Advanced Electron Paramagnetic Resonance Techniques Research Articles

Investigation of resonance-stabilized radicals associated with soot particle inception using advanced electron paramagnetic resonance techniques

In order to tackle the climate emergency, it is imperative to advance cleaner technologies to reduce pollutant emission as soot particles. However, there is still a lack of complete understanding of the mechanisms responsible for their formation. In this work, we performed an investigation devoted to the study of persistent radicals potentially involved in the formation of soot particles, by continuous wave and pulsed electron paramagnetic resonance. This work provides experimental evidence of the presence in nascent soot of highly branched, resonance-stabilized aromatic radicals bearing aliphatic groups, linked together by short carbon chains, and reinforced by non-covalent π-π interactions. These radicals appear to be highly specific of nascent soot and quickly disappear with the increasing soot maturity. Their presence in nascent soot could represent an underestimated health risk factor in addition to the already well documented effect of the high specific surface and the presence of harmful adsorbates.

A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy

A novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytical techniques such as Electron Paramagnetic Resonance (EPR) and solid state Dynamic Nuclear Polarization (DNP). The necessary spin-label was synthesized and inserted into the disulfide bridge of eptifibatide via reductive followed by insertion by a double Michael addition under physiological conditions. This procedure is universally applicable for disulfide containing biomolecules and is expected to preserve their tertiary structure with minimal change due to the small size of the label and restoring of the previous disulfide connection. HPLC and MS analysis show the successful introduction of the spin label and EPR spectroscopy confirms its activity. DNP-enhanced solid state NMR experiments show signal enhancement factors of up to 19 in 13C CP MAS experiments which corresponds to time saving factors of up to 361. This clearly shows the high potential of our new spin labeling strategy for the introduction of site selective radical spin labels into biomolecules and biosolids without compromising its conformational integrity for structural investigations employing solid-state DNP or advanced EPR techniques.

Extending electron paramagnetic resonance to nanoliter volume protein single crystals using a self-resonant microhelix.

Electron paramagnetic resonance (EPR) spectroscopy on protein single crystals is the ultimate method for determining the electronic structure of paramagnetic intermediates at the active site of an enzyme and relating the magnetic tensor to a molecular structure. However, crystals of dimensions typical for protein crystallography (0.05 to 0.3mm) provide insufficient signal intensity. In this work, we present a microwave self-resonant microhelix for nanoliter samples that can be implemented in a commercial X-band (9.5 GHz) EPR spectrometer. The self-resonant microhelix provides a measured signal-to-noise improvement up to a factor of 28 with respect to commercial EPR resonators. This work opens up the possibility to use advanced EPR techniques for studying protein single crystals of dimensions typical for x-ray crystallography. The technique is demonstrated by EPR experiments on single crystal [FeFe]-hydrogenase (Clostridium pasteurianum; CpI) with dimensions of 0.3 mm by 0.1 mm by 0.1 mm, yielding a proposed g-tensor orientation of the Hox state.

Analysis of Spin Probe Viability for Protein Structure Investigation Using Advanced EPR Techniques

An accurate depiction of protein structure and its mobility is necessary to understanding protein function and many methods exist to determine this. Obtaining protein structural characteristic by advanced electron paramagnetic resonance (EPR) spectroscopy techniques like double electron‐electron resonance (DEER) is emerging as a powerful technique that is complementary to other well established structural characterization tools. Such studies most commonly require the ligation of “spin labels” to different sites on the protein, often by a site‐specific reaction between a stable molecule containing a nitroxide radical and an amino acid residue. We seek to develop a robust set of tools for predicting structures of hard‐to‐characterize proteins by combining the distance information obtained from advanced EPR techniques with mobility information obtained from room temperature continuous wave EPR (CW EPR). Many methods of spin‐labeling a protein exist; however, the viability of these specific approaches for performing structural determination techniques like DEER has not yet been explored. The most common spin‐labeling methodology conjugates (1‐oxyl‐2,2,5,5‐tetramethylpyrroline‐3‐methyl) methanethiosulfonate (MTSSL) to a protein via a disulfide bond with a cysteine residue. To achieve this conjugation site‐specifically, all native surface‐located cysteine residues must be removed, which may not be desirable from a structural integrity standpoint. Utilizing unnatural amino acids can alleviate this issue and the spin‐label can be added to the protein via techniques such as click chemistry, a rapid cycloaddition that can occur at physiological conditions. In this work, we compare how different spin labeling techniques perform from an experimental point of view. We seek, overall, to determine a powerful and easy‐to‐use protocol for structural determination. Here, we use T4 Lysozyme as a well‐characterized protein model and compare 4‐alkyl TEMPO installed on the unnatural amino acid p‐‐azidophenylalanine (pAzF) with the aforementioned MTSSL/cysteine label. For this purpose, we use a cysteine‐free version of T4 Lysozyme and genetically encode either cysteine or pAzF to specific positions. The results presented focus on the structural rigidity of the spin labels, a key factor defining the flexibility of certain areas of the protein and the certainty of the distance determination. Moreover, as different spin probes display different spectroscopic characteristics, each spin label is evaluated in terms of DEER sensitivity and efficiency. Finally, we compare different advanced EPR techniques (DEER, DQC, relaxation enhancement) to determine the most robust in distance predictions.Support or Funding InformationThe work is funded by the Pennsylvania State University startup fund of Alexey Silakov.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The cyanide ligands of [FeFe] hydrogenase: pulse EPR studies of (13)C and (15)N-labeled H-cluster.

The two cyanide ligands in the assembled cluster of [FeFe] hydrogenase originate from exogenous l-tyrosine. Using selectively labeled tyrosine substrates, the cyanides were isotopically labeled via a recently developed in vitro maturation procedure allowing advanced electron paramagnetic resonance techniques to probe the electronic structure of the catalytic core of the enzyme. The ratio of the isotropic 13C hyperfine interactions for the two CN– ligands—a reporter of spin density on their respective coordinating iron ions—collapses from ≈5.8 for the Hox form of hydrogenase to <2 for the CO-inhibited form. Additionally, when the maturation was carried out using [15N]-tyrosine, no features previously ascribed to the nitrogen of the bridging dithiolate ligand were observed suggesting that this bridge is not sourced from tyrosine.

Pro‐ and Antioxidative Effect of α‐Tocopherol on Edible Oils, Triglycerides and Fatty Acids

Using advanced electron paramagnetic resonance techniques (EPR), oxidation of crude vegetable oils and their components (fatty acids and triglycerides) by radicals generated from hydrogen peroxide was investigated. The correlation rotational times were determined allowing us to characterize radicals formed during edible oils oxidation. Additionally 1H- and 14N-hyperfine coupling constants differentiate the fatty acids dependently on their unsaturation. The acids with a higher number of unsaturated bonds exhibit higher AN values of PBN/·lipid adduct. The waste oil with high free fatty acids content underwent the oxidation reaction more efficiently, however due to saturation and the high content of the fatty acids the carbon-centered radicals formed (upon hydrogen peroxide radicals) and their PBN (N-tert-butyl-α-phenylnitrone) adducts were less stable. The antioxidant effect was dependent on the amount of α-tocopherol added. In small amounts of up to 0.35 mg/1 g of fatty acid or triglyceride, it inhibited the creation of PBN/·lipid adducts while with higher amounts it intensified adduct formation. The α-tocopherol (AT) addition influence was also studied as spin scavenging dependence and indicated that any addition of the antioxidant in the investigated samples led to free radical scavenging and the effect increased with the increase in AT content.

ENDOR and ESEEM investigation of the Ni-containing superoxide dismutase

Superoxide dismutases (SODs) protect cells against oxidative stress by disproportionating O2(-) to H(2)O(2) and O(2). The recent finding of a nickel-containing SOD (Ni-SOD) has widened the diversity of SODs in terms of metal contents and SOD catalytic mechanisms. The coordination and geometrical structure of the metal site and the related electronic structure are the keys to understanding the dismutase mechanism of the enzyme. We performed Q-band (14)N,(1/2)H continuous wave (CW) and pulsed electron-nuclear double resonance (ENDOR) and X-band (14)N electron spin echo envelope modulation (ESEEM) on the resting-state Ni-SOD extracted from Streptomyces seoulensis. In-depth analysis of the data obtained from the multifrequency advanced electron paramagnetic resonance techniques detailed the electronic structure of the active site of Ni-SOD. The analysis of the field-dependent Q-band (14)N CW ENDOR yielded the nuclear hyperfine and quadrupole coupling tensors of the axial N(delta) of the His-1 imidazole ligand. The tensors are coaxial with the g-tensor frame, implying the g-tensor direction is modulated by the imidazole plane. X-band (14)N ESEEM characterized the hyperfine coupling of N(epsilon) of His-1 imidazole. The nuclear quadrupole coupling constant of the nitrogen suggests that the hydrogen-bonding between N(epsilon)-H and O(Glu-17) present for the reduced-state Ni-SOD is weakened or broken upon oxidizing the enzyme. Q-band (1)H CW ENDOR and pulsed (2)H Mims ENDOR showed a strong hyperfine coupling to the protons(s) of the equatorially coordinated His-1 amine and a weak hyperfine coupling to either the proton(s) of a water in the pocket at the side opposite the axial N(delta) or the proton of a water hydrogen-bonded to the equatorial thiolate ligand.

Photochemical processes in photosynthesis studied by advanced electron paramagnetic resonance techniques

Various continuous-wave and pulse electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) experiments performed on the radical species occurring in photosynthetic reaction centers of plants and bacteria during light-induced charge separation are reviewed here. Emphasis is placed on time-resolved experiments performed on short-lived intermediate states such as radical pairs and triplet states for which also hyperfine information can be obtained from pulse ENDOR spectroscopy. Detailed insight into the electronic structure of these intermediates and their interaction with the protein environment is now becoming available.

Pulsed EPR spectroscopy: biological applications.

Pulsed electron paramagnetic resonance (EPR) methods such as ESEEM, PELDOR, relaxation time measurements, transient EPR, high-field/high-frequency EPR, and pulsed ENDOR, have been used successfully to investigate the local structure and dynamics of paramagnetic centers in biological samples. These methods allow different contributions to the EPR spectra to be distinguished and can help unravel complicated EPR spectra consisting of overlapping resonance lines, as are often found in disordered protein samples. The basic principles, specific potentials, technical requirements, and limitations of these advanced EPR techniques will be reviewed together with recent applications to metal centers, organic radicals, and spin labels in proteins.

Advanced electron paramagnetic resonance techniques for the study of coal structure

A combination of advanced transient electron paramagnetic resonance (e.p.r.) techniques have been applied to the study of the chemistry of coals of widely varying rank. These pulsed e.p.r. experiments allow detailed studies of the relaxation behaviour of the carbon radicals in coals, including the phase memory time ( T m ) and the electron spin lattice relaxation time ( T 1 e ) which significantly augment the information obtained by conventional constant wave (c.w.) e.p.r. methods. Pulsed electron-nuclear double resonance allows quantitative determination of the various hyperfine interactions which the radicals have with protons on the same and adjacent molecules. Another particularly striking extension of previous e.p.r. techniques is to use the dynamics of the electron spins to track the relaxation properties of protons in their vicinity — the first instance of probing these nuclei which are inaccessible by n.m.r. methods.