118 results on '"Marina Bennati"'
Search Results
2. Overhauser enhanced liquid state nuclear magnetic resonance spectroscopy in one and two dimensions
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Marcel Levien, Luming Yang, Alex van der Ham, Maik Reinhard, Michael John, Armin Purea, Jürgen Ganz, Thorsten Marquardsen, Igor Tkach, Tomas Orlando, and Marina Bennati
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Science - Abstract
Abstract Nuclear magnetic resonance (NMR) is fundamental in the natural sciences, from chemical analysis and structural biology, to medicine and physics. Despite its enormous achievements, one of its most severe limitations is the low sensitivity, which arises from the small population difference of nuclear spin states. Methods such as dissolution dynamic nuclear polarization and parahydrogen induced hyperpolarization can enhance the NMR signal by several orders of magnitude, however, their intrinsic limitations render multidimensional hyperpolarized liquid-state NMR a challenge. Here, we report an instrumental design for 9.4 Tesla liquid-state dynamic nuclear polarization that enabled enhanced high-resolution NMR spectra in one and two-dimensions for small molecules, including drugs and metabolites. Achieved enhancements of up to two orders of magnitude translate to signal acquisition gains up to a factor of 10,000. We show that hyperpolarization can be transferred between nuclei, allowing DNP-enhanced two-dimensional 13C–13C correlation experiments at 13C natural abundance. The enhanced sensitivity opens up perspectives for structural determination of natural products or characterization of drugs, available in small quantities. The results provide a starting point for a broader implementation of DNP in liquid-state NMR.
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- 2024
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3. Mechanoradicals in tensed tendon collagen as a source of oxidative stress
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Christopher Zapp, Agnieszka Obarska-Kosinska, Benedikt Rennekamp, Markus Kurth, David M. Hudson, Davide Mercadante, Uladzimir Barayeu, Tobias P. Dick, Vasyl Denysenkov, Thomas Prisner, Marina Bennati, Csaba Daday, Reinhard Kappl, and Frauke Gräter
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Science - Abstract
The existence, nature and biological relevance of mechanoradicals in proteins are unknown. Here authors show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species and suggest that collagen I evolved as a radical sponge against mechano-oxidative damage.
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- 2020
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4. Structural heterogeneity of α-synuclein fibrils amplified from patient brain extracts
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Timo Strohäker, Byung Chul Jung, Shu-Hao Liou, Claudio O. Fernandez, Dietmar Riedel, Stefan Becker, Glenda M. Halliday, Marina Bennati, Woojin S. Kim, Seung-Jae Lee, and Markus Zweckstetter
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Science - Abstract
Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are characterized by the pathological accumulation of α-synuclein. Here the authors employ fluorescent probes, electron microscopy and NMR spectroscopy to study the properties of α-synuclein aggregates that were amplified from patient brain extracts and observe a greater structural diversity among PD patients compared to MSA patients.
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- 2019
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5. Large P-31-NMR enhancements in liquid state dynamic nuclear polarization through radical/target molecule non-covalent interaction
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Maik Reinhard, Marcel Levien, Marina Bennati, and Tomas Orlando
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General Physics and Astronomy ,Physical and Theoretical Chemistry - Abstract
Dynamic nuclear polarization (DNP) is a method to enhance the low sensitivity of nuclear magnetic resonance (NMR) via spin polarization transfer from electron spins to nuclear spins. In the liquid state, this process is mediated by fast modulations of the electron-nuclear hyperfine coupling and its efficiency depends strongly on the applied magnetic field. A peculiar case study is triphenylphosphine (PPh3) dissolved in benzene and doped with BDPA radical because it gives 31P-NMR signal enhancements of two orders of magnitude up to a magnetic field of 14.1 T. Here we show that the large 31P enhancements of BDPA/PPh3 in benzene at 1.2 T (i) decrease when the moieties are dissolved in other organic solvents, (ii) are strongly reduced when using a nitroxide radical, and (iii) vanish with pentavalent 31P triphenylphosphine oxide. Those experimental observations are rationalized with numerical calculations based on density functional theory that show the tendency of BDPA and PPh3 to form a weak complex via non-covalent interaction that leads to large hyperfine couplings to 31P (ΔAiso ≥ 13 MHz). This mechanism is hampered in other investigated systems. The case study of 31P-DNP in PPh3 is an important example that extends the current understanding of DNP in the liquids state: non-covalent interactions between radical and target can be particularly effective to obtain large NMR signal enhancements.
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- 2023
6. 17 O Hyperfine Spectroscopy Reveals Hydration Structure of Nitroxide Radicals in Aqueous Solutions
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Fabian Hecker, Lisa Fries, Markus Hiller, Mario Chiesa, and Marina Bennati
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General Chemistry ,General Medicine ,Catalysis - Abstract
The hydration structure of nitroxide radicals in aqueous solutions is elucidated by advanced 17O hyperfine (hf) spectroscopy with support of quantum chemical calculations and MD simulations. A piperidine and a pyrrolidine-based nitroxide radical are compared and show clear differences in the preferred directionality of H-bond formation. We demonstrate that these scenarios are best represented in 17O hf spectra, where in-plane coordination over σ-type H-bonding leads to little spin density transfer on the water oxygen and small hf couplings, whereas π-type perpendicular coordination generates much larger hf couplings. Quantitative analysis of the spectra based on MD simulations and DFT predicted hf parameters is consistent with a distribution of close solvating water molecules, in which directionality is influenced by subtle steric effects of the ring and the methyl group substituents.
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- 2022
7. Large
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Maik, Reinhard, Marcel, Levien, Marina, Bennati, and Tomas, Orlando
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Dynamic nuclear polarization (DNP) is a method to enhance the low sensitivity of nuclear magnetic resonance (NMR)
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- 2022
8. Distribution of Hβ hyperfine couplings in a tyrosyl radical revealed by 263 GHz ENDOR spectroscopy
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Benjamin Eltzner, H. Wiechers, Markus Hiller, Marina Bennati, Igor Tkach, Yvo Pokern, and Stephan Huckemann
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Physics ,Crystallography ,Deuterium ,law ,Radical ,Dihedral angle ,Ring (chemistry) ,Electron paramagnetic resonance ,Spectroscopy ,Hyperfine structure ,Atomic and Molecular Physics, and Optics ,Spectral line ,law.invention - Abstract
$$^1$$ 1 H ENDOR spectra of tyrosyl radicals (Y$$^\bullet$$ ∙ ) have been the subject of numerous EPR spectroscopic studies due to their importance in biology. Nevertheless, assignment of all internal $$^1$$ 1 H hyperfine couplings has been challenging because of substantial spectral overlap. Recently, using 263 GHz ENDOR in conjunction with statistical analysis, we could identify the signature of the H$$^{\upbeta _2}$$ β 2 coupling in the essential Y$$_{122}$$ 122 radical of Escherichia coli ribonucleotide reductase, and modeled it with a distribution of radical conformations. Here, we demonstrate that this analysis can be extended to the full-width $$^1$$ 1 H ENDOR spectra that contain the larger H$$^{\upbeta _1}$$ β 1 coupling. The H$$^{\upbeta _2}$$ β 2 and H$$^{\upbeta _1}$$ β 1 couplings are related to each other through the ring dihedral and report on the amino acid conformation. The 263 GHz ENDOR data, acquired in batches instead of averaging, and data processing by a new “drift model” allow reconstructing the ENDOR spectra with statistically meaningful confidence intervals and separating them from baseline distortions. Spectral simulations using a distribution of ring dihedral angles confirm the presence of a conformational distribution, consistent with the previous analysis of the H$$^{\upbeta _2}$$ β 2 coupling. The analysis was corroborated by 94 GHz $$^2$$ 2 H ENDOR of deuterated Y$$_{122}^\bullet$$ 122 ∙ . These studies provide a starting point to investigate low populated states of tyrosyl radicals in greater detail.
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- 2022
9. Detection of Water Molecules on the Radical Transfer Pathway of Ribonucleotide Reductase by 17O Electron–Nuclear Double Resonance Spectroscopy
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JoAnne Stubbe, Marina Bennati, and Fabian Hecker
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Free Radicals ,Electrons ,Oxygen Isotopes ,010402 general chemistry ,Photochemistry ,01 natural sciences ,Biochemistry ,Catalysis ,Electron Transport ,Electron transfer ,Colloid and Surface Chemistry ,Ribonucleotide Reductases ,Escherichia coli ,Molecule ,Hyperfine structure ,Density Functional Theory ,chemistry.chemical_classification ,Chemistry ,Communication ,Electron Spin Resonance Spectroscopy ,Water ,General Chemistry ,Resonance (chemistry) ,0104 chemical sciences ,Ribonucleotide reductase ,Enzyme ,Electron nuclear double resonance spectroscopy ,Tyrosine ,Proton-coupled electron transfer - Abstract
The role of water in biological proton-coupled electron transfer (PCET) is emerging as a key for understanding mechanistic details at atomic resolution. Here we demonstrate 17O high-frequency electron–nuclear double resonance (ENDOR) in conjunction with H217O-labeled protein buffer to establish the presence of ordered water molecules at three radical intermediates in an active enzyme complex, the α2β2E. coli ribonucleotide reductase. Our data give unambiguous evidence that all three, individually trapped, intermediates are hyperfine coupled to one water molecule with Tyr-O···17O distances in the range 2.8–3.1 Å. The availability of this structural information will allow for quantitative models of PCET in this prototype enzyme. The results also provide a spectroscopic signature for water H-bonded to a tyrosyl radical.
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- 2021
10. Comment on mr-2022-12
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Marina Bennati
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- 2022
11. Spin density localization and accessibility of organic radicals affect liquid-state DNP efficiency
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Tomas Orlando, Maik Reinhard, Marina Bennati, Markus Hiller, Igor Tkach, and Marcel Levien
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Liquid state ,010405 organic chemistry ,Chemistry ,Radical ,General Physics and Astronomy ,Molecule ,chemical and pharmacologic phenomena ,Physical and Theoretical Chemistry ,Spin density ,010402 general chemistry ,Photochemistry ,01 natural sciences ,0104 chemical sciences - Abstract
We report a large variation in liquid DNP performance of up to a factor of about five in coupling factor among organic radicals commonly used as polarizing agents. A comparative study of 1H and 13C DNP in model systems shows the impact of the spin density distribution and accessibility of the radical site by the target molecule.
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- 2021
12. Mechanoradicals in tensed tendon collagen as a source of oxidative stress
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Vasyl Denysenkov, Uladzimir Barayeu, Marina Bennati, Csaba Daday, Agnieszka Obarska-Kosinska, Benedikt Rennekamp, Markus Kurth, Christopher Zapp, Tobias P. Dick, Frauke Gräter, David M. Hudson, Thomas F. Prisner, Davide Mercadante, and Reinhard Kappl
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0301 basic medicine ,Biomaterials - proteins ,General Physics and Astronomy ,Biocompatible Materials ,medicine.disease_cause ,01 natural sciences ,law.invention ,Tendons ,chemistry.chemical_compound ,Biopolymers ,law ,Hydrogen peroxide ,Electron paramagnetic resonance ,lcsh:Science ,chemistry.chemical_classification ,Condensed Matter - Materials Science ,Multidisciplinary ,Homolysis ,Dihydroxyphenylalanine ,Biological Physics (physics.bio-ph) ,Collagen ,Oxidation-Reduction ,Free Radicals ,Radical ,Science ,FOS: Physical sciences ,010402 general chemistry ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,medicine ,Molecule ,Animals ,Physics - Biological Physics ,Biopolymers in vivo ,Bond cleavage ,Reactive oxygen species ,Electron Spin Resonance Spectroscopy ,Materials Science (cond-mat.mtrl-sci) ,General Chemistry ,0104 chemical sciences ,Rats ,Oxidative Stress ,030104 developmental biology ,chemistry ,Biophysics ,lcsh:Q ,Reactive Oxygen Species ,Oxidative stress - Abstract
As established nearly a century ago, mechanoradicals originate from homolytic bond scission in polymers. The existence, nature and biological relevance of mechanoradicals in proteins, instead, are unknown. We here show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species, essential biological signaling molecules. Electron-paramagnetic resonance (EPR) spectroscopy of stretched rat tail tendon, atomistic molecular dynamics simulations and quantum-chemical calculations show that the radicals form by bond scission in the direct vicinity of crosslinks in collagen. Radicals migrate to adjacent clusters of aromatic residues and stabilize on oxidized tyrosyl radicals, giving rise to a distinct EPR spectrum consistent with a stable dihydroxyphenylalanine (DOPA) radical. The protein mechanoradicals, as a yet undiscovered source of oxidative stress, finally convert into hydrogen peroxide. Our study suggests collagen I to have evolved as a radical sponge against mechano-oxidative damage and proposes a mechanism for exercise-induced oxidative stress and redox-mediated pathophysiological processes., The existence, nature and biological relevance of mechanoradicals in proteins are unknown. Here authors show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species and suggest that collagen I evolved as a radical sponge against mechano-oxidative damage.
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- 2020
13. 19F Electron-nuclear double resonance reveals interaction between redox-active tyrosines across the α/β interface of E. coli ribonucleotide reductase
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Andreas Meyer, Annemarie Kehl, Chang Cui, Fehmke A. K. Reichardt, Fabian Hecker, Lisa-Marie Funk, Manas K. Ghosh, Kuan-Ting Pan, Henning Urlaub, Kai Tittmann, JoAnne Stubbe, and Marina Bennati
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Colloid and Surface Chemistry ,General Chemistry ,Biochemistry ,Catalysis - Abstract
Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α2β2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and β, to initiate, using its metallo-cofactor in β2, nucleotide reduction in α2. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y356(β) and Y731(α) across the α/β interface. Using 2,3,5-F3Y122-β2 with 3,5-F2Y731-α2, GDP (substrate) and TTP (allosteric effector), a Y356• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron–electron double resonance spectroscopies. 94 GHz 19F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y356• and the 19F nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the double mutant E52Q/F3Y122-β2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F2Y731. Remarkably, one conformation is consistent with 3,5-F2Y731 within the H-bond distance to Y356•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y356 and Y731. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.
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- 2022
14. Measurement of Angstrom to Nanometer Molecular Distances with 19 F Nuclear Spins by EPR/ENDOR Spectroscopy
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Andreas Meyer, Sebastian Dechert, Surjendu Dey, Claudia Höbartner, and Marina Bennati
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010405 organic chemistry ,General Medicine ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences - Published
- 2019
15. Measurement of Angstrom to Nanometer Molecular Distances with 19F Nuclear Spins by EPR/ENDOR Spectroscopy
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Marina Bennati, Surjendu Dey, Sebastian Dechert, Andreas Meyer, and Claudia Höbartner
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Models, Molecular ,Nitroxide mediated radical polymerization ,Materials science ,Structure Determination ,Gyromagnetic ratio ,chemistry.chemical_element ,010402 general chemistry ,01 natural sciences ,Molecular physics ,Catalysis ,law.invention ,law ,RNA ,fluorine ,high field ENDOR ,spin labelling ,structural biology ,Humans ,Spectroscopy ,Electron paramagnetic resonance ,Research Articles ,Spins ,010405 organic chemistry ,Electron Spin Resonance Spectroscopy ,Resonance ,General Chemistry ,0104 chemical sciences ,Structural biology ,chemistry ,Fluorine ,Spin Labels ,Research Article - Abstract
Spectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio‐macromolecules using 19F nuclear spins and nitroxide radicals in combination with high‐frequency (94 GHz/3.4 T) electron–nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the 19F label enables to access distances up to about 1.5 nm with an accuracy of 0.1–1 Å. The experiment is not limited by the size of the bio‐macromolecule. Performance is illustrated on synthesized fluorinated model compounds as well as spin‐labelled RNA duplexes. The results demonstrate that our simple but strategic spin‐labelling procedure combined with state‐of‐the‐art spectroscopy accesses a distance range crucial to elucidate active sites of nucleic acids or proteins in the solution state., What did the NO say to the 19 F? Dipolar couplings between nitroxide spin labels and 19F nuclear spins report inter‐spin distances in the range of ≲15 Å at sub‐angstrom accuracy. These couplings are measured with sub‐nanomole spin sensitivity by high frequency (94 GHz) electron–nuclear double resonance (ENDOR) and can be employed to obtain detailed structural insights into biomolecules.
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- 2019
16. Resolution of chemical shift anisotropy in
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Annemarie, Kehl, Markus, Hiller, Fabian, Hecker, Igor, Tkach, Sebastian, Dechert, Marina, Bennati, and Andreas, Meyer
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Electron Spin Resonance Spectroscopy ,Anisotropy ,Computer Simulation - Abstract
Pulsed
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- 2021
17. Statistical analysis of ENDOR spectra
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JoAnne Stubbe, Stephan Huckemann, Igor Tkach, Marina Bennati, Benjamin Eltzner, Markus Hiller, Clemens Beeken, and Yvo Pokern
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Statistics as Topic ,statistical tests ,010402 general chemistry ,01 natural sciences ,Molecular physics ,Spectral line ,law.invention ,tyrosyl radical ,law ,Ribonucleotide Reductases ,Escherichia coli ,Computer Simulation ,Amino Acids ,bootstrap ,Electron paramagnetic resonance ,Spectroscopy ,Hyperfine structure ,Physics ,Larmor precession ,Multidisciplinary ,010405 organic chemistry ,Electron Spin Resonance Spectroscopy ,Resonance ,Statistical model ,ENDOR ,0104 chemical sciences ,Biophysics and Computational Biology ,Protein Subunits ,Physical Sciences ,Density functional theory ,error model - Abstract
Significance Statistical modeling of experimental data is gaining increasing importance in biological science due to the availability of large datasets. Here we present a statistical analysis of electron–nuclear double resonance, a technique that delivers information on the angstrom to nanometer scale around paramagnetic centers in proteins. The described method allows for recognizing experimental artifacts and provides the most probable signal as well as its uncertainty. Application to representative high-field electron–nuclear double resonance spectra of a prototype tyrosyl radical in a protein, the β2 subunit of Escherichia coli ribonucleotide reductase, demonstrates that subtle information can be uncovered, such as a distribution of molecular orientations relevant for the biological function of this essential radical., Electron–nuclear double resonance (ENDOR) measures the hyperfine interaction of magnetic nuclei with paramagnetic centers and is hence a powerful tool for spectroscopic investigations extending from biophysics to material science. Progress in microwave technology and the recent availability of commercial electron paramagnetic resonance (EPR) spectrometers up to an electron Larmor frequency of 263 GHz now open the opportunity for a more quantitative spectral analysis. Using representative spectra of a prototype amino acid radical in a biologically relevant enzyme, the Y122• in Escherichia coli ribonucleotide reductase, we developed a statistical model for ENDOR data and conducted statistical inference on the spectra including uncertainty estimation and hypothesis testing. Our approach in conjunction with 1H/2H isotopic labeling of Y122• in the protein unambiguously established new unexpected spectral contributions. Density functional theory (DFT) calculations and ENDOR spectral simulations indicated that these features result from the beta-methylene hyperfine coupling and are caused by a distribution of molecular conformations, likely important for the biological function of this essential radical. The results demonstrate that model-based statistical analysis in combination with state-of-the-art spectroscopy accesses information hitherto beyond standard approaches.
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- 2021
18. Semi-Rigid Nitroxide Spin Label for Long-Range EPR Distance Measurements of Lipid Bilayer Embedded β-Peptides
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Marina Bennati, Brigitte Worbs, Janine Wegner, Ulf Diederichsen, Gabriele Valora, Karin Halbmair, and Annemarie Kehl
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Nitroxide mediated radical polymerization ,010405 organic chemistry ,Chemistry ,Lipid Bilayers ,Organic Chemistry ,Electron Spin Resonance Spectroscopy ,Resonance ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Protein Structure, Secondary ,Catalysis ,0104 chemical sciences ,law.invention ,Turn (biochemistry) ,Crystallography ,law ,Helix ,Nitrogen Oxides ,Spin Labels ,Peptides ,Lipid bilayer ,Spectroscopy ,Spin label ,Electron paramagnetic resonance - Abstract
β-Peptides are an interesting new class of transmembrane model peptides based on their conformationally stable and well-defined secondary structures. Herein, we present the synthesis of the paramagnetic β-amino acid β3 -hTOPP (4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-d-β3 -homophenylglycine) that enables investigations of β-peptides by EPR spectroscopy. This amino acid adds to the, to date, sparse number of β-peptide spin labels. Its performance was evaluated by investigating the helical turn of a 314 -helical transmembrane model β-peptide. Nanometer distances between two incorporated β3 -hTOPP labels in different environments were measured by using pulsed electron/electron double resonance (PELDOR/DEER) spectroscopy. Due to the semi-rigid conformational design, the label delivers reliable distances and sharp (one-peak) distance distributions even in the lipid bilayer. The results indicate that the investigated β-peptide folds into a 3.2514 helix and maintains this conformation in the lipid bilayer.
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- 2019
19. Dynamic Nuclear Polarization of 13 C Nuclei in the Liquid State over a 10 Tesla Field Range
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Tomas Orlando, Rıza Dervişoğlu, Marcel Levien, Igor Tkach, Thomas F. Prisner, Loren B. Andreas, Vasyl P. Denysenkov, and Marina Bennati
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010405 organic chemistry ,General Medicine ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences - Published
- 2018
20. Dynamic Nuclear Polarization of 13 C Nuclei in the Liquid State over a 10 Tesla Field Range
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Thomas F. Prisner, Riza Dervisoglu, Marcel Levien, Tomas Orlando, Vasyl Denysenkov, Igor Tkach, Loren B. Andreas, and Marina Bennati
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Materials science ,Spins ,010405 organic chemistry ,General Chemistry ,Nuclear Overhauser effect ,Electron ,010402 general chemistry ,Polarization (waves) ,01 natural sciences ,Catalysis ,3. Good health ,0104 chemical sciences ,law.invention ,Paramagnetism ,Chemical physics ,law ,Hyperpolarization (physics) ,Electron paramagnetic resonance ,Hyperfine structure - Abstract
Nuclear magnetic resonance (NMR) techniques play an essential role in natural science and medicine. In spite of the tremendous utility associated with the small energies detected, the most severe limitation is the low signal-to-noise ratio. Dynamic nuclear polarization (DNP), a technique based on transfer of polarization from electron to nuclear spins, has emerged as a tool to enhance sensitivity of NMR. However, the approach in liquids still faces several challenges. Herein we report the observation of room-temperature, liquid DNP 13 C signal enhancements in organic small molecules as high as 600 at 9.4 Tesla and 800 at 1.2 Tesla. A mechanistic investigation of the 13 C-DNP field dependence shows that DNP efficiency is raised by proper choice of the polarizing agent (paramagnetic center) and by halogen atoms as mediators of scalar hyperfine interaction. Observation of sizable DNP of 13 CH2 and 13 CH3 groups in organic molecules at 9.4 T opens perspective for a broader application of this method.
- Published
- 2018
21. Studies of transmembrane peptides by pulse dipolar spectroscopy with semi-rigid TOPP spin labels
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Igor Tkach, Ulf Diederichsen, and Marina Bennati
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Spin label ,Transmembrane peptide ,Materials science ,DEER ,Biophysics ,Review ,SDSL ,010402 general chemistry ,01 natural sciences ,law.invention ,Paramagnetism ,PDS ,PELDOR ,Pulsed ESR ,β-peptide ,law ,Spectroscopy ,Spin (physics) ,Electron paramagnetic resonance ,010405 organic chemistry ,Resolution (electron density) ,Cell Membrane ,Electron Spin Resonance Spectroscopy ,General Medicine ,Dipolar spectroscopy ,α-TOPP ,0104 chemical sciences ,Membrane ,Chemical physics ,Helix ,Spin Labels ,Peptides ,β-TOPP - Abstract
Electron paramagnetic resonance (EPR)-based pulsed dipolar spectroscopy measures the dipolar interaction between paramagnetic centers that are separated by distances in the range of about 1.5–10 nm. Its application to transmembrane (TM) peptides in combination with modern spin labelling techniques provides a valuable tool to study peptide-to-lipid interactions at a molecular level, which permits access to key parameters characterizing the structural adaptation of model peptides incorporated in natural membranes. In this mini-review, we summarize our approach for distance and orientation measurements in lipid environment using novel semi-rigid TOPP [4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-L-phenylglycine] labels specifically designed for incorporation in TM peptides. TOPP labels can report single peak distance distributions with sub-angstrom resolution, thus offering new capabilities for a variety of TM peptide investigations, such as monitoring of various helix conformations or measuring of tilt angles in membranes. Graphical Abstract
- Published
- 2021
22. Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules
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Stefan Stoll, Elena G. Bagryanskaya, Gunnar Jeschke, Sharon Ruthstein, Sunil Saxena, Marina Bennati, Malte Drescher, Burkhard Endeward, Olav Schiemann, Marilena Di Valentin, Ilya Kuprov, Janet E. Lovett, Laura Galazzo, Jack H. Freed, Katrin Ackermann, Mykhailo Azarkh, Christiane R. Timmel, Dinar Abdullin, Laura Esteban Hofer, Daniella Goldfarb, Thomas F. Prisner, Hassane S. Mchaourab, Eric J. Hustedt, Svetlana Kucher, Luis Fábregas Ibáñez, Enrica Bordignon, Andreas Meyer, Caspar A. Heubach, Tobias Hett, Bela E. Bode, BBSRC, The Leverhulme Trust, The Royal Society, University of St Andrews. EaSTCHEM, University of St Andrews. Biomedical Sciences Research Complex, University of St Andrews. Centre of Magnetic Resonance, University of St Andrews. School of Chemistry, and University of St Andrews. School of Physics and Astronomy
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Spin label ,DEER ,Good practice ,Peptides and proteins ,Biochemistry ,Resonance (particle physics) ,Quantum mechanics ,Catalysis ,Cyclic N-Oxides ,Colloid and Surface Chemistry ,PELDOR ,Labeling ,Benchmark study ,QD ,Monomers ,Crystal structure ,Conformational ensembles ,chemistry.chemical_classification ,Ground truth ,Biomolecule ,Integrated structural biology ,Nitroxide ,Electron Spin Resonance Spectroscopy ,Proteins ,Reproducibility of Results ,3rd-DAS ,General Chemistry ,QD Chemistry ,Benchmarking ,chemistry ,Structural biology ,ddc:540 ,Data analysis ,Benchmark (computing) ,Spin Labels ,EPR ,Biological system - Abstract
Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron-electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases., Journal of the American Chemical Society, 143 (43), ISSN:0002-7863, ISSN:1520-5126
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- 2021
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23. Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets
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Marina Bennati, Brandon L. Greene, Gyunghoon Kang, Daniel G. Nocera, Catherine L. Drennan, Chang Cui, and JoAnne Stubbe
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Biochemistry & Molecular Biology ,Ribonucleotide ,Antineoplastic Agents ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Medical and Health Sciences ,Article ,Protein Structure, Secondary ,Nucleic acid metabolism ,Small Molecule Libraries ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,Neoplasms ,Drug Discovery ,Ribonucleotide Reductases ,Escherichia coli ,therapeutics ,Humans ,Nucleotide ,Enzyme Inhibitors ,Escherichia coli Infections ,ribonucleotide reductases ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,mechanisms ,Nucleotides ,Drug discovery ,DNA replication ,Biological Sciences ,Small molecule ,structures ,Anti-Bacterial Agents ,3. Good health ,0104 chemical sciences ,Molecular Docking Simulation ,Protein Subunits ,Enzyme ,chemistry ,Biocatalysis ,Oxidation-Reduction ,Function (biology) - Abstract
© 2020 Annual Reviews Inc.. All rights reserved. Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRscentral role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.
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- 2020
24. Nitroxide derivatives for dynamic nuclear polarization in liquids: The role of rotational diffusion
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Marina Bennati, Tomas Orlando, Markus Hiller, Igor Tkach, and M. Levien
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Nitroxide mediated radical polymerization ,Materials science ,Fermi contact interaction ,Letter ,Doping ,Physics::Medical Physics ,Rotational diffusion ,02 engineering and technology ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Ring (chemistry) ,01 natural sciences ,Toluene ,0104 chemical sciences ,Magnetic field ,chemistry.chemical_compound ,chemistry ,Chemical physics ,General Materials Science ,Physical and Theoretical Chemistry ,0210 nano-technology ,Polarization (electrochemistry) - Abstract
Polarization transfer efficiency in liquid-state dynamic nuclear polarization (DNP) depends on the interaction between polarizing agents (PAs) and target nuclei modulated by molecular motions. We show how translational and rotational diffusion differently affect the DNP efficiency. These contributions were disentangled by measuring 1H-DNP enhancements of toluene and chloroform doped with nitroxide derivatives at 0.34 T as a function of either the temperature or the size of the PA. The results were employed to analyze 13C-DNP data at higher fields, where the polarization transfer is also driven by the Fermi contact interaction. In this case, bulky nitroxide PAs perform better than the small TEMPONE radical due to structural fluctuations of the ring conformation. These findings will help in designing PAs with features specifically optimized for liquid-state DNP at various magnetic fields.
- Published
- 2020
25. Cross-polarisation ENDOR for spin-1 deuterium nuclei
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Robert Zeier, Isabel Bejenke, Steffen J. Glaser, Roberto Rizzato, and Marina Bennati
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Materials science ,010304 chemical physics ,Spins ,Nuclear Theory ,Biophysics ,macromolecular substances ,Electron ,010402 general chemistry ,Condensed Matter Physics ,01 natural sciences ,0104 chemical sciences ,law.invention ,Deuterium ,law ,0103 physical sciences ,Condensed Matter::Strongly Correlated Electrons ,ddc:530 ,Sensitivity (control systems) ,Physical and Theoretical Chemistry ,Atomic physics ,Nuclear Experiment ,Spin (physics) ,Electron paramagnetic resonance ,Molecular Biology - Abstract
Efficient transfer of spin polarisation from electron to nuclear spins is emerging as a common target of several advanced spectroscopic experiments, ranging from sensitivity enhancement in nuclear magnetic resonance (NMR) and methods for the detection of single molecules based on optically detected magnetic resonance (ODMR) to hyperfine spectroscopy. Here, we examine the feasibility of electron-nuclear cross-polarisation at a modified Hartmann-Hahn condition (called eNCP) for applications in ENDOR experiments with spin-1 deuterium nuclei, which are important targets in studies of hydrogen bonds in biological systems and materials. We have investigated a two-spin model system of deuterated malonic acid radicals in a single crystal. Energy matching conditions as well as ENDOR signal intensities were determined for a spin Hamiltonian under the effect of microwave and radiofrequency irradiation. The results were compared with numerical simulations and 94-GHz ENDOR experiments. The compelling agreement between theoretical predictions and experimental results demonstrates that spin density operator formalism in conjunction with suitable approximations in regard to spin relaxation provides a satisfactory description of the polarisation transfer effect. The results establish a basis for future numerical optimizations of polarisation transfer experiments using multiple-pulse sequences or shaped pulses and for moving from model systems to real applications in disordered systems.
- Published
- 2020
26. Understanding Overhauser Dynamic Nuclear Polarisation through NMR relaxometry
- Author
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Claudio Luchinat, Enrico Ravera, Marina Bennati, and Giacomo Parigi
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Relaxometry ,Materials science ,010304 chemical physics ,Dynamic nuclear polarisation ,Biophysics ,Condensed Matter::Mesoscopic Systems and Quantum Hall Effect ,010402 general chemistry ,Condensed Matter Physics ,01 natural sciences ,0104 chemical sciences ,Nuclear magnetic resonance ,Solid-state nuclear magnetic resonance ,0103 physical sciences ,Sensitivity (control systems) ,Dipolar relaxation ,Physical and Theoretical Chemistry ,Molecular Biology - Abstract
Overhauser dynamic nuclear polarisation (DNP) represents a potentially outstanding tool to increase the sensitivity of solution and solid state NMR experiments, as well as of magnetic resonance ima...
- Published
- 2018
27. One-thousand-fold enhancement of high field liquid nuclear magnetic resonance signals at room temperature
- Author
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Marina Bennati, Niels Karschin, Marcel Levien, Guoquan Liu, Claudio Luchinat, and Giacomo Parigi
- Subjects
Nitroxide mediated radical polymerization ,010405 organic chemistry ,Chemistry ,General Chemical Engineering ,Analytical chemistry ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,Carbon-13 NMR ,Polarizer ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences ,law.invention ,Nuclear magnetic resonance ,Solid-state nuclear magnetic resonance ,law ,Molecule ,Chemistry (all) ,Chemical Engineering (all) ,Two-dimensional nuclear magnetic resonance spectroscopy ,Earth's field NMR - Abstract
Nuclear magnetic resonance (NMR) is a fundamental spectroscopic technique for the study of biological systems and materials, molecular imaging and the analysis of small molecules. It detects interactions at very low energies and is thus non-invasive and applicable to a variety of targets, including animals and humans. However, one of its most severe limitations is its low sensitivity, which stems from the small interaction energies involved. Here, we report that dynamic nuclear polarization in liquid solution and at room temperature can enhance the NMR signal of 13C nuclei by up to three orders of magnitude at magnetic fields of ∼3 T. The experiment can be repeated within seconds for signal averaging, without interfering with the sample magnetic homogeneity. The method is therefore compatible with the conditions required for high-resolution NMR. Enhancement of 13C signals on various organic compounds opens up new perspectives for dynamic nuclear polarization as a general tool to increase the sensitivity of liquid NMR.
- Published
- 2017
28. Pulse EPR Measurements of Intramolecular Distances in a TOPP-Labeled Transmembrane Peptide in Lipids
- Author
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Marina Bennati, Karin Halbmair, Ulf Diederichsen, and Janine Wegner
- Subjects
Models, Molecular ,Protein Conformation, alpha-Helical ,Lipid Bilayers ,Glycine ,Biophysics ,Diketopiperazines ,010402 general chemistry ,01 natural sciences ,law.invention ,Cyclic N-Oxides ,chemistry.chemical_compound ,Nuclear magnetic resonance ,law ,0103 physical sciences ,Amino Acid Sequence ,Spin label ,Lipid bilayer ,Electron paramagnetic resonance ,POPC ,010304 chemical physics ,Chemistry ,Pulsed EPR ,Biophysical Letter ,Bilayer ,Cell Membrane ,Electron Spin Resonance Spectroscopy ,Resonance ,0104 chemical sciences ,Intramolecular force ,Spin Labels ,Peptides - Abstract
We present the performance of nanometer-range pulse electron paramagnetic resonance distance measurements (pulsed electron-electron double resonance/double electron-electron resonance, PELDOR/DEER) on a transmembrane WALP24 peptide labeled with the semirigid unnatural amino acid 4-(3,3,5,5-tetra-methyl-2,6-dioxo-4-oxylpiperazin-1-yl)-l-phenylglycine (TOPP). Distances reported by the TOPP label are compared to the ones reported by the more standard MTSSL spin label, commonly employed in protein studies. Using high-power pulse electron paramagnetic resonance spectroscopy at Q-band frequencies (34 GHz), we show that in contrast to MTSSL, our label reports one-peak, sharp (Δr ≤ 0.4 nm) intramolecular distances. Orientational selectivity is not observed. When spin-labeled WALP24 was inserted in two representative lipid bilayers with different bilayer thickness, i.e., DMPC and POPC, the intramolecular distance reported by TOPP did not change with the bilayer environment. In contrast, the distance measured with MTSSL was strongly affected by the hydrophobic thickness of the lipid. The results demonstrate that the TOPP label is well suited to study the intrinsic structure of peptides immersed in lipids.
- Published
- 2016
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29. Structural heterogeneity of α-synuclein fibrils amplified from patient brain extracts
- Author
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Markus Zweckstetter, Timo Strohäker, Shu-Hao Liou, Byung Chul Jung, Glenda M. Halliday, Woojin S. Kim, Seung-Jae Lee, Marina Bennati, Claudio O. Fernández, Dietmar Riedel, and Stefan Becker
- Subjects
0301 basic medicine ,Male ,Models, Molecular ,Synucleinopathies ,Protein Conformation ,Parkinson's disease ,General Physics and Astronomy ,Protein aggregation ,Biochemistry ,law.invention ,0302 clinical medicine ,Protein structure ,law ,metabolism [alpha-Synuclein] ,lcsh:Science ,metabolism [Synucleinopathies] ,Aged, 80 and over ,Multidisciplinary ,Chemistry ,Brain ,Parkinson Disease ,Nuclear magnetic resonance spectroscopy ,Structural heterogeneity ,3. Good health ,diagnosis [Multiple System Atrophy] ,alpha-Synuclein ,Female ,ddc:500 ,diagnosis [Parkinson Disease] ,α-synuclein fibrils ,Science ,metabolism [Parkinson Disease] ,diagnosis [Synucleinopathies] ,Fibril ,Protein Aggregation, Pathological ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Atrophy ,metabolism [Protein Aggregation, Pathological] ,mental disorders ,metabolism [Multiple System Atrophy] ,medicine ,Humans ,metabolism [Tissue Extracts] ,Aged ,Tissue Extracts ,General Chemistry ,Multiple System Atrophy ,medicine.disease ,In vitro ,nervous system diseases ,030104 developmental biology ,chemistry [alpha-Synuclein] ,nervous system ,metabolism [Brain] ,Biophysics ,lcsh:Q ,Electron microscope ,Solution-state NMR ,030217 neurology & neurosurgery - Abstract
Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are clinically distinctive diseases that feature a common neuropathological hallmark of aggregated α-synuclein. Little is known about how differences in α-synuclein aggregate structure affect disease phenotype. Here, we amplified α-synuclein aggregates from PD and MSA brain extracts and analyzed the conformational properties using fluorescent probes, NMR spectroscopy and electron paramagnetic resonance. We also generated and analyzed several in vitro α-synuclein polymorphs. We found that brain-derived α-synuclein fibrils were structurally different to all of the in vitro polymorphs analyzed. Importantly, there was a greater structural heterogeneity among α-synuclein fibrils from the PD brain compared to those from the MSA brain, possibly reflecting on the greater variability of disease phenotypes evident in PD. Our findings have significant ramifications for the use of non-brain-derived α-synuclein fibrils in PD and MSA studies, and raise important questions regarding the one disease-one strain hypothesis in the study of α-synucleinopathies., Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are characterized by the pathological accumulation of α-synuclein. Here the authors employ fluorescent probes, electron microscopy and NMR spectroscopy to study the properties of α-synuclein aggregates that were amplified from patient brain extracts and observe a greater structural diversity among PD patients compared to MSA patients.
- Published
- 2019
30. 1H high field electron-nuclear double resonance spectroscopy at 263 GHz/9.4 T
- Author
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Müge Kasanmascheff, Igor Tkach, Fabian Hecker, Markus Hiller, Peter Höfer, Marina Bennati, Isabel Bejenke, Ion Prisecaru, Igor Gromov, and Annemarie Kehl
- Subjects
Nuclear and High Energy Physics ,Materials science ,Proton ,Resolution (electron density) ,Biophysics ,Resonance ,010402 general chemistry ,Condensed Matter Physics ,01 natural sciences ,Biochemistry ,Molecular physics ,Optical spectrometer ,Spectral line ,030218 nuclear medicine & medical imaging ,0104 chemical sciences ,Characterization (materials science) ,law.invention ,03 medical and health sciences ,0302 clinical medicine ,law ,High field ,Spectroscopy - Abstract
We present and discuss the performance of 1H electron-nuclear double resonance (ENDOR) at 263 GHz/9.4 T by employing a prototype, commercial quasi optical spectrometer. Basic instrumental features of the setup are described alongside a comprehensive characterization of the new ENDOR probe head design. The performance of three different ENDOR pulse sequences (Davies, Mims and CP-ENDOR) is evaluated using the 1H BDPA radical. A key feature of 263 GHz spectroscopy – the increase in orientation selectivity in comparison with 94 GHz experiments – is discussed in detail. For this purpose, the resolution of 1H ENDOR spectra at 263 GHz is verified using a representative protein sample containing approximately 15 picomoles of a tyrosyl radical. Davies ENDOR spectra recorded at 5 K reveal previously obscured spectral features, which are interpreted by spectral simulations aided by DFT calculations. Our analysis shows that seven internal proton couplings are detectable for this specific radical if sufficient orientation selectivity is achieved. The results prove the fidelity of 263 GHz experiments in reporting orientation-selected 1H ENDOR spectra and demonstrate that new significant information can be uncovered in complex molecular systems, owing to the enhanced resolution combined with high absolute sensitivity and no compromise in acquisition time.
- Published
- 2019
31. Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-α mutant unmasks a new conformation of the pathway residue 731
- Author
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JoAnne Stubbe, Müge Kasanmascheff, Wankyu Lee, Marina Bennati, and Thomas U. Nick
- Subjects
chemistry.chemical_classification ,Conformational change ,Ribonucleotide ,biology ,Chemistry ,Stereochemistry ,Allosteric regulation ,Active site ,02 engineering and technology ,General Chemistry ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,0104 chemical sciences ,E. coli ribonucleotide ,RNRs ,Crystallography ,Electron transfer ,Ribonucleotide reductase ,Catalytic cycle ,biology.protein ,Nucleotide ,0210 nano-technology - Abstract
Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all living organisms. The catalytic cycle of E. coli RNR involves a long-range proton-coupled electron transfer (PCET) from a tyrosyl radical (Y122c) in subunit b2 to a cysteine (C439) in the active site of subunit a2, which subsequently initiates nucleotide reduction. This oxidation occurs over 35 °A and involves a specific pathway of redox active amino acids (Y1224[W48?]4Y356 in b2 to Y7314Y7304C439 in a2). The mechanisms of the PCET steps at the interface of the a2b2 complex remain puzzling due to a lack of structural information for this region. Recently, DFT calculations on the 3-aminotyrosyl radical (NH2Y731c)-a2 trapped by incubation of NH2Y731-a2/b2/CDP(substrate)/ATP(allosteric effector) suggested that R411-a2, a residue close to the a2b2 interface, interacts with NH2Y731c and accounts in part for its perturbed EPR parameters. To examine its role, we further modified NH2Y731-a2 with a R411A substitution. NH2Y731c/ R411A generated upon incubation of NH2Y731/R411A-a2/b2/CDP/ATP was investigated using multifrequency (34, 94 and 263 GHz) EPR, 34 GHz pulsed electron–electron double resonance (PELDOR) and electron–nuclear double resonance (ENDOR) spectroscopies. The data indicate a large conformational change in NH2Y731c/R411A relative to the NH2Y731c single mutant. Particularly, the inter-spin distance from NH2Y731c/R411A in one ab pair to Y122c in a second ab pair decreases by 3 °A in the presence of the R411A mutation. This is the first experimental evidence for the flexibility of pathway residue Y731-a2 in an a2b2 complex and suggests a role for R411 in the stacked Y731/Y730 conformation involved in collinear PCET. Furthermore, NH2Y731c/R411A serves as a probe of the PCET process across the subunit interface. peerReviewed
- Published
- 2016
32. Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm
- Author
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Wankyu Lee, Müge Kasanmascheff, Daniel G. Nocera, Cecilia Tommos, JoAnne Stubbe, Michael Huynh, Marina Bennati, Anthony Quartararo, Cyrille Costentin, and Isabel Bejenke
- Subjects
0301 basic medicine ,Stereochemistry ,010402 general chemistry ,medicine.disease_cause ,01 natural sciences ,Biochemistry ,Redox ,Article ,law.invention ,03 medical and health sciences ,law ,Ribonucleotide Reductases ,medicine ,Escherichia coli ,Electron paramagnetic resonance ,chemistry.chemical_classification ,Aqueous solution ,Molecular Structure ,Chemistry ,0104 chemical sciences ,Amino acid ,030104 developmental biology ,Ribonucleotide reductase ,Enzyme ,Transfer RNA ,Tyrosine ,Oxidation-Reduction - Abstract
3-Aminotyrosine (NH2Y) has been a useful probe to study the role of redox active tyrosines in enzymes. This report describes properties of NH2Y of key importance for its application in mechanistic studies. By combining the tRNA/NH2Y-RS suppression technology with a model protein tailored for amino acid redox studies (α3X, X = NH2Y), the formal reduction potential of NH2Y32(O•/OH) (E°’ = 395 ± 7 mV at pH 7.08 ± 0.05) could be determined using protein film voltammetry. We find that the ΔE°’ between NH2Y32(O•/OH) and Y32(O•/OH) when measured under reversible conditions is ~300 – 400 mV larger than earlier estimates based on irreversible voltammograms obtained on aqueous NH2Y and Y. We have also generated D6-NH2Y731-α2 of RNR, which when incubated with β2/CDP/ATP generates the D6-NH2Y731•-α2/β2 complex. By multi-frequency EPR (35, 94 and 263 GHz) and 34 GHz 1H ENDOR spectroscopies, we determined the hyperfine coupling (hfc) constants of the amino protons that establishes RNH2• planarity and thus minimal perturbation of the reduction potential by the protein environment. The amount of Y in the isolated NH2Y-RNR incorporated by infidelity of the NH2YRS/tRNA pair was determined by a generally useful LC-MS method. This information is essential to the usefulness of this NH2Y probe to study any protein of interest and is employed to address our previously reported activity associated with NH2Y-substituted RNRs.
- Published
- 2018
33. Photo-induced radical polarization and liquid-state dynamic nuclear polarization using fullerene nitroxide derivatives
- Author
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Marina Bennati, Nikolay Enkin, Igor Tkach, Shu-Hao Liou, and Guoquan Liu
- Subjects
Nitroxide mediated radical polymerization ,Fullerene ,010405 organic chemistry ,Chemistry ,Radical ,General Physics and Astronomy ,010402 general chemistry ,Photochemistry ,01 natural sciences ,Toluene ,0104 chemical sciences ,Solvent ,chemistry.chemical_compound ,Physics::Atomic and Molecular Clusters ,Moiety ,Physical and Theoretical Chemistry ,Physics::Chemical Physics ,Polarization (electrochemistry) ,Excitation - Abstract
We report on radical polarization and optically-driven liquid DNP using nitroxide radicals functionalized by photoexcitable fullerene derivatives. Pulse laser excitation of the fullerene moiety leads to transient nitroxide radical polarization that is one order of magnitude larger than that at the Boltzmann equilibrium. The life time of the radical polarization increases with the size of the fullerene derivative and is correlated with the electronic spin-lattice relaxation time T1e. Overhauser NMR signal enhancements of toluene solvent protons were observed under steady-state illumination, which replaced microwave irradiation.
- Published
- 2017
34. Spectroscopic evidence for a H bond network at Y356 located at the subunit interface of active E. coli ribonucleotide reductase
- Author
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Müge Kasanmascheff, JoAnne Stubbe, Marina Bennati, Kanchana R. Ravichandran, and Thomas U. Nick
- Subjects
chemistry.chemical_classification ,biology ,Stereochemistry ,Chemistry ,Protein subunit ,02 engineering and technology ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,Biochemistry ,Cofactor ,0104 chemical sciences ,law.invention ,Amino acid ,Catalysis ,Electron transfer ,Crystallography ,Ribonucleotide reductase ,law ,biology.protein ,High field ,0210 nano-technology ,Electron paramagnetic resonance - Abstract
The reaction catalyzed by E. coli ribonucleotide reductase (RNR) composed of α and β subunits that form an active α2β2 complex is a paradigm for proton-coupled electron transfer (PCET) processes in biological transformations. β2 contains the diferric tyrosyl radical (Y122·) cofactor that initiates radical transfer (RT) over 35 Å via a specific pathway of amino acids (Y122· ⇆ [W48] ⇆ Y356 in β2 to Y731 ⇆ Y730 ⇆ C439 in α2). Experimental evidence exists for colinear and orthogonal PCET in α2 and β2, respectively. No mechanistic model yet exists for the PCET across the subunit (α/β) interface. Here, we report unique EPR spectroscopic features of Y356·-β, the pathway intermediate generated by the reaction of 2,3,5-F3Y122·-β2/CDP/ATP with wt-α2, Y731F-α2, or Y730F-α2. High field EPR (94 and 263 GHz) reveals a dramatically perturbed g tensor. [1H] and [2H]-ENDOR reveal two exchangeable H bonds to Y356·: a moderate one almost in-plane with the π-system and a weak one. DFT calculation on small models of Y· indicates that two in-plane, moderate H bonds (rO–H ∼1.8–1.9 Å) are required to reproduce the gx value of Y356· (wt-α2). The results are consistent with a model, in which a cluster of two, almost symmetrically oriented, water molecules provide the two moderate H bonds to Y356· that likely form a hydrogen bond network of water molecules involved in either the reversible PCET across the subunit interface or in H+ release to the solvent during Y356 oxidation.
- Published
- 2017
35. Spectroscopic Evidence for a H Bond Network at Y
- Author
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Thomas U, Nick, Kanchana R, Ravichandran, JoAnne, Stubbe, Müge, Kasanmascheff, and Marina, Bennati
- Subjects
Electron Transport ,Models, Molecular ,Protein Subunits ,Ribonucleotide Reductases ,Electron Spin Resonance Spectroscopy ,Escherichia coli ,Water ,Hydrogen Bonding - Abstract
The reaction catalyzed by E. coli ribonucleotide reductase (RNR) composed of α and β subunits that form an active α2β2 complex is a paradigm for proton-coupled electron transfer (PCET) processes in biological transformations. β2 contains the diferric tyrosyl radical (Y
- Published
- 2017
36. EPR Interactions - Hyperfine Couplings
- Author
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Marina Bennati
- Subjects
Materials science ,Condensed matter physics ,010405 organic chemistry ,law ,Atomic physics ,010402 general chemistry ,Electron paramagnetic resonance ,01 natural sciences ,Hyperfine structure ,0104 chemical sciences ,law.invention - Published
- 2017
37. Advanced electron paramagnetic resonance on the catalytic iron–sulfur cluster bound to the CCG domain of heterodisulfide reductase and succinate: quinone reductase
- Author
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Ulrich Ermler, Bernhard Jaun, Alistair J. Fielding, Silvan Scheller, Kristian Parey, and Marina Bennati
- Subjects
Iron-Sulfur Proteins ,Methanobacteriaceae ,Molecular Sequence Data ,ved/biology.organism_classification_rank.species ,Succinic Acid ,Iron–sulfur cluster ,Reductase ,010402 general chemistry ,Photochemistry ,01 natural sciences ,Biochemistry ,law.invention ,Inorganic Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,law ,Methanothermobacter marburgensis ,NAD(P)H Dehydrogenase (Quinone) ,Amino Acid Sequence ,Electron paramagnetic resonance ,030304 developmental biology ,0303 health sciences ,biology ,Chemistry ,ved/biology ,Sulfolobus solfataricus ,Electron Spin Resonance Spectroscopy ,Substrate (chemistry) ,biology.organism_classification ,Protein Structure, Tertiary ,0104 chemical sciences ,Quinone ,Crystallography ,Oxidoreductases ,Protein Binding - Abstract
Heterodisulfide reductase (Hdr) is a key enzyme in the energy metabolism of methanogenic archaea. The enzyme catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) to the thiol coenzymes M (CoM-SH) and B (CoB-SH). Cleavage of CoM-S-S-CoB at an unusual FeS cluster reveals unique substrate chemistry. The cluster is fixed by cysteines of two cysteine-rich CCG domain sequence motifs (CX_(31–39)CCX_(35–36)CXXC) of subunit HdrB of the Methanothermobacter marburgensis HdrABC complex. We report on Q-band (34 GHz) ^(57)Fe electron-nuclear double resonance (ENDOR) spectroscopic measurements on the oxidized form of the cluster found in HdrABC and in two other CCG-domain-containing proteins, recombinant HdrB of Hdr from M. marburgensis and recombinant SdhE of succinate: quinone reductase from Sulfolobus solfataricus P2. The spectra at 34 GHz show clearly improved resolution arising from the absence of proton resonances and polarization effects. Systematic spectral simulations of 34 GHz data combined with previous 9 GHz data allowed the unambiguous assignment of four ^(57)Fe hyperfine couplings to the cluster in all three proteins. ^(13)C Mims ENDOR spectra of labelled CoM-SH were consistent with the attachment of the substrate to the cluster in HdrABC, whereas in the other two proteins no substrate is present. ^(57)Fe resonances in all three systems revealed unusually large ^(57)Fe ENDOR hyperfine splitting as compared to known systems. The results infer that the cluster’s unique magnetic properties arise from the CCG binding motif.
- Published
- 2013
38. Long-Range Distances in Amyloid Fibrils of α-Synuclein from PELDOR Spectroscopy
- Author
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Marina Bennati, Soraya Pornsuwan, Stefan Becker, Karin Giller, Dietmar Riedel, and Christian Griesinger
- Subjects
fibrils ,Amyloid ,Biophysics Very Important Paper ,Molecular Sequence Data ,Fibril ,Catalysis ,law.invention ,chemistry.chemical_compound ,law ,biophysics ,Amino Acid Sequence ,Electron paramagnetic resonance ,Peptide sequence ,Alpha-synuclein ,chemistry.chemical_classification ,Electron Spin Resonance Spectroscopy ,General Medicine ,General Chemistry ,Communications ,proteins ,Amino acid ,Crystallography ,Monomer ,chemistry ,Electron diffraction ,alpha-Synuclein ,distance measurement ,EPR spectroscopy - Abstract
α-Synuclein (aS), a small protein containing 140 amino acids, undergoes self-assembly into amyloid fibrils and plaques (Lewy bodies), which are pathological hallmarks of Parkinson’s disease (PD) as well as other neurodegenerative diseases.[1] While oligomeric species of aS are considered to exert the neurotoxic activity[2] that can be rescued by reducing those oligomers either by diversion to smaller oligomers[3] or acceleration of fibril formation, cell-to-cell transmission in nontransgenic mice points to a direct role of the fibrils in spreading the disease from peripheral to central neurons.[4] Understanding the molecular interactions that lead to misfolding strongly relies on the availability of suited biophysical methods that can access the structure of these states. For the monomeric form, magnetic resonance techniques gave evidence for a natively disordered yet partially folded protein[5a,b] that upon binding to lipid vesicles adopts an α-helical structure.[6–8] When aS is aggregated into fibrils, X-ray and electron diffraction studies[9a,b] revealed that it arranges in a classical cross-β conformation, where the individual β-sheets arrange perpendicularly to the fibril axis with spacing of 4.7–4.8 A along the fibril axis and 10–11 A perpendicular to the axis. In particular, aS stacks in a parallel, in-register arrangement as revealed by continuous-wave (CW) electron paramagnetic resonance (EPR)[10] and more recently supported by solid-state nuclear magnetic resonance (ss-NMR) spectroscopy.[11]
- Published
- 2013
39. High-resolution measurement of long-range distances in RNA: pulse EPR spectroscopy with TEMPO-labeled nucleotides
- Author
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Igor Tkach, Deniz Sezer, Marina Bennati, Jan Seikowski, Karin Halbmair, and Claudia Höbartner
- Subjects
0301 basic medicine ,Chemistry ,Pulsed EPR ,Base pair ,RNA ,General Chemistry ,Site-directed spin labeling ,010402 general chemistry ,01 natural sciences ,Molecular physics ,behavioral disciplines and activities ,humanities ,0104 chemical sciences ,Nucleobase ,law.invention ,03 medical and health sciences ,Crystallography ,030104 developmental biology ,law ,Nucleic acid structure ,Electron paramagnetic resonance ,long-range distances ,EPR spectroscopy ,QD001-65 General ,Macromolecule - Abstract
Distance measurements in RNAs by pulse EPR with TEMPO-labeled nucleotides allow for model free conversion of distances into base-pair separation., Structural information at atomic resolution of biomolecular assemblies, such as RNA and RNA protein complexes, is fundamental to comprehend biological function. Modern spectroscopic methods offer exceptional opportunities in this direction. Here we present the capability of pulse EPR to report high-resolution long-range distances in RNAs by means of a recently developed spin labeled nucleotide, which carries the TEMPO group directly attached to the nucleobase and preserves Watson–Crick base-pairing. In a representative RNA duplex with spin-label separations up to 28 base pairs (≈8 nm) we demonstrate that the label allows for a model-free conversion of inter-spin distances into base-pair separation (Δbp) if broad-band pulse excitation at Q band frequencies (34 GHz) is applied. The observed distance distribution increases from ±0.2 nm for Δbp = 10 to only ±0.5 nm for Δbp = 28, consistent with only small deviations from the “ideal” A-form RNA structure. Molecular dynamics (MD) simulations conducted at 20 °C show restricted conformational freedom of the label. MD-generated structural deviations from an “ideal” A-RNA geometry help disentangle the contributions of local flexibility of the label and its neighboring nucleobases and global deformations of the RNA double helix to the experimental distance distributions. The study demonstrates that our simple but strategic spin labeling procedure can access detailed structural information on RNAs at atomic resolution over distances that match the size of macromolecular RNA complexes.
- Published
- 2015
40. Linoleic acid positioning in psi factor producing oxygenase A, a fusion protein with an atypical cytochrome P450 activity
- Author
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Florian Brodhun, Ivo Feussner, Marina Bennati, Alistair J. Fielding, and Christian Koch
- Subjects
0303 health sciences ,Oxygenase ,Stereochemistry ,030302 biochemistry & molecular biology ,Substrate (chemistry) ,Cell Biology ,Biology ,Biochemistry ,Fusion protein ,03 medical and health sciences ,Protein structure ,Dioxygenase ,Phosphofructokinase 2 ,Asparagine ,Binding site ,Molecular Biology ,030304 developmental biology - Abstract
Psi factor producing oxygenases (Ppos) are fusion proteins consisting of a peroxidase-like functionality in the N-terminus and a P450-fold in the C-terminal part of the polypeptide chain. It was shown that they are responsible for the production of oxidized fatty acids that play a pivotal role in the control of fungal colonization of plant and mammalian hosts. The similarity of the primary structure of the single domains to various host-derived oxylipin-forming enzymes and functional conservation of these enzymatic activities was the basis for prediction of the 3D conformations of the single domains of a prototype Ppo enzyme. We were able to predict a putative substrate binding pocket in the N-terminal domain of the enzyme and support this finding by site-directed mutagenesis. With the proposed substrate binding mode all known determinants of oxygen insertion are in a reasonable spatial arrangement for catalysis. Additionally, we could identify an arginine and show its involvement in substrate binding by kinetic analysis of the respective variant. While substrate position in the dioxygenase domain is well defined, our results indicate that the substrate binding to the P450 domain is rather unconstrained. Nevertheless an asparagine residue within the I-helix is shown to be involved in catalysis and promotes a shortcut of the typical P450 reaction cycle. Taken together, the results presented here exemplify that fatty acids are oxidized in all kingdoms of life by structural and functional highly conserved enzymes.
- Published
- 2011
41. TOPP - eine Aminosäure mit Nitroxid-Spinmarkierungen für EPR-Abstandsmessungen
- Author
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Giuseppe Sicoli, Marina Bennati, Ulf Diederichsen, Tatiana Y. Baranova, and Sven Stoller
- Subjects
Materials science ,010405 organic chemistry ,General Medicine ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences - Published
- 2011
42. Multifrequency Electron Paramagnetic Resonance Characterization of PpoA, a CYP450 Fusion Protein that Catalyzes Fatty Acid Dioxygenation
- Author
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Christian Koch, Florian Brodhun, Marina Bennati, Ivo Feussner, Alistair J. Fielding, Vasyl Denysenkov, and Roberta Pievo
- Subjects
Reaction mechanism ,Free Radicals ,Stereochemistry ,Recombinant Fusion Proteins ,Radical ,Heme ,010402 general chemistry ,Photochemistry ,01 natural sciences ,Biochemistry ,Catalysis ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Cytochrome P-450 Enzyme System ,Dioxygenase ,law ,Histidine ,Electron paramagnetic resonance ,030304 developmental biology ,0303 health sciences ,Pulsed EPR ,Reaction step ,Fatty Acids ,Electron Spin Resonance Spectroscopy ,General Chemistry ,0104 chemical sciences ,Oxygen ,chemistry ,Mutation ,Biocatalysis ,Tyrosine - Abstract
PpoA is a fungal dioxygenase that produces hydroxylated fatty acids involved in the regulation of the life cycle and secondary metabolism of Aspergillus nidulans . It was recently proposed that this novel enzyme employs two different heme domains to catalyze two separate reactions: within a heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid [(8R)-HPODE]; in the second reaction step (8R)-HPODE is isomerized within a P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. In the present study, pulsed EPR methods were applied to find spectroscopic evidence for the reaction mechanism, thought to involve paramagnetic intermediates. We observe EPR resonances of two distinct heme centers with g-values typical for Fe(III) S = (5)/(2) high-spin (HS) and Fe(III) S = (1)/(2) low-spin (LS) hemes. (14)N ENDOR spectroscopy on the S = (5)/(2) signal reveals resonances consistent with an axial histidine ligation. Reaction of PpoA with the substrate leads to the formation of an amino acid radical on the early millisecond time scale concomitant to a substantial reduction of the S = (5)/(2) heme signal. High-frequency EPR (95- and 180-GHz) unambiguously identifies the new radical as a tyrosyl, based on g-values and hyperfine couplings from spectral simulations. The radical displays enhanced T(1)-spin-lattice relaxation due to the proximity of the heme centers. Further, EPR distance measurements revealed that the radical is distributed among the monomeric subunits of the tetrameric enzyme at a distance of approximately 5 nm. The identification of three active paramagnetic centers involved in the reaction of PpoA supports the previously proposed reaction mechanism based on radical chemistry.
- Published
- 2011
43. A dual-mode microwave resonator for double electron–electron spin resonance spectroscopy at W-band microwave frequencies
- Author
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Claudia Höbartner, Giuseppe Sicoli, Igor Tkach, and Marina Bennati
- Subjects
Electromagnetic field ,Nuclear and High Energy Physics ,Chemistry ,Electron Spin Resonance Spectroscopy ,Biophysics ,Analytical chemistry ,Equipment Design ,Electron ,Condensed Matter Physics ,Polarization (waves) ,Biochemistry ,Resonator ,Electromagnetic Fields ,W band ,Frequency separation ,Computer Simulation ,Atomic physics ,Microwaves ,Spectroscopy ,Software ,Microwave - Abstract
We present a dual-mode resonator operating at/near 94 GHz (W-band) microwave frequencies and supporting two microwave modes with the same field polarization at the sample position. Numerical analysis shows that the frequencies of both modes as well as their frequency separation can be tuned in a broad range up to GHz. The resonator was constructed to perform pulsed ELDOR experiments with a variable separation of "pump" and "detection" frequencies up to Δν=350 MHz. To examine its performance, test ESE/PELDOR experiments were performed on a representative biradical system.
- Published
- 2011
44. Sekundärstrukturanalyse von spinmarkierter RNA mit Puls-EPR-Spektroskopie
- Author
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Giuseppe Sicoli, Marina Bennati, Falk Wachowius, and Claudia Höbartner
- Subjects
Stereochemistry ,Chemistry ,RNA ,General Medicine - Published
- 2010
45. Probing Secondary Structures of Spin-Labeled RNA by Pulsed EPR Spectroscopy
- Author
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Giuseppe Sicoli, Marina Bennati, Falk Wachowius, and Claudia Höbartner
- Subjects
Riboswitch ,Base Sequence ,Chemistry ,Guanine ,Stereochemistry ,RNA Conformation ,Electron Spin Resonance Spectroscopy ,RNA ,General Chemistry ,Site-directed spin labeling ,Catalysis ,Nucleobase ,chemistry.chemical_compound ,Crystallography ,Nucleic acid ,Nucleic Acid Conformation ,Spin Labels ,Oligonucleotide Probes ,DNA - Abstract
The ability of RNA to interconvert between multiple conformational states is essential for the diversity of biological functions that have been discovered in the recent past. For example, the correct operation of regulatory RNA elements, such as riboswitches, is based on the precise interplay of alternative RNA conformations. Studying the molecular mechanisms of RNA function entails probing RNA-folding intermediates on the energy landscape. EPR spectroscopy, in particular, has been increasingly applied to obtain structural information on nucleic acids, including local conformational changes in RNA and the identification of metal-ion binding sites. Pulsed EPR techniques (PELDOR/ DEER) have been used to determine distances between paramagnetic centers in specifically modified RNA. PELDOR should therefore be suitable for the detection of alternative RNA conformations that involve distinct changes in base-pairing patterns. The accessibility of spin-labeled RNA still poses the major challenge for the widespread applicability of powerful EPR techniques. Nitroxide radicals are the most commonly used type of paramagnetic labels for nucleic acids. Several methods have been reported for attaching nitroxide groups at internal positions at the ribose, the phosphate backbone, or at nucleobases, often by means of multiatom linkers that provide several unwanted degrees of rotational freedom. Rigid nitroxide spin labels conjugated to the nucleobase or to nucleobase analogues have been reported for DNA. Our RNA spin-labeling approach addresses the direct attachment of nitroxide labels onto RNA nucleobases, such that conformational changes can be directly detected by PELDOR (i.e., by the change in distance between two labeled nucleotides). The nucleobase spin labels used in this study are also designed to preserve the Watson–Crick base-pairing capability of labeled nucleotides and not to interfere with alternative base-pairing patterns in different RNA conformations. Here, we describe the installation of nitroxide spin labels on exocyclic amino groups of the RNA nucleobases guanine, adenine, and cytosine (Figure 1) with unprecedented effi
- Published
- 2010
46. Structural Examination of the Transient 3-Aminotyrosyl Radical on the PCET Pathway of E. coli Ribonucleotide Reductase by Multifrequency EPR Spectroscopy
- Author
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Mohammad R. Seyedsayamdost, Tomislav Argirević, JoAnne Stubbe, Ellen Catherine Minnihan, and Marina Bennati
- Subjects
Models, Molecular ,Protein Conformation ,Stereochemistry ,Protein subunit ,Allosteric regulation ,Photochemistry ,Biochemistry ,Article ,Catalysis ,law.invention ,Electron transfer ,Colloid and Surface Chemistry ,law ,Ribonucleotide Reductases ,Escherichia coli ,Nucleotide ,Electron paramagnetic resonance ,chemistry.chemical_classification ,Pulsed EPR ,Electron Spin Resonance Spectroscopy ,General Chemistry ,Ribonucleotide reductase ,chemistry ,Isotope Labeling ,Transfer RNA ,Tyrosine - Abstract
E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 A from a tyrosyl radical (Y(122)*) within the beta2 subunit to a cysteine residue (C(439)) within the alpha2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y(122) --W(48) --Y(356) in beta2, across the subunit interface to Y(731) --Y(730) --C(439) in alpha2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in alpha2. Incubation of this mutant with beta2, substrate, and allosteric effector resulted in loss of the Y(122)* and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH(2)Y*). In the current study [(15)N]- and [(14)N]-NH(2)Y(730)* have been generated in H(2)O and D(2)O and characterized by continuous wave 9 GHz EPR and pulsed EPR spectroscopies at 9, 94, and 180 GHz. The data give insight into the electronic and molecular structure of NH(2)Y(730)*. The g tensor (g(x) = 2.0052, g(y) = 2.0042, g(z) = 2.0022), the orientation of the beta-protons, the hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the aromatic ring were determined. The hyperfine coupling constants and geometry of the NH(2) moiety are consistent with an intramolecular hydrogen bond within NH(2)Y(730)*. This analysis is an essential first step in using the detailed structure of NH(2)Y(730)* to formulate a model for a PCET mechanism within alpha2 and for use of NH(2)Y in other systems where transient Y*s participate in catalysis.
- Published
- 2009
47. Cross-Polarization Electron-Nuclear Double Resonance Spectroscopy
- Author
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Marina Bennati and Roberto Rizzato
- Subjects
Electron nuclear double resonance ,Spins ,Pulsed EPR ,Chemistry ,Electron Spin Resonance Spectroscopy ,Resonance ,Atomic and Molecular Physics, and Optics ,law.invention ,Paramagnetism ,Ribonucleotide reductase ,Nuclear magnetic resonance ,law ,Ribonucleotide Reductases ,Escherichia coli ,Physical and Theoretical Chemistry ,Spectroscopy ,Electron paramagnetic resonance - Abstract
Magnetic nuclei in the proximity of a paramagnetic center can be polarized through electron-nuclear cross-polarization and detected in electron-nuclear double resonance (ENDOR) spectroscopy. This principle is demonstrated in a single-crystal model sample as well as on a protein, the β2 subunit of E.coli ribonucleotide reductase (RNR), which contains an essential tyrosyl radical. ENDOR is a fundamental technique to detect magnetic nuclei coupled to paramagnetic centers. It is widely employed in biological and materials sciences. Despite its utility, its sensitivity in real samples is about one to two orders of magnitude lower than conventional electron paramagnetic resonance, thus restricting its application potential. Herein, we report the performance of a recently introduced concept to polarize nuclear spins and detect their ENDOR spectrum, which is based on electron-nuclear cross polarization (eNCP). A single-crystal study permits us to disentangle eNCP conditions and CP-ENDOR intensities, providing the experimental foundation in agreement with the theoretical prediction. The CP-ENDOR performance on a real protein sample is best demonstrated with the spectra of the essential tyrosyl radical in the β2 subunit of E.coli RNR.
- Published
- 2015
48. High-Field Electron Paramagnetic Resonance and Density Functional Theory Study of Stable Organic Radicals in Lignin: Influence of the Extraction Process, Botanical Origin, and Protonation Reactions on the Radical g Tensor
- Author
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Christian Bährle, Frédéric Vogel, Gunnar Jeschke, Thomas U. Nick, and Marina Bennati
- Subjects
chemistry.chemical_classification ,Models, Molecular ,Resolution (mass spectrometry) ,Chemistry ,Radical ,Extraction (chemistry) ,Electron Spin Resonance Spectroscopy ,Protonation ,Polymer ,Lignin ,Wood ,law.invention ,chemistry.chemical_compound ,law ,Benzoquinones ,Organic chemistry ,Physical chemistry ,Density functional theory ,Physical and Theoretical Chemistry ,Protons ,Electron paramagnetic resonance - Abstract
The radical concentrations and g factors of stable organic radicals in different lignin preparations were determined by X-band EPR at 9 GHz. We observed that the g factors of these radicals are largely determined by the extraction process and not by the botanical origin of the lignin. The parameter mostly influencing the g factor is the pH value during lignin extraction. This effect was studied in depth using high-field EPR spectroscopy at 263 GHz. We were able to determine the gxx, gyy, and gzz components of the g tensor of the stable organic radicals in lignin. With the enhanced resolution of high-field EPR, distinct radical species could be found in this complex polymer. The radical species are assigned to substituted o-semiquinone radicals and can exist in different protonation states SH3+, SH2, SH1-, and S2-. The proposed model structures are supported by DFT calculations. The g principal values of the proposed structure were all in reasonable agreement with the experiments.
- Published
- 2015
49. Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli α2 ribonucleotide reductase
- Author
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Marina Bennati, Frank Neese, Wankyu Lee, Simone Koßmann, JoAnne Stubbe, and Thomas U. Nick
- Subjects
Models, Molecular ,Ribonucleotide ,Free Radicals ,Stereochemistry ,Molecular Conformation ,Biochemistry ,Deoxyribonucleotides ,Catalysis ,Article ,Electron Transport ,Colloid and Surface Chemistry ,Ribonucleotide Reductases ,Escherichia coli ,Nucleotide ,Amino Acids ,chemistry.chemical_classification ,biology ,Chemistry ,Active site ,Hydrogen Bonding ,General Chemistry ,Electron transport chain ,Amino acid ,Ribonucleotide reductase ,13. Climate action ,biology.protein ,Proton-coupled electron transfer ,Protons - Abstract
Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y122•, Escherichia coli) in subunit β of a cysteine (C439) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y122 ⇆ Y356 in β to Y731 ⇆ Y730 ⇆ C439 in α); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y356-β, Y731- and Y730-α, and each protein was incubated with the appropriate second subunit β(α), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y•) in the active α2β2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH2Y•s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. (2)H electron-nuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH2Y730•, one to NH2Y731• and none to NH2Y356•. Similar experiments with double mutants α-NH2Y730/C439A and α-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within α. The implications of these observations for the PCET process within α and at the interface are discussed.
- Published
- 2014
50. High-frequency 180 GHz PELDOR
- Author
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Thomas F. Prisner, JoAnne Stubbe, Marina Bennati, and Vasyl Denysenkov
- Subjects
Spectrometer ,Solid-state physics ,Pulsed EPR ,Chemistry ,Analytical chemistry ,Resonance ,Molecular physics ,Atomic and Molecular Physics, and Optics ,law.invention ,Magnetic field ,Dipole ,Paramagnetism ,law ,Electron paramagnetic resonance - Abstract
For aromatic organic radicals, pulsed electron-electron double resonance (PELDOR) experiments at high magnetic fields offer the possibility to achieve orientation-selective pumping and detection that could allow one not only to determine the distance between paramagnetic species but also their relative orientation with respect to the interconnecting dipolar axis. We present a PELDOR two-frequency setup that was introduced into our homebuilt 180 GHz pulsed electron paramagnetic resonance (EPR) spectrometer and we discuss its technical and experimental features. The capability of 180 GHz PELDOR has been tested using the three-pulse ELDOR sequence on the protein RNR-R2 (ribonucleotide reductase) fromEscherichia coli, which contains two tyrosyl radicals at a distance of 3.3 nm. At 180 GHz, orientation selectivity is observed and the modulation frequency was found in good agreement with theoretical predictions, which take into account the relative orientation of the radicals from X-ray data.
- Published
- 2005
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