RESUMO
Tunneling of methyl rotors coupled to an electron spin causes magnetic field independent electron spin echo envelope modulation (ESEEM) at low temperatures. For nitroxides containing alkyl substituents, we observe this effect as a contribution at the beginning of the Hahn echo decay signal occurring on a faster time scale than the matrix-induced decoherence. The tunneling ESEEM contribution includes information on the local environment of the methyl rotors, which manifests as a distribution of rotation barriers P(V3) when measuring the paramagnetic species in a glassy matrix. Here, we investigate the differences in tunneling behaviour of geminal methyl and ethyl group rotors in nitroxides while exploring different levels of theory in our previously introduced methyl quantum rotor (MQR) model. Moreover, we extend the MQR model to analyze the tunneling ESEEM originating from two different rotor types coupled to the same electron spin. We find that ethyl groups in nitroxides give rise to stronger tunneling ESEEM contributions than methyl groups because the difference between hyperfine couplings of their methyl protons better matches the tunneling frequency. The methyl rotors of both ethyl and propyl groups exhibit distributions at lower rotation barriers compared to geminal methyl groups. This is in good agreement with density functional theory (DFT) calculations of their rotation barriers and showcases that conformational flexibility impacts the hindrance of rotation. Using Monte-Carlo based fitting in combination with an identifiability analysis of the MQR model parameter space, we extract rotation barrier distributions for the individual rotor types in mixed-rotor nitroxides as well as identify which rotors dominate the observed tunneling contribution in the Hahn echo decay signal.
RESUMO
We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 × 106 M-1, is at least 1 order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase in the rotational correlation time, τrot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τrot and electron spin coherence time, Tm, along with the narrowing of interspin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4-9, compared to the common spin label such as MTSL, which is not affected by CB-7. Interspin distances of 3 nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating an undisturbed structure and conformation of the protein.
Assuntos
Proteínas , Água , Marcadores de Spin , Espectroscopia de Ressonância de Spin Eletrônica , Água/químicaRESUMO
The electron paramagnetic resonance (EPR) spectra of lanthanide(III) ions besides Gd3+ , bound to small-molecule and protein chelators, are uncharacterized. Here, the EPR properties of 7 lanthanide(III) ions bound to the natural lanthanide-binding protein, lanmodulin (LanM), and the synthetic small-molecule chelator, 3,4,3-LI(1,2-HOPO) ("HOPO"), were systematically investigated. Echo-detected pulsed EPR spectra reveal intense signals from ions for which the normal continuous-wave first-derivative spectra are negligibly different from zero. Spectra of Kramers lanthanide ions Ce3+ , Nd3+ , Sm3+ , Er3+ , and Yb3+ , and non-Kramers Tb3+ and Tm3+ , bound to LanM are more similar to the ions in dilute aqueous:ethanol solution than to those coordinated with HOPO. Lanmodulins from two bacteria, with distinct metal-binding sites, had similar spectra for Tb3+ but different spectra for Nd3+ . Spin echo dephasing rates (1/Tm ) are faster for lanthanides than for most transition metals and limited detection of echoes to temperatures below ~6 to 12â K. Dephasing rates were environment dependent and decreased in the order water:ethanol>LanM>HOPO, which is attributed to decreasing librational motion. These results demonstrate that the EPR spectra and relaxation times of lanthanide(III) ions are sensitive to coordination environment, motivating wider application of these methods for characterization of both small-molecule and biomolecule interactions with lanthanides.
RESUMO
We describe the synthesis, characterization, and application of an isotopologue of the trityl radical OX071, labeled with 13C at the central carbon (13C1). This spin probe features large anisotropy of the hyperfine coupling with the 13C1 (I = 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 in vivo measurement of tissue microviscosity by EPR.
Assuntos
Compostos de Tritil , Espectroscopia de Ressonância de Spin EletrônicaRESUMO
A perchlorotriarylmethyl tricarboxylic acid radical 99% enriched in 13C at the central carbon (13C1-PTMTC) was characterized in phosphate buffered saline solution (pH = 7.2) (PBS) at ambient temperature. Samples immobilized in 1:1 PBS:glycerol or in 9:1 trehalose:sucrose were studied as a function of temperature. Isotope enrichment at C1 creates a trityl that can be used to accurately measure microscopic viscosity. Understanding of the impact of the 13C hyperfine interaction on electron spin relaxation is important for application of this trityl in oximetry and distance measurements. The anisotropic 13C1 hyperfine couplings (Ax = Ay = 24 ± 2 MHz, Az = 200 ± 1 MHz) are larger than for the related 13C1-perdeuterated Finland trityl (13C1-dFT) and the g anisotropy (gx = 2.0013, gy = 2.0016, gz = 2.0042) is slightly larger than for 13C1-dFT. The tumbling correlation times (τR) for 13C1-PTMTC are 0.20 ± 0.02 ns in PBS and 0.40 ± 0.05 ns in 3:1 PBS:glycerol, which are shorter than for 13C1-dFT in the same solutions. T1 for 13C1-PTMTC is 3.5 ± 0.5 µs in PBS and 5.3 ± 0.4 µs in 3:1 PBS:glycerol, which are shorter than for 13C1-dFT due to faster tumbling, larger anisotropy of the 13C1 hyperfine, and about 30% larger contribution from the local mode. In immobilized samples T1 for 13C1-PTMTC is similar to that for 13C1-dFT and other trityls without chlorine or 13C1 substituents, indicating that the 13C1 and Cl substituents on the phenyl rings have little impact on T1. The temperature dependence of T1 was modeled with contributions from the direct, Raman, and local mode processes. Broadening of CW linewidths of about 0.6 G in fluid solution and about 2 G in rigid lattice is attributed to unresolved 35,37Cl hyperfine couplings.
RESUMO
PURPOSE: In dynamic nuclear polarization (DNP), the solution needs to form a glass to attain significant levels of polarization in reasonable time periods. Molecules that do not form glasses by themselves are often mixed with glass forming excipients. Although glassing agents are often essential in DNP studies, they have the potential to perturb the metabolic measurements that are being studied. Glycerol, the glassing agent of choice for in vivo DNP studies, is effective in reducing ice crystal formation during freezing, but is rapidly metabolized, potentially altering the redox and adenosine triphosphate balance of the system. METHODS: DNP buildup curves of 13 C urea and alanine with OX063 in the presence of trehalose, glycerol, and other polyol excipients were measured as a function of concentration. T1 and Tm relaxation times for OX063 in the presence of trehalose were measured by EPR. RESULTS: Approximately 15-20 wt% trehalose gives a glass that polarizes samples more rapidly than the commonly used 60%-wt formulation of glycerol and yields similar polarization levels within clinically relevant timeframes. CONCLUSIONS: Trehalose may be an attractive biologically inert alternative to glycerol for situations where there may be concerns about glycerol's glucogenic potential and possible alteration of the adenosine triphosphate/adenosine diphosphate and redox balance.
Assuntos
Glicerol , Compostos Heterocíclicos , Trealose , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância MagnéticaRESUMO
Synthesis of bis-spiro-oxetane and bis-spiro-tetrahydrofuran pyrroline nitroxide radicals relies on the Mitsunobu reaction-mediated double cyclizations of N-Boc protected pyrroline tetraols. Structures of the nitroxide radicals are supported by X-ray crystallography. In a trehalose/sucrose matrix at room temperature, the bis-spiro-oxetane nitroxide radical possesses electron spin coherence time, Tm ≈ 0.7 µs. The observed enhanced Tm is most likely associated with strong hydrogen bonding of oxetane moieties to the trehalose/sucrose matrix.
Assuntos
Elétrons , Furanos , Espectroscopia de Ressonância de Spin Eletrônica , Éteres Cíclicos , Óxidos de Nitrogênio , PirróisRESUMO
X-band (ca. 9â GHz) fluid solution rapid-scan electron paramagnetic resonance spectra are reported for radicals with multiline spectra and resolution of hyperfine lines as narrow as 30â mG. Highly-resolved spectra of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy, diphenylnitroxide, galvinoxyl, and perylene cation radical with excellent signal-to-noise are shown, demonstrating the capabilities of the rapid-scan technique to characterize very small, well-resolved hyperfine couplings. To acquire high resolution spectra the signal bandwidth must be less than the resonator bandwidth. Signal bandwidth is inversely proportional to linewidth and proportional to scan rate. Resonator bandwidth is inversely proportional to resonator Q. Proper selection of scan rate and resonator Q is needed to achieve resolution of closely-spaced narrow EPR lines.
Assuntos
Compostos Benzidrílicos/química , Óxidos N-Cíclicos/química , Óxidos de Nitrogênio/química , Perileno/química , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Estrutura MolecularRESUMO
Electron spin relaxation times T1 and Tm of Tb3+ and Tm3+ in 1:1 water:ethanol and of Tb3+ doped (2%) in crystalline La2(oxalate)3 decahydrate were measured between about 4.2 and 10 K. Both cations are non-Kramers ions and have J = 6 ground states. Echo-detected spectra are compared with CW spectra and with field-stepped direct-detected EPR spectra. Due to the strong temperature dependence of T1, measurements were not made above 10 K. Between about 4.2 and 6 K T1 is strongly concentration dependent between 1 and ~50 mM. T1 values at 4.2 K are in the µs range which is orders of magnitude faster than for 3d transition metals. Phase memory times, Tm, are less than 500 ns, which is short relative to values observed for 3d transition metals and organic radicals at 4 K. Tm is longer in the oxalate lattice which is attributed to the lower proton concentration in oxalate than in the organic solvent, which decreases nuclear spin diffusion. The rigidity of the crystalline lattice also may contribute to longer Tm.
RESUMO
Mycofactocin is a putative redox cofactor and is classified as a ribosomally synthesized and post-translationally modified peptide (RiPP). Some RiPP natural products, including mycofactocin, rely on a radical S-adenosylmethionine (RS, SAM) protein to modify the precursor peptide. Mycofactocin maturase, MftC, is a unique RS protein that catalyzes the oxidative decarboxylation and C-C bond formation on the precursor peptide MftA. However, the number, chemical nature, and catalytic roles for the MftC [Fe-S] clusters remain unknown. Here, we report that MftC binds a RS [4Fe-4S] cluster and two auxiliary [4Fe-4S] clusters that are required for MftA modification. Furthermore, electron paramagnetic resonance spectra of MftC suggest that SAM and MftA affect the environments of the RS and Aux I cluster, whereas the Aux II cluster is unaffected by the substrates. Lastly, reduction potential assignments of individual [4Fe-4S] clusters by protein film voltammetry show that their potentials are within 100 mV of each other.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Bactérias/genética , Catálise , Domínio Catalítico , Cisteína/química , Técnicas Eletroquímicas , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas Ferro-Enxofre/genética , Mycobacterium ulcerans/química , Oxirredução , S-Adenosilmetionina/metabolismo , Espectroscopia de MossbauerRESUMO
A 25 mm diameter 250 MHz crossed-loop resonator was designed for rapid scan electron paramagnetic resonance imaging. It has a saddle coil for the driven resonator and a fine wire, loop gap resonator for the sample resonator. There is good separation of E and B fields and high isolation between the two resonators, permitting a wide range of sample types to be measured. Applications to imaging of nitroxide, trityl, and LiPc samples illustrate the utility of the resonator. Using this resonator and a trityl sample the signal-to-noise of a rapid scan absorption spectrum is about 20 times higher than for a first-derivative CW spectrum.
RESUMO
The radical formed by reduction of 5-bromo-6-oxo-6-phenylhexyl methanesulfonate, an α-bromoketone, with SmI2 was spin trapped with 2-methyl-2-nitrosopropane. Electron paramagnetic resonance spectra of the spin adduct and the adduct formed in the analogous reaction with selectively deuterated substrate identify the radical intermediate in this SmI2 reduction as a carbon-centered radical. This result supports the proposal that the formation of reactive Sm-enolates arises from reduction of the carbon-bromine bond rather than a ketyl radical anion.
Assuntos
Iodetos/química , Compostos Nitrosos/química , Samário/química , Detecção de Spin , Ácidos Sulfônicos/química , Espectroscopia de Ressonância de Spin Eletrônica , OxirreduçãoRESUMO
Low frequency electron paramagnetic resonance imaging is a powerful tool to non-invasively measure the physiological status of tumors. Here, we report on the design and functionality of a rapid scan and pulse table-top imaging spectrometer based around an arbitrary waveform generator and 25mm cross-loop resonator operating at 700 MHz. Two and four-dimensional rapid scan spectral-spatial images are presented. This table-top imager is a prototype for future pre-clinical imagers.
RESUMO
Nature utilizes [FeFe]-hydrogenase enzymes to catalyze the interconversion between H2 and protons and electrons. Catalysis occurs at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated 2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize the nonprotein ligands of the H-cluster. These enzymes interact with HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe subcluster assembly. Prior characterization of HydF demonstrated the protein exists in both dimeric and tetrameric states and coordinates both [4Fe-4S]2+/+ and [2Fe-2S]2+/+ clusters [Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick, J. B. (2016) Biochemistry 55, 3514-3527]. Herein, electron paramagnetic resonance (EPR) is utilized to characterize the [2Fe-2S]+ and [4Fe-4S]+ clusters bound to HydF. Examination of spin relaxation times using pulsed EPR in HydF samples exhibiting both [4Fe-4S]+ and [2Fe-2S]+ cluster EPR signals supports a model in which the two cluster types either are bound to widely separated sites on HydF or are not simultaneously bound to a single HydF species. Gel filtration chromatographic analyses of HydF spectroscopic samples strongly suggest the [2Fe-2S]+ and [4Fe-4S]+ clusters are coordinated to the dimeric form of the protein. Lastly, we examined the 2Fe subcluster-loaded form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase activation. Together, the results indicate a specific role for the HydF dimer in the H-cluster biosynthesis pathway.
Assuntos
Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/química , S-Adenosilmetionina/química , Enxofre/química , Catálise , Domínio Catalítico , Clostridium/enzimologia , Espectroscopia de Ressonância de Spin Eletrônica , Hidrogenase/química , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Oxirredução , Conformação Proteica , S-Adenosilmetionina/metabolismo , Enxofre/metabolismoRESUMO
Tau fibrils are pathological aggregates that can transfer between neurons and then recruit soluble Tau monomers by template-assisted conversion. The propagation of different fibril polymorphs is thought to be a contributing factor to phenotypic diversity in Alzheimer disease and other Tauopathies. We found that a homogeneous population of Tau fibrils composed of the truncated version K18 (residues 244-372) gradually converted to a new set of fibril conformers when subjected to multiple cycles of seeding and growth. Using double electron-electron resonance (DEER) spectroscopy, we observed that the distances between spin labels at positions 311 and 328 in the fibril core progressively decreased. The findings were corroborated by changes in turbidity, morphology, and protease sensitivity. Fibrils that were initially formed under stirring conditions exhibited an increased fragility compared with fibrils formed quiescently after multiple cycles of seeding. The quiescently formed fibrils were marked by accelerated growth. The difference in fragility and growth between the different conformers explains how the change in incubation condition could lead to the amplification of a minor subpopulation of fibrils. Under quiescent conditions where fibril breakage is minimal, faster growing fibrils have a selective advantage. The findings are of general importance as they suggest that changes in selective pressures during fibril propagation in the human brain could result in the emergence of new fibril conformers with varied clinicopathological consequences.
Assuntos
Mutação , Conformação Proteica , Proteínas tau/química , Proteínas tau/genética , Sequência de Aminoácidos , Espectroscopia de Ressonância de Spin Eletrônica , Eletroforese em Gel de Poliacrilamida , Humanos , Microscopia Eletrônica , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Tauopatias/genética , Tauopatias/metabolismo , Proteínas tau/ultraestruturaRESUMO
We report the design, synthesis, and electron spin relaxation properties of hydrophilic tetracarboxylate ester pyrroline nitroxides 1 and 2, which serve as models in the search for new spin labels for DEER distance measurement at room temperature. The nitroxides are designed to have the methyl groups further away from the N-O spin site to decrease the inequivalent couplings of the unpaired electron to the methyl protons that shorten Tm at T > 70 K in currently used labels. The key step in the synthesis of 1 and 2 is the reaction of the dianion of pyrrole-1,2,5-tricarboxylic acid tert-butyl ester dimethyl ester with electrophiles such as methyl chloroformate and methyl bromoacetate. Structures of 1 and 2 are confirmed by X-ray crystallography. Studies of electron spin relaxation rates in rigid trehalose/sucrose matrices reveal approximately temperature independent values of 1/Tm for 1 and 2 up to about 160 K and modest temperature dependence up to 295 K, demonstrating that increasing the distance between the nitroxide moiety and methyl groups is effective in lengthening Tm at T > 70 K.
Assuntos
Ácidos Carboxílicos/química , Óxidos de Nitrogênio/química , Pirróis/química , Ácidos Carboxílicos/síntese química , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares , Estrutura Molecular , Óxidos de Nitrogênio/síntese química , Pirróis/síntese químicaRESUMO
The triarylmethyl radical OX063d24 is currently used for pulsed electron paramagnetic resonance oximetry at 250 MHz. Both 1/T 1 and 1/T 2 increase with increasing oxygen concentration. The dependence of 1/T 1 on probe concentration is smaller than for 1/T 2. To inform the selection of the optimum frequency for in vivo oximetry 1/T 1, 1/T 2 and signal-to-noise were measured as a function of frequency between 400 and 1000 MHz on a variable-frequency spectrometer with an adjustable-frequency cross-loop resonator. 1/T 1 and 1/T 2 decrease with increasing frequency and signal-to-noise increases with increasing frequency, which are all favourable for imaging at higher frequencies. However, depth of penetration of the radio frequency (RF) into an animal decreases with increasing frequency. Assuming that the RF loss in the animal to be studied determines the resonator Q, our results indicate that the optimum frequency for in vivo imaging will be determined by the desired depth of penetration in the tissue.
Assuntos
Elétrons , Oximetria/métodos , Compostos de Sulfidrila/química , Deutério/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Indenos/química , Ondas de Rádio , Razão Sinal-Ruído , Compostos de Tritil/químicaRESUMO
A copper X-band (9.22 GHz) cross loop resonator has been constructed for use with 4 mm sample tubes. The Q for the two resonators are 380 and 350, respectively. The resonator efficiency is about 1 G per square root of watt. Operation has been demonstrated with measurement of T1 by saturation recovery for samples of coal and an immobilized nitroxide radical.
RESUMO
Resonators for preclinical electron paramagnetic resonance imaging have been designed primarily for rodents and rabbits and have internal diameters between 16 and 51 mm. Lumped circuit resonators include loop-gap, Alderman-Grant, and saddle coil topologies and surface coils. Bimodal resonators are useful for isolating the detected signal from incident power and reducing dead time in pulse experiments. Resonators for continuous wave, rapid scan, and pulse experiments are described. Experience at the University of Chicago and University of Denver in design of resonators for in vivo imaging is summarized.
RESUMO
In vivo oximetry by pulsed electron paramagnetic resonance is based on measurements of changes in electron spin relaxation rates of probe molecules, such as the triarylmethyl radicals. A series of experiments was performed at frequencies between 250 MHz and 1.5 GHz to assist in the selection of an optimum frequency for oximetry. Electron spin relaxation rates for the triarylmethyl radical OX063 as a function of radical concentration, salt concentration, and resonance frequency were measured by electron spin echo 2-pulse decay and 3-pulse inversion recovery in the frequency range of 250 MHz-1.5 GHz. At constant OX063 concentration, 1/T1 decreases with increasing frequency because the tumbling dependent processes that dominate relaxation at 250 MHz are less effective at higher frequency. 1/T2 also decreases with increasing frequency because 1/T1 is a significant contribution to 1/T2 for trityl radicals in fluid solution. 1/T2-1/T1, the incomplete motional averaging contribution to 1/T2, increases with increasing frequency. At constant frequency, relaxation rates increase with increasing radical concentration due to contributions from collisions that are more effective for 1/T2 than 1/T1. The collisional contribution to relaxation increases as the concentration of counter-ions in solution increases, which is attributed to interactions of cations with the negatively charged radicals that decrease repulsion between trityl radicals. The Signal-to-Noise ratio (S/N) of field-swept echo-detected spectra of OX063 were measured in the frequency range of 400 MHz-1 GHz. S/N values, normalized by âQ, increase as frequency increases. Adding salt to the radical solution decreased S/N because salt lowers the resonator Q. Changing the temperature from 19 to 37 °C caused little change in S/N at 700 MHz. Both slower relaxation rates and higher S/N at higher frequencies are advantageous for oximetry. The potential disadvantage of higher frequencies is the decreased depth of penetration into tissue.