Dipolar pathways in dipolar EPR spectroscopy

Dipolar electron paramagnetic resonance (EPR) experiments such as double electron--electron resonance (DEER) measure distributions of nanometer-scale distances between unpaired electrons, which provide valuable information for structural characterization of proteins and...

Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR

Labeling of biomolecules with a paramagnetic probe for nuclear magnetic resonance (NMR) spectroscopy enables determining long-range distance restraints, which are otherwise not accessible by classically used dipolar coupling-based NMR approaches. Distance restraints derived from paramagnetic relaxation enhancements (PREs) can facilitate the structure determination of large proteins and protein complexes. We herein present the site-directed labeling of the large oligomeric bacterial DnaB helicase from Helicobacter pylori with cysteine-reactive maleimide tags carrying either a nitroxide radical or a lanthanide ion. The success of the labeling reaction was followed by quantitative continuous-wave electron paramagnetic resonance (EPR) experiments performed on the nitroxide-labeled protein. PREs were extracted site-specifically from 2D and 3D solid-state NMR spectra. A good agreement with predicted PRE values, derived by computational modeling of nitroxide and Gd3+ tags in the low-resolution DnaB crystal structure, was found. Comparison of experimental PREs and model-predicted spin label-nucleus distances indicated that the size of the "blind sphere" around the paramagnetic center, in which NMR resonances are not detected, is slightly larger for Gd3+ (~14 Å) than for nitroxide (~11 Å) in 13C-detected 2D spectra of DnaB. We also present Gd3+-Gd3+ dipolar electron-electron resonance EPR experiments on DnaB supporting the conclusion that DnaB was present as a hexameric assembly.

Study on spinel - typed catalyst NiCo2O4 for total oxidation of toluene

Toluene is a component of volatile organic compounds which need to be converted into non-poisonous one. Thus, we study the spinel-typed catalyst as NiCo2O4 for total oxidation of toluene because the advantage of spinel catalyst is multi- defective. Based on the sol-gel method, the catalysts NiCo2O4 were successfully prepared with different ratios of Ni/Co. The characteristic properties of catalysts were evaluated by XRD, BET, H2-TPR and EPR experiments. Amongst these ratios, the catalyst with ratio Ni/Co as 0.5 had the best activity in the total oxidation of toluene, as 100% conversion reached at 250 oC.

Synthesis of Novel Kaolin-Supported g-C3N4/CeO2 Composites with Enhanced Photocatalytic Removal of Ciprofloxacin

Herein, novel ternary kaolin/CeO2/g-C3N4 composite was prepared by sol-gel method followed by hydrothermal treatment. The self-assembled 3D “sandwich” structure consisting of kaolin, CeO2 and g-C3N4 nanosheets, was systematically characterized by appropriate techniques to assess its physicochemical properties. In the prerequisite of visible-light irradiation, the removal efficiency of ciprofloxacin (CIP) over the kaolin/CeO2/g-C3N4 composite was about 90% within 150 min, 2-folds higher than those of pristine CeO2 and g-C3N4. The enhanced photocatalytic activity was attributed to the improved photo-induced charge separation efficiency and the large specific surface area, which was determined by electrochemical measurements and N2 physisorption methods, respectively. The synergistic effect between the kaolin and CeO2/g-C3N4 heterostructure improved the photocatalytic performance of the final solid. The trapping and electron paramagnetic resonance (EPR) experiments demonstrated that the hole (h+) and superoxide radicals (•O2−) played an important role in the photocatalytic process. The photocatalytic mechanism for CIP degradation was also proposed based on experimental results. The obtained results revealed that the kaolin/CeO2/g-C3N4 composite is a promising solid catalyst for environmental remediation.

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.

Expanding the Genetic Code for Site-Directed Spin-Labeling

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy enables studies of the structure, dynamics, and interactions of proteins in the noncrystalline state. The scope and analytical value of SDSL–EPR experiments crucially depends on the employed labeling strategy, with key aspects being labeling chemoselectivity and biocompatibility, as well as stability and spectroscopic properties of the resulting label. The use of genetically encoded noncanonical amino acids (ncAA) is an emerging strategy for SDSL that holds great promise for providing excellent chemoselectivity and potential for experiments in complex biological environments such as living cells. We here give a focused overview of recent advancements in this field and discuss their potentials and challenges for advancing SDSL–EPR studies.

The pivotal role of MBD4–ATP7B in the human Cu(i) excretion path as revealed by EPR experiments and all-atom simulations

Atox1–MBD4 interaction mediates the in-cell Cu(i) concentration.

Biomolecular EPR Meets NMR at High Magnetic Fields

In this review on advanced biomolecular EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR regarding the measurement of interactions and dynamics of large molecules embedded in fluid-solution or solid-state environments. Our focus is on the characterization of protein structure, dynamics and interactions, using sophisticated EPR spectroscopy methods. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy to new horizons reaching millimeter and sub-millimeter wavelengths and 15 T Zeeman fields. Expanding traditional applications to paramagnetic systems, spin-labeling of biomolecules has become a mainstream multifrequency approach in EPR spectroscopy. In the high-frequency/high-field EPR region, sub-micromolar concentrations of nitroxide spin-labeled molecules are now sufficient to characterize reaction intermediates of complex biomolecular processes. This offers promising analytical applications in biochemistry and molecular biology where sample material is often difficult to prepare in sufficient concentration for NMR characterization. For multifrequency EPR experiments on frozen solutions typical sample volumes are of the order of 250 μL (S-band), 150 μL (X-band), 10 μL (Q-band) and 1 μL (W-band). These are orders of magnitude smaller than the sample volumes required for modern liquid- or solid-state NMR spectroscopy. An important additional advantage of EPR over NMR is the ability to detect and characterize even short-lived paramagnetic reaction intermediates (down to a lifetime of a few ns). Electron–nuclear and electron–electron double-resonance techniques such as electron–nuclear double resonance (ENDOR), ELDOR-detected NMR, PELDOR (DEER) further improve the spectroscopic selectivity for the various magnetic interactions and their evolution in the frequency and time domains. PELDOR techniques applied to frozen-solution samples of doubly spin-labeled proteins allow for molecular distance measurements ranging up to about 100 Å. For disordered frozen-solution samples high-field EPR spectroscopy allows greatly improved orientational selection of the molecules within the laboratory axes reference system by means of the anisotropic electron Zeeman interaction. Single-crystal resolution is approached at the canonical g-tensor orientations—even for molecules with very small g-anisotropies. Unique structural, functional, and dynamic information about molecular systems is thus revealed that can hardly be obtained by other analytical techniques. On the other hand, the limitation to systems with unpaired electrons means that EPR is less widely used than NMR. However, this limitation also means that EPR offers greater specificity, since ordinary chemical solvents and matrices do not give rise to EPR in contrast to NMR spectra. Thus, multifrequency EPR spectroscopy plays an important role in better understanding paramagnetic species such as organic and inorganic radicals, transition metal complexes as found in many catalysts or metalloenzymes, transient species such as light-generated spin-correlated radical pairs and triplets occurring in protein complexes of photosynthetic reaction centers, electron-transfer relays, etc. Special attention is drawn to high-field EPR experiments on photosynthetic reaction centers embedded in specific sugar matrices that enable organisms to survive extreme dryness and heat stress by adopting an anhydrobiotic state. After a more general overview on methods and applications of advanced multifrequency EPR spectroscopy, a few representative examples are reviewed to some detail in two Case Studies: (I) High-field ELDOR-detected NMR (EDNMR) as a general method for electron–nuclear hyperfine spectroscopy of nitroxide radical and transition metal containing systems; (II) High-field ENDOR and EDNMR studies of the Oxygen Evolving Complex (OEC) in Photosystem II, which performs water oxidation in photosynthesis, i.e., the light-driven splitting of water into its elemental constituents, which is one of the most important chemical reactions on Earth.

Cu(ii)–tBu–PHOX catalyzed enantioselective malonate addition onto 3-hydroxy 2-oxindoles: application in the synthesis of dimeric pyrroloindoline alkaloids

Cu(ii)–PHOX-catalyzed malonate addition onto 3-hydroxy 3-indolyl-2-oxindoles afforded products in >99% ee. A Cu(ii)-species has been identified as an active species based on detailed X-ray, NMR, CV and EPR experiments.