroxidative reactions can be halted.

Electrogeneration of Hydroxyl Radicals on Boron-Doped Diamond Electrodes

2003-01-01

Béatrice Marselli , J. Garcı́a-Gómez , Pierre Michaud +2 more

The electrogeneration of hydroxyl radicals was studied at a synthetic boron-doped diamond (BDD) thin film electrode. Spin trapping was used for detection of hydroxyl radicals with 5,5-dimethyl-1-pyrroline-N-oxide and with salicylic … The electrogeneration of hydroxyl radicals was studied at a synthetic boron-doped diamond (BDD) thin film electrode. Spin trapping was used for detection of hydroxyl radicals with 5,5-dimethyl-1-pyrroline-N-oxide and with salicylic acid using electron spin resonance and liquid chromatography measurements, respectively. The production of hydrogen peroxide and competitive oxidation of formic and oxalic acids were also investigated using bulk electrolysis. The results have shown that oxidation of salicylic acid leads to the production of hydroxylated products (2,3- and 2,5-dihydroxybenzoic acids). These results demonstrate that the oxidation process on BDD electrodes involves hydroxyl radicals as electrogenerated intermediates. © 2003 The Electrochemical Society. All rights reserved.

Simultaneousin vivo spectral editing and water suppression

1998-10-01

Marlene Mescher , Hellmut Merkle , Jonathan Kirsch +2 more

Water suppression is typically performed in vivo by exciting the longitudinal magnetization in combination with dephasing, or by using frequency-selective coherence generation. MEGA, a frequency-selective refocusing technique, can be placed … Water suppression is typically performed in vivo by exciting the longitudinal magnetization in combination with dephasing, or by using frequency-selective coherence generation. MEGA, a frequency-selective refocusing technique, can be placed into any pulse sequence element designed to generate a Hahn spin-echo or stimulated echo, to dephase transverse water coherences with minimal spectral distortions. Water suppression performance was verified in vivo using stimulated echo acquisition mode (STEAM) localization, which provided water suppression comparable with that achieved with four selective pulses in 3,1-DRYSTEAM. The advantage of the proposed method was exploited for editing J-coupled resonances. Using a double-banded pulse that selectively inverts a J-coupling partner and simultaneously suppresses water, efficient metabolite editing was achieved in the point resolved spectroscopy (PRESS) and STEAM sequences in which MEGA was incorporated. To illustrate the efficiency of the method, the detection of γ-aminobutyric acid (GABA) was demonstrated, with minimal contributions from macromolecules and overlying singlet peaks at 4 T. The estimated occipital GABA concentration was consistent with previous reports, suggesting that editing for GABA is efficient when based on MEGA at high field strengths. © 1998 John Wiley & Sons, Ltd.

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Electron spin resonance—Elementary theory and practical applications

1972-12-01

Ralph Livingston

EPR of exchange-coupled systems

1990-12-01

W. Weltner

Polymer NSR spectroscopy. II. The high resolution spectra of methyl methacrylate polymers prepared with free radical and anionic initiators

1960-05-01

F. A. Bovey , George Van Dyke Tiers

Abstract Spectra of methyl methacrylate polymers in chloroform solution at 90° show three α‐methyl proton peaks. Measurements of their areas in spectra of polymers prepared with free radical and anionic … Abstract Spectra of methyl methacrylate polymers in chloroform solution at 90° show three α‐methyl proton peaks. Measurements of their areas in spectra of polymers prepared with free radical and anionic initiators indicate that these may be attributed to (1) isotactic sequences (ddd or lll), (2) syndiotactic sequences (ldl or dld) , and (3) heterotactic sequences (ldd, dll, ddl , or lld) . The conformation and stereochemical configuration of the chains may thus be examined with considerable discrimination; degrees of regularity, block sizes, etc. appear to be determinable. Free radical polymers of methyl methacrylate are found to be predominantly syndiotactic, whereas those produced with anionic initiators. such as n ‐butyllithium, are shown to be predominantly isotactic, in agreement with the findings of others. The backbone methylene resonance also shows striking changes with chain stereochemical configuration, such as to provide absolute, independent confirmation of the assigned structures.

Calculation of NMR and EPR Parameters

2004-05-25

Martin Kaupp , Michæl Bühl , Vladimir G. Malkin

Abnormally Long O–O Bond in trans-HOON: An Exemplary Charge-Shift Bond

2025-06-25

Huaiyu Zhang , Jia Wei , Rui Ma +3 more | The Journal of Physical Chemistry A

Nitrous acid (HONO) plays a significant role in atmospheric and combustion chemistry. While extensive attention has been devoted to the study of HONO, its isomer (HOON) has remained relatively unexplored … Nitrous acid (HONO) plays a significant role in atmospheric and combustion chemistry. While extensive attention has been devoted to the study of HONO, its isomer (HOON) has remained relatively unexplored until recent experimental and theoretical analyses revealed its unusually long and weak O-O bond. In contrast, its sulfur-substituted analogue, HOSN, exhibits a normal O-S bond. Here, we explored the intriguing bonding nature of trans-HOXN (X = O, S) from the perspective of the ab initio valence bond (VB) theory in order to elucidate the different behaviors of the O-O and O-S bonds therein. Our results demonstrated that the bonding in trans-HOON can be described as a three-center four-electron charge-shift bond, where the ON moiety most closely resembles nitric oxide, with some nitrene characters. Since the π bond in ON is a dative bond resulting from one lone pair on the oxygen atom, the accumulated negative charge on N enhances the hyperconjugation from the nitrogen lone pair of σ symmetry to the σ*O-O orbital. Ultimately, it is the enhanced hyperconjugative interaction that plays a dominating role in the elongation and weakening of the O-O bond. In contrast, trans-HOSN is characterized as a two-center two-electron charge-shift bond. Compared with H2O2 which takes a skew geometry, both trans-HOXN (X = O, S) prefer a planar geometry. While geometric relaxation provides the primary stabilizing force for trans-HOON, the planarity of trans-HOSN arises dominantly from the conjugation and hyperconjugation effects.

Small Protein Domains as Potential Spin Labels for In Vitro, Cellular, and Light-Induced Dipolar EPR Spectroscopy

2025-06-23

Andreas Günter , Susanna Ciuti , Lukas Denkhaus +7 more | Journal of the American Chemical Society

This study explores the potential of small light-oxygen-voltage (LOV) domains for utilization as protein spin labels in different dipolar EPR spectroscopy methods. The distinctive photochemical properties of selected LOV domain … This study explores the potential of small light-oxygen-voltage (LOV) domains for utilization as protein spin labels in different dipolar EPR spectroscopy methods. The distinctive photochemical properties of selected LOV domain variants are exploited to generate a variety of (meta)stable flavin mononucleotide (FMN) radicals upon blue light absorption. Three different radicals, FMN·-, FMNH·, and an FMN-methionine radical, and an excited FMN triplet species, were generated. The FMN radicals were generated in LOV single domains and two model LOV1-LOV2 fusion proteins, and the latter proteins demonstrated that simple and effective orthogonal spin labeling can be performed. Subsequently, dipolar EPR experiments were conducted in aqueous solution and in cells with and without additional light excitation, in order to measure the distances between the FMN cofactor radicals, and to infer the structure and dynamics of the LOV domain proteins. Interestingly, all LOV1-LOV2 fusion proteins exhibit defined but largely distinct distances. This can be attributed to two factors: the respective LOV domains have different interactions with each other, and the presence of neutral FMN radicals leads to dimerization of the LOV1 domains. Nevertheless, using LOV domains as genetically encoded spin labels could offer numerous advantages. As a true molecular biology concept, labeling and measurements can be performed in any accessible cell type using light as the only stimulus. Additionally, the various paramagnetic FMN states enable the measurement of distances between two radicals, as well as between a radical and a triplet state.

Decoherence of Hyperfine Coupled 19F and 1H Nuclei in Gadolinium(III) Model Complexes

2025-06-21

Alexey V. Bogdanov , Veronica Frydman , Xun‐Cheng Su +1 more | The Journal of Physical Chemistry B

Time-domain electron-nuclear double resonance (ENDOR) was employed to determine the nuclear decoherence rate (1/T2n) of fluorine nuclei in four model Gd(III) chelates of varying structures, over the temperature range 3.7-10 … Time-domain electron-nuclear double resonance (ENDOR) was employed to determine the nuclear decoherence rate (1/T2n) of fluorine nuclei in four model Gd(III) chelates of varying structures, over the temperature range 3.7-10 K. These complexes are derivatives of Gd(III) spin labels used in 19F-ENDOR distance measurements, which emerged as an efficient method for measuring short-range distances in biomolecules. It was found that the 19F decoherence originates primarily from electron spin flips, following the relationship T2n ≈ 2T1e, where T1e is the Gd(III) spin-lattice relaxation time. The obtained 19F decoherence rates (1-8 kHz) indicate that the intrinsic 19F-ENDOR line widths are small relative to other broadening contributions, and therefore do not significantly limit distance resolution of 19F ENDOR. Proton decoherence measurements were carried out for comparison, revealing two 1H populations with different relaxation rates; the faster component was sensitive to the matrix protonation and attributed to dipolar flip-flops among weakly coupled protons, a mechanism absent for 19F. These results elucidate key factors affecting ENDOR resolution and provide new insights into the nuclear spin relaxation mechanism in paramagnetic systems.

Revealing the Impact of g-Tensor Anisotropy on the Charge Recombination in Donor–Acceptor Dyads Under High Magnetic Fields

2025-06-18

Paul Mentzel , Luca Gerhards , Denise Koppenhöfer +6 more | Journal of the American Chemical Society

Four new donor-acceptor dyads, featuring triarylamine donors and perylene diimide acceptors, were synthesized to investigate the influence of the g-tensor on the magnetic field-dependent spin dynamics of the resulting radical … Four new donor-acceptor dyads, featuring triarylamine donors and perylene diimide acceptors, were synthesized to investigate the influence of the g-tensor on the magnetic field-dependent spin dynamics of the resulting radical pairs. These pairs are characterized by the exchange interaction 2J being larger than the effective isotropic hyperfine coupling aeff. To control the isotropic g-factor and g-tensor anisotropy of the radical anion generated via photoinduced electron transfer, the perylene diimides were functionalized with phenyl chalcogen ethers. In dyads containing oxygen, sulfur, and selenium ether substituents, not only was the characteristic 2J-resonance observed but also a pronounced high-field effect in the charge recombination kinetics, extending up to B = 10 T. Quantum dynamics simulations based on the stochastic Liouville equation revealed that this effect is primarily driven by g-tensor anisotropy-induced relaxation, which increases along the chalcogen ether series. Additionally, we derived an exact analytical solution describing the impact of g-factor differences and g-tensor anisotropy on spin relaxation in the high-field limit. These findings highlight the critical role of g-tensor-induced relaxation in radical pairs at high magnetic fields, offering new insights for the molecular design of materials with potential applications in quantum information science, where incoherent relaxation processes should be avoided.

Observation of spectrally continuous resonance enhancement by mode coalescence

2025-06-18

Nitzan Shani , Amit Kumar Shakya , Fan Cheng +3 more | Optics Express

Proton transfer through a charged conduit in respiratory complex I - long range effects and conformational gating

2025-06-17

Luka Simsive , Oleksii Zdorevskyi , Vivek Sharma | bioRxiv (Cold Spring Harbor Laboratory)

Energy coupling processes in respiratory complex I - a large redox-driven proton pump in the inner mitochondrial membrane - remains one of the most enigmatic problems in modern bioenergetics. Recent … Energy coupling processes in respiratory complex I - a large redox-driven proton pump in the inner mitochondrial membrane - remains one of the most enigmatic problems in modern bioenergetics. Recent high-resolution cryo EM structures of complex I revealed extensive hydration in the interior of the protein, including the buried E channel, which is an acidic charged conduit that bridges the quinone binding cavity with the extended membrane domain of the enzyme. Despite the general agreement that E channel participates in proton transfer, absence of proton density in the cryo-EM maps pose a significant challenge to develop viable models of proton pumping. By adhering to the hypothesis that E channel catalyzes transfer of proton(s) from the quinone binding cavity to the membrane-bound proton pumping site(s), we performed hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations using the ~2.4 A cryo-EM structure of mitochondrial complex I from Mus musculus. By combining classical atomistic MD simulations with the hybrid QM/MM free energy calculations, we identify several energetically favorable Grotthuss-competent proton transfer paths in the E channel region. As part of the long-range coupling in complex I, our calculations show that protonation of a single acidic amino acid residue in the distal MM surroundings can alter the dynamics of proton transfer in the E channel region. Additionally, we pinpoint the gating function of a highly conserved tyrosine residue in the E channel, which undergoes conformational flipping to establish an energetically favorable proton transfer path. In the context of the redox-coupled proton pumping mechanism of complex I, we propose a stepping-stone model of proton transfer through the E channel.

Domain-Specific EPR Spectroscopy to Monitor Facilitated Dissociation of a DNA-Transcription Factor Complex

2025-06-17

Ludovica M. Epasto , Mahdi Khalil , Giuseppe Sicoli +1 more | bioRxiv (Cold Spring Harbor Laboratory)

Abstract Transcription factors (TFs) regulate gene expression by interacting with specific DNA sequences in various manners, yet the mechanisms by which they dissociate from high-affinity DNA sites under physiological conditions … Abstract Transcription factors (TFs) regulate gene expression by interacting with specific DNA sequences in various manners, yet the mechanisms by which they dissociate from high-affinity DNA sites under physiological conditions remain not completely understood. Here, we employ continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy in native solution state to resolve, with domain-level precision, the dissociation dynamics of the MYC-associated factor X (MAX) from its cognate EBOX DNA motif. Site-directed spin labeling reveals that in the absence of DNA, MAX undergoes a stepwise dissociation process—beginning with melting of the N-terminal disordered region (NTD), followed by the helix-loop-helix (HLH) domain, and culminating in leucine zipper (LZ) dimer dissociation. DNA binding reorganizes this process into a cooperative all-or-none transition, wherein destabilization of the LZ triggers concerted collapse of the entire trimeric MAX:MAX–DNA complex. Strikingly, the addition of a disordered BRCA1 fragment (residues 219–504) disrupts this cooperativity by selectively destabilizing DNA contacts in the NTD and HLH regions, without perturbing the LZ. This results in facilitated dissociation via competitive DNA binding, observed here directly at domain-level resolution. Our findings establish EPR spectroscopy as a uniquely sensitive tool for dissecting TF–DNA dynamics under physiological conditions, offering mechanistic insight into regulated unbinding processes inaccessible to ensemble methods.

Road Map for the Use of Electron Spin Resonance Spectroscopy in the Study of Functionalized Magnetic Nanoparticles

2025-06-16

Tomasz Kubiak , Bernadeta Dobosz | Materials

Electron paramagnetic resonance (EPR) spectroscopy is gaining increasing recognition in research on various nanostructures. In the case of iron oxide nanoparticles, EPR measurements offer the possibility of determining the magnetic … Electron paramagnetic resonance (EPR) spectroscopy is gaining increasing recognition in research on various nanostructures. In the case of iron oxide nanoparticles, EPR measurements offer the possibility of determining the magnetic phase and the exact type (Fe3O4, γ-Fe2O3, α-Fe2O3, or a combination) of the core material. Furthermore, the EPR technique enables the study of relaxation processes, estimation of the effective and surface anisotropy constants, and assessment of the influence of sample aging on the magnetic properties of nanoparticles. The scope of the information obtained can be further expanded by utilizing spin labeling of polymer-coated nanoparticles. By analyzing the signals from the attached nitroxide, one can determine certain properties of the coating and its interactions with the environment (e.g., body fluids, cells, tissues) and also perform imaging of nanoparticles in various media. In some cases, EPR can help monitor the encapsulation of active substances and their release processes. Unfortunately, despite the enormous potential, not all of the possibilities offered by EPR are routinely used in nanoscience. Therefore, the present article aims not only to present the current applications and existing trends but also to indicate directions for future EPR research, constituting a road map.

Effects of Methionine and Glutathione on Acute Ototoxicity Induced by Amikacin and Furosemide in an Animal Model of Hearing Threshold Decrease

2025-06-15

Marek Zadrożniak , Marcin Szymański , Jarogniew J. Łuszczki | Biomedicines

Background/Objectives: Aminoglycoside antibiotics and loop diuretics are still the main causes of hearing loss in patients, and no specific prevention is available for this drug-induced ototoxicity. The aim of this … Background/Objectives: Aminoglycoside antibiotics and loop diuretics are still the main causes of hearing loss in patients, and no specific prevention is available for this drug-induced ototoxicity. The aim of this study was to compare the protective effects of methionine (MET) and glutathione (GLUT) in terms of the reduction in ototoxicity induced by mixtures of amikacin (AMI, an aminoglycoside antibiotic) and furosemide (FUR, a loop diuretic) in a mouse model in which the hearing threshold decreased by 20% and 50%, respectively. Methods: To compare the otoprotective effects of MET and GLUT on AMI- and FUR-induced ototoxicity in mice, an isobolographic transformation of interactions was applied. Results: MET, but not GLUT, mitigated the AMI- and FUR-induced hearing threshold changes in mice. Additionally, MET exerted an antagonistic interaction with a combination of FUR+AMI, as the hearing threshold decreased by 50%, and an additive interaction, with a tendency toward antagonism in the model of hearing threshold decreased by 20%. In contrast, GLUT exerted only additive interactions when combined with FUR+AMI for both variant hearing thresholds decreased by 20% and 50%, respectively. Only MET could be a potential otoprotective drug in further prevention of hearing loss induced by AMI and FUR. Conclusions: MET is superior to GLUT in mitigating AMI- and FUR-induced hearing threshold decreases in mice. MET could be recommended as an otoprotectant in the prevention of hearing loss in patients receiving AMI and FUR.

Experience-dependent sharp-wave ripple deficits in an Alzheimer's disease mouse model

2025-06-13

Paulina Schnur , Nicole Byron , Symeon Gerasimou +4 more | bioRxiv (Cold Spring Harbor Laboratory)

Amyloid pathology is a hallmark of Alzheimer's disease (AD). Hippocampal sharp-wave ripples (SWRs) play a role in memory consolidation and are impaired in various AD mouse models. However, it remains … Amyloid pathology is a hallmark of Alzheimer's disease (AD). Hippocampal sharp-wave ripples (SWRs) play a role in memory consolidation and are impaired in various AD mouse models. However, it remains unclear how experience affects SWRs and how extrinsic signals contribute to SWR generation in AD. Here, by combining behavioral, in vivo electrophysiological and fiber photometry approaches, we show that an experience-dependent increase in hippocampal SWRs is disrupted in male and female 5xFAD mice. In wild-type mice, SWRs during non-rapid eye movement sleep (NREMS) increased after exploring a novel environment and the SWR rate gradually decreased with NREMS episodes and multiple behavioral sessions. However, 5xFAD mice did not show such experience-dependent SWR rate changes. A similar deficit was observed after a novel object recognition test. On the other hand, sleep spindles were intact in 5xFAD mice under all conditions. Since deficits in basal forebrain cholinergic neurons have been implicated in 5xFAD mice and SWRs are regulated by hippocampal cholinergic tone, we examined if hippocampal cholinergic signals could explain experience-dependent SWR deficits. Using fiber photometry and expressing a genetically encoded acetylcholine (ACh) sensor in the hippocampus, we found that ACh dynamics in the hippocampus were intact in 5xFAD mice across sleep-wake cycles, including NREMS, while we also found a negative correlation of infraslow cortical signal power dynamics with hippocampal ACh signals during NREMS regardless of genotypes. These results suggest that experience-dependent SWR deficits stem from non-cholinergic pathological changes.

Design and characterization of a tunable open TE011 resonator for Q-band pulse EPR experiments

2025-06-08

Kyle Jorgensen , Alexey Silakov | Journal of Magnetic Resonance

Effect of Hydrostatic Pressure on the g Tensor and Hyperfine Coupling Constants of the Nitroxide Radical Characterized by Ab Initio Calculations

2025-06-05

Ana Gurgenidze , Anna I. Krylov , Susumu Takahashi | The Journal of Physical Chemistry Letters

We present a computational study characterizing the effect of hydrostatic pressure on magnetic spin parameters that are used to analyze the electron paramagnetic resonance (EPR) spectra. Site-directed spin labeling (SDSL) … We present a computational study characterizing the effect of hydrostatic pressure on magnetic spin parameters that are used to analyze the electron paramagnetic resonance (EPR) spectra. Site-directed spin labeling (SDSL) in combination with EPR spectroscopy is a powerful tool for investigating the structure and dynamics of biological molecules. In studies using SDSL-based EPR spectroscopy, it is essential to know the spin parameters, such as the g factor and the hyperfine constants, precisely. However, the experimental characterization of these spin parameters under extreme conditions is often challenging. We report quantum chemistry calculations of g tensors and hyperfine coupling tensors (A tensors) for the nitroxide radical spin label in the pressure range of 0-15 GPa. The hydrostatic pressure causes structural changes, which, in turn, result in linear changes of the g and A tensors. The observed linear dependence of the g and A tensors suggests that these quantities can serve as reporters of local pressure in complex environments. The corresponding simulated EPR spectra at 9 and 230 GHz reveal that the changes of the EPR spectrum are more pronounced in the former. Our results indicate that the computational approach can address the challenge of determining magnetic spin parameters under extreme conditions, such as under high hydrostatic pressure.

Nitrite attenuates oxidative burst in brain tissue following ischemia-reperfusion via modulation of complex I-linked reversed electron transfer

2025-05-30

Cândida Dias , Cátia F. Lourenço , João Laranjinha +1 more | Free Radical Biology and Medicine

New insights in “Catecholaldehyde hypotehsis” in Parkinson’s disease: The glutathionylated-aldehyde reductases as crosslink between 4-hydroxy-2-nonenal and 3,4-dihydroxyphenylacetaldehyde detoxification

2025-05-30

Rosella Rotondo , Ivana Baldassarre , Lorenza Leonardi +2 more | Free Radical Biology and Medicine

Glutathione peroxidases as redox sensors in plants

2025-05-30

Thomaz Stumpf Trenz , Sophie Hendrix , Camila Luiza Delaix +5 more | Free Radical Biology and Medicine

Reduced basis method for few-body bound state emulation

2025-05-30

NULL AUTHOR_ID | Physical review. C

Analytic continuation in coupling constant applied to two-proton emitters

2025-05-29

NULL AUTHOR_ID | Physical review. C

5.1 Ergebnisse der Resonanzcodierung

2025-05-28

Electron-Molecule Elastic Scattering: A Study Using the Configuration Interaction Method and Model Potentials

2025-05-27

Maria Cristina Costa dos Santos , Aline de Almeida Simões , Leandro C. Santos +2 more

Abstract In this work, the theory of electron-molecule scattering and the influence of model potentials on improving differential cross-section (DCS) results are addressed. Unlike the treatment that uses the Hartree-Fock … Abstract In this work, the theory of electron-molecule scattering and the influence of model potentials on improving differential cross-section (DCS) results are addressed. Unlike the treatment that uses the Hartree-Fock (HF) approximation, the Configuration Interaction method (CI) is employed to treat the target and obtain the static and exchange potentials. Using CI, the influence and behavior of the correlation-polarization model potential proposed by Padial and Norcross, as well as the potential called free parameter B, are analyzed in the calculation of DCS for elastic e–N 2 scattering, with in the energy range of 5 eV to 20 eV. By comparing the results with experimental data and other theoretical results, it is shown that, although the Padial and Norcross potential is widely used in conjunction with HF and leads to satisfactory results in this case, its use with the CI method leads to unsatisfactory results, particularly for low scattering angles. On the other hand, the free parameter B potential appears to be more appropriate for including polarization effects, with no need for corrections related to electron correlation. The Lippmann-Schwinger equation, applied to scattering theory, is solved using the iterative variational method of Schwinger (SVIM), and in the CI calculation, single and double excitations are included.

Comparative Analysis of Förster Resonance Energy Transfer (FRET) in Spherical and Planar Geometries

2025-05-23

Onur İnan

This research explores the influence of geometric configuration on Förster Resonance Energy Transfer (FRET) efficiency. Specifically, it compares spherical arrangements (relevant to structures like nanoparticles) with planar arrangements (found in … This research explores the influence of geometric configuration on Förster Resonance Energy Transfer (FRET) efficiency. Specifically, it compares spherical arrangements (relevant to structures like nanoparticles) with planar arrangements (found in systems like cell membranes). A key goal is to clarify the interplay between FRET efficiency, inter-molecular distances, and the characteristic Förster distance. By employing both mathematical models and visual representations, the study seeks to provide a detailed understanding of how FRET operates under these distinct geometric constraints. The findings are intended to be broadly applicable, offering valuable insights for the design and analysis of FRET-based experimental work across diverse scientific disciplines.

The Challenges and Strategies in Assessing Frequency of Micturitions

2025-05-23

Shunqi Chen , XiaoLi Wang , XiangKun Li +2 more

Modulation of Doxorubicin-Induced ROS Accumulation in Cardiomyocytes Using Ibuprofen-Conjugated Synthetic Lipids as Carriers

2025-05-23

Soumya Saroj , Akshaya Kana Veedu , Chandrasekhar Reddy U +3 more

Conjugation of an NSAID such as ibuprofen to the head group of oxanorbornane-based lipids and the use of their aggregates as carriers for doxorubicin (Dox) are discussed here. These conjugates … Conjugation of an NSAID such as ibuprofen to the head group of oxanorbornane-based lipids and the use of their aggregates as carriers for doxorubicin (Dox) are discussed here. These conjugates were characterized by various spectroscopic techniques, including 2D-NMR, and insights into their assembly were gathered through PXRD, AFM, SEM, DLS, and qNano techniques. Free lipids as well as their formulations (lipid:cholesterol:Dox in a 3:1.5:2 molar ratio) showed a high tendency to form solid lipid particles, which was verified by TEM analysis. The presence of the ibuprofen unit led to an increase in interlipid spacing and a characteristic change in their packing. Active loading through a pH gradient allowed us to achieve high drug entrapment and a controlled release profile. The formulation AT3.3, prepared by this method, showed a Dox entrapment of ∼90%, with a controlled release of ∼18% by the end of 24 h; only ∼66% of the entrapped Dox was released by the end of 5 days. Cytotoxicity studies in NIH3T3 cells and hemolytic assay results showed that these lipids and their formulations have a good safety profile. Results from flow cytometry experiments in A549 cells revealed that the formulation AT3.3 induces effects similar to free Dox, with cell cycle arrest predominantly at the S phase and G2/M phase. At the same time, the response from the blank formulation was comparable to that of the control. Confocal microscopy studies in NIH3T3 and A549 cells showed that free Dox gets localized mainly in the nucleus, while the use of the carrier (AT3.3) causes significant localization of the drug on the cytoplasmic side as well. ROS induction due to free Dox and its formulation (AT3.3) in cardiomyocytes and A549 cells was also compared, and the results showed a protective effect in cardiomyocytes when using this formulation.

Evaluating the effect of 1H decoupling on 19F longitudinal relaxation and signal line shape in 5-Fluorotryptophan

2025-05-21

Yuki Toyama , Koh Takeuchi , Ichio Shimada

Amide proton transfer-weighted magnetic resonance imaging for the evaluation of testicular spermatogenic function: a preliminary study

2025-05-20

Guanglei Tang , Shu‐Lin Ma , Wenhao Fu +3 more

To determine the amide proton transfer-weighted (APTw) imaging features in testes with age, and to assess the feasibility of APTw magnetic resonance imaging (MRI) in assessing testicular spermatogenic function. A … To determine the amide proton transfer-weighted (APTw) imaging features in testes with age, and to assess the feasibility of APTw magnetic resonance imaging (MRI) in assessing testicular spermatogenic function. A total of 23 male patients with clinically confirmed hypospermatogenesis caused by epididymo-orchitis were included in the case group (group A) and another 93 men (age range, 20-80 years) were included in the control group. The control group was divided into four subgroups: group B1 (20-34 years, n = 25), group B2 (35-49 years, n = 23), group B3 (50-64 years, n = 21), and group B4 (65-80 years, n = 24). All participants underwent 3.0T MRI scan, and the APT signal intensity (SI) and apparent diffusion coefficient (ADC) value of each testis were examined. The ADC and APT SI were independently measured by two radiologists blinded to clinical data, and average values were calculated. A power analysis was conducted to determine the required sample size. APT SI was negatively correlated with age (r = -0.510, P < 0.001), whereas ADC was positively correlated with age (r = 0.317, P = 0.006). The APT SI was significantly higher in group A (1.77 ± 0.41) than in group B1 (1.43 ± 0.21), group B2 (1.37 ± 0.31), group B3 (1.30 ± 0.35), and group B4 (1.20 ± 0.35) (all P < 0.01). The ADC was significantly higher in group A [(0.549 ± 0.091) × 10-3 mm2/s] compared with group B1 [(0.449 ± 0.047) × 10-3 mm2/s], group B2 [(0.475 ± 0.022) × 10-3 mm2/s], and group B3 [(0.488 ± 0.051) × 10-3 mm2/s] (all P < 0.01), whereas no statistically significant difference was found between group A and group B4 (P > 0.05). The APT SI of the normal testes decreased with age, whereas a significant elevation of APT SI was detected in patients with hypospermatogenesis caused by epididymo-orchitis. Hypospermatogenesis caused by degeneration or inflammation can be differentiated by APT quantity combined with ADC value.

ER‑Localized Ceramide Accumulation Due to Disrupted CERT Transport Contributes to Replicative Senescence

2025-05-19

Shweta Chitkara , Natasha Gozali , Andrey N. Kuzmin +4 more

Ceramides are bioactive sphingolipids that are central to cellular metabolism, signaling, and stress responses. Synthesized de novo in the endoplasmic reticulum (ER) through a tightly regulated enzymatic pathway, ceramides can … Ceramides are bioactive sphingolipids that are central to cellular metabolism, signaling, and stress responses. Synthesized de novo in the endoplasmic reticulum (ER) through a tightly regulated enzymatic pathway, ceramides can also be generated from hydrolysis complex sphingolipids, including hexosylceramides and sphingomyelins, and the salvage pathway. Their dynamic turnover across distinct cellular compartments results in spatial regulation of ceramide levels and its functional consequences. Mitochondrial ceramide accumulation, in particular, is a critical factor of apoptosis, where ceramides can disrupt membrane integrity. In addition to apoptosis, ceramides are implicated in replicative senescence. However, the mechanisms of ceramide regulation and function in senescence remain incompletely understood. In this study, we employed comparative lipid profiling, gene expression analysis and Raman spectroscopy to dissect sphingolipid regulatory networks that might be responsible for ceramide regulation and localization in senescence. We found that while both apoptotic and senescent cells exhibit elevated ceramide levels, only senescent cells display a marked depletion of sphingomyelins, especially very-long-chain species. Despite transcriptional upregulation of sphingomyelin synthase 1, enzymatic activity remained unchanged, suggesting impaired ceramide transport. Pharmacological inhibition of Ceramide Transfer Protein (CERT), the ER to Golgi ceramide transporter, phenocopied the senescent sphingolipid profile and enhanced senescence markers. Furthermore, Raman spectroscopy revealed ceramide accumulation at the ER but not the mitochondria in senescent cells, consistent with a non-apoptotic phenotype. Together, our results identify disrupted ceramide turnover to sphingomyelin as a key feature of senescence and highlight the importance of subcellular ceramide localization in determining cell fate.

Formation of Singlet Oxygen in Addition to Hydroxyl Radical via the Fenton Reaction

2025-05-17

Ryōsuke Shimizu , Haruki Watanabe , Sayaka Iida +2 more

We established an LC-MS/MS method for detecting uric acid oxidation metabolites to evaluate reactive oxygen and nitrogen species, as uric acid gives specific products. Parabanic acid was identified during attempts … We established an LC-MS/MS method for detecting uric acid oxidation metabolites to evaluate reactive oxygen and nitrogen species, as uric acid gives specific products. Parabanic acid was identified during attempts to detect hydroxyl radical-specific products in the Fenton reaction. As parabanic acid is a singlet oxygen-specific product of uric acid, this indicates the Fenton system, which is known for the generation of hydroxyl radicals, also forms singlet oxygen products. This notion was confirmed by replacing uric acid with tryptophan, which resulted in the formation of singlet oxygen-specific oxidation products (cis- and trans-WOOH) and their reductants, cis- and trans-WOH. Product amounts were reduced in a dose-dependent manner by the addition of the singlet oxygen quenchers sodium azide or 1,4-diazabicyclo[2.2.2]octane. Surprisingly, the estimated amount of singlet oxygen produced was 50- to 70-fold greater than that of hydroxyl radical, considering the quantum yield of the reaction between uric acid and singlet oxygen. The formation of singlet oxygen under anaerobic conditions suggested it was derived from hydrogen peroxide. The production of non-labeled parabanic acid, even in an 18O2 atmosphere or the presence of H218O, supported this hypothesis. These results confirmed that singlet oxygen was derived from hydrogen peroxide. The proposed mechanism of singlet oxygen formation is as follows. Two hydrogen peroxyl radicals formed by the reaction of hydrogen peroxide and ferric ion or hydroxyl radical are coupled to form a hydrogen tetraoxide, which subsequently decomposes to form singlet oxygen and hydrogen peroxide via a Russell-like mechanism. Finally, it was observed that significantly more singlet oxygen was generated in whole human blood compared with red blood cell-depleted blood during pseudo-inflammation initiated by lipopolysaccharide addition, suggesting that singlet oxygen formation was due to the Fenton reaction. Thus, the Fenton reaction may be a novel pathway for singlet oxygen production.

Investigating low-barrier hydrogen-bonded-assisted proton tunneling in Fujikurin A–D molecules: a computational study

2025-05-16

Luca Nanni

Nitroxides: Chemistry, Antioxidant Properties, and Biomedical Applications

2025-05-14

Krzysztof Gwoździński , Anna Pieniążek , Łukasz Gwoździński

Nitroxides are stable organic free radicals with a wide range of applications. They have found applications in chemistry, biochemistry, biophysics, molecular biology, and biomedicine as EPR/NMR imaging techniques. As spin … Nitroxides are stable organic free radicals with a wide range of applications. They have found applications in chemistry, biochemistry, biophysics, molecular biology, and biomedicine as EPR/NMR imaging techniques. As spin labels and probes, they are used in electron paramagnetic resonance (EPR) spectroscopy in the study of proteins, lipids, nucleic acids, and enzymes, as well as for measuring oxygen concentration in cells and cellular organelles, as well as tissues and intracellular pH. Their unique redox properties have allowed them to be used as exogenous antioxidants. In this review, we have discussed the chemical properties of nitroxides and their antioxidant properties. Furthermore, we have considered their use as radioprotectors and protective agents in ischemia/reperfusion in vivo and in vitro. We also presented other applications of nitroxides in protecting cells and tissues from oxidative stress and in protein studies and discussed their use in EPR/MRI.

Investigating the Conformational Diversity of the TMR-3 Aptamer

2025-05-12

Maximilian Gauger , Elke Duchardt‐Ferner , Anna‐Lena J. Halbritter +4 more

Aptamers are a class of in vitro selected small RNA motifs that bind a small-molecule ligand with high affinity and specificity. They are promising candidates for the regulation of gene … Aptamers are a class of in vitro selected small RNA motifs that bind a small-molecule ligand with high affinity and specificity. They are promising candidates for the regulation of gene expression in vivo and can aid in further understanding the interaction of RNA with small molecules and conformational changes that may occur upon ligand binding. The TMR-3 aptamer was selected via systematic evolution of ligands by exponential enrichment (SELEX) and binds the fluorophores tetramethylrhodamine (TMR) and 5-carboxy-tetramethylrhodamine (5-TAMRA) with nanomolar affinity. The three-dimensional structure of the TMR-3 aptamer complex with 5-TAMRA was previously determined using liquid-state NMR. By combining the existing NMR restraints with long-range PELDOR distance and orientation information, a broad structural ensemble was generated. From this broad ensemble, a subset of structures was selected by globally fitting orientation-selective PELDOR data from multiple frequency bands. The subensemble represents the conformational variety resulting from the dynamics of the complex. The overall structure of the three-way junction, previously reported by NMR experiments, is retained in the ensemble of the bound state and we were additionally able to characterize the fluctuation of the different stems of the aptamer. Furthermore, in addition to the ligand-bound state we could access the unbound state of the TMR-3 aptamer which was previously uncharacterized. The unbound state of the aptamer is much more structurally diverse, compared to the ligand-bound state. A significant fraction of the ensemble of the unbound state strongly resembles the ligand-bound state, indicating that the ligand-bound state is preformed, which further suggests a conformational-capture ligand-binding mechanism. Apart from the conformations that resemble the ligand-bound state, distinct conformational states which are not present in the presence of the ligand, were successfully identified.