87 results on '"Marina Bennati"'
Search Results
2. 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
3. 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
4. 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
5. 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
6. 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
7. 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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8. 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
9. 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.
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- 2020
10. One-thousand-fold enhancement of high field liquid nuclear magnetic resonance signals at room temperature
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Marina Bennati, Niels Karschin, Marcel Levien, Guoquan Liu, Claudio Luchinat, and Giacomo Parigi
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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.
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- 2017
11. Pulse EPR Measurements of Intramolecular Distances in a TOPP-Labeled Transmembrane Peptide in Lipids
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Marina Bennati, Karin Halbmair, Ulf Diederichsen, and Janine Wegner
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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.
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- 2016
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12. Structural heterogeneity of α-synuclein fibrils amplified from patient brain extracts
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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
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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.
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- 2019
13. Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-α mutant unmasks a new conformation of the pathway residue 731
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JoAnne Stubbe, Müge Kasanmascheff, Wankyu Lee, Marina Bennati, and Thomas U. Nick
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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
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- 2016
14. Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm
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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
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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
15. Photo-induced radical polarization and liquid-state dynamic nuclear polarization using fullerene nitroxide derivatives
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Marina Bennati, Nikolay Enkin, Igor Tkach, Shu-Hao Liou, and Guoquan Liu
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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
16. Spectroscopic evidence for a H bond network at Y356 located at the subunit interface of active E. coli ribonucleotide reductase
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Müge Kasanmascheff, JoAnne Stubbe, Marina Bennati, Kanchana R. Ravichandran, and Thomas U. Nick
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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.
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- 2017
17. Advanced electron paramagnetic resonance on the catalytic iron–sulfur cluster bound to the CCG domain of heterodisulfide reductase and succinate: quinone reductase
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Ulrich Ermler, Bernhard Jaun, Alistair J. Fielding, Silvan Scheller, Kristian Parey, and Marina Bennati
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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
18. 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
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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
19. 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
20. 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
21. A dual-mode microwave resonator for double electron–electron spin resonance spectroscopy at W-band microwave frequencies
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Claudia Höbartner, Giuseppe Sicoli, Igor Tkach, and Marina Bennati
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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
22. 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
23. 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
24. 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
25. 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
26. 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
27. 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
28. 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
29. Pulsed 180-GHz EPR/ENDOR/PELDOR spectroscopy
- Author
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Thomas F. Prisner, Marina Bennati, M. M. Hertel, and Vasyl Denysenkov
- Subjects
Manganese ,Electron nuclear double resonance ,Free Radicals ,Spectrometer ,Chemistry ,Pulsed EPR ,Electron Spin Resonance Spectroscopy ,Resonance ,General Chemistry ,Electron ,Guanosine Diphosphate ,Spectral line ,law.invention ,Cyclic N-Oxides ,Nuclear magnetic resonance ,law ,Ribonucleotide Reductases ,Escherichia coli ,ras Proteins ,Polystyrenes ,Tyrosine ,General Materials Science ,Spectroscopy ,Electron paramagnetic resonance - Abstract
Within this review, we describe a home-built pulsed electron paramagnetic resonance (EPR) spectrometer operating at 180 GHz as well as the incorporation of two double resonance techniques, electron nuclear double resonance (ENDOR) and pulsed electron double resonance (PELDOR), along with first applications. Hahn-echo decays on a TEMPO/polystyrene sample are presented, demonstrating that the observation of anisotropic librational motions is possible in a very precise manner at high magnetic fields. Bisdiphenylene-phenyl-allyl is used as a model system to illustrate the performance of the setup for 1H-ENDOR using the Mims as well as the Davies sequence. Furthermore, first 1H-Mims and Davies ENDOR spectra on a biological sample, the wild-type Ras*Mn2+*GDP protein, are reported. The capability of the 180-GHz PELDOR setup is demonstrated using the three-pulse ELDOR sequence on the protein ribonucleotide reductase (RNR) subunit R2 from Escherichia coli, which contains two tyrosyl radicals at a 33 angstroms distance. At 180 GHz, orientation selectivity is observed and the modulation frequency is found to be in good agreement with theoretical predictions.
- Published
- 2005
30. Molecular dynamics of nitroxides in glasses as studied by multi-frequency EPR
- Author
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Martin Fuchs, Evgeniya Kirilina, Thomas F. Prisner, Klaus Möbius, Burkhard Endeward, Sergei A. Dzuba, Alexander Schnegg, and Marina Bennati
- Subjects
Nitroxide mediated radical polymerization ,Chemistry ,Analytical chemistry ,Resonance ,General Chemistry ,Molecular physics ,Spectral line ,law.invention ,Spin probe ,Molecular dynamics ,law ,Relaxation (physics) ,General Materials Science ,Glass transition ,Electron paramagnetic resonance - Abstract
Pulsed multi-frequency EPR was used to investigate orientational molecular motion of the nitroxide spin probe (Fremy's salt) in glycerol glass near the glass transition temperature. By measuring echo-detected EPR spectra at different pulse separation times at resonance frequencies of 3, 9.5, 95 and 180 GHz, we were able to discriminate between different relaxation mechanisms and characterize the timescale of molecular reorientations (10(-7)-10(-10) s). We found that near the glass transition temperature, the orientation-dependent transverse relaxation is dominated by fast reorientational fluctuations, which may be overlapped with fast modulations of the canonical g-matrix values. The data was interpreted using a new simulation program for the orientation-dependent transverse relaxation rate 1/T2 of nitroxides based on different models for the molecular motion. The validity of the different models was assessed by comparing least-square fits of the simulated relaxation behaviour to the experimental data.
- Published
- 2005
31. End-to-End Distance Determination in a Cucurbit[6]uril-Based Rotaxane by PELDOR Spectroscopy
- Author
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Elisabetta Mezzina, Marco Lucarini, Paola Franchi, Costanza Casati, Marina Bennati, Roberta Pievo, R. Pievo, C. Casati, P. Franchi, E. Mezzina, M. Bennati, and M. Lucarini
- Subjects
Bridged-Ring Compounds ,NITROXIDE ,Rotaxane ,Rotaxanes ,Chemistry ,Electron Spin Resonance Spectroscopy ,Imidazoles ,Water ,Molecular Dynamics Simulation ,Photochemistry ,ELECTRON DOUBLE-RESONANCE ,Atomic and Molecular Physics, and Optics ,law.invention ,Crystallography ,law ,PULSED ELECTRON ,RADICALS ,Water chemistry ,SPIN-ECHO ,Physical and Theoretical Chemistry ,Electron paramagnetic resonance ,Spectroscopy - Abstract
Distance determination: The use of pulsed electron–electron double resonance (PELDOR/DEER) spectroscopy to determine the distances among the end units of a paramagnetic cucurbit[6]uril-based rotaxane is demonstrated.
- Published
- 2012
32. Electron Paramagnetic Resonance Spectroscopy Measures the Distance between the External β-Strands of Folded α-Synuclein in Amyloid Fibrils
- Author
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Marina Bennati, Thomas M. Jovin, Dietmar Riedel, Christian Griesinger, Karin Giller, Stefan Becker, and Irina Karyagina
- Subjects
Electron paramagnetic resonance spectroscopy ,Alpha-synuclein ,Amyloid ,Protein Folding ,Biophysical Letter ,Scattering ,Molecular Sequence Data ,Electron Spin Resonance Spectroscopy ,Biophysics ,Amyloid fibril ,Protein Structure, Secondary ,chemistry.chemical_compound ,Nuclear magnetic resonance ,chemistry ,Intramolecular force ,alpha-Synuclein ,Humans ,Mutant Proteins ,Protein folding ,α synuclein ,Amino Acid Sequence - Abstract
The misfolding of α-synuclein (αS) to a cross-β-sheet amyloid structure is associated with pathological conditions in Parkinson's and other neurodegenerative diseases. Using pulse electron paramagnetic resonance spectroscopy combined with a cross-labeling strategy involving four double mutants, we were able to determine the intramolecular distance between the extremal β-strands. The distance of 4.5 ± 0.5 nm is in good agreement with the dimensions of a protofilament reported by other low-resolution techniques, such as x-ray scattering and atomic force microscopy.
- Published
- 2011
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33. High-frequency EPR and ENDOR: Time-domain spectroscopy of ribonucleotide reductase
- Author
-
Marina Bennati, Robert G. Griffin, and JoAnne Stubbe
- Subjects
Electron nuclear double resonance ,Nuclear magnetic resonance ,Ribonucleotide reductase ,law ,Chemistry ,Relaxation (NMR) ,Electron paramagnetic resonance ,Spectroscopy ,Photochemistry ,Deoxyribonucleotides ,Atomic and Molecular Physics, and Optics ,Volume concentration ,law.invention - Abstract
This paper discusses time-domain electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) experiments aimed at elucidating the enzymatic mechanism of ribonucleotide reductase (RNR), the enzyme responsible for the conversion of ribonucleotides to deoxyribonucleotides. The article begins with a discussion of the current state of the art of instrumentation for high-frequency EPR and ENDOR and some suggestions as to future developments. We then provide an introduction to the chemistry of RNR and a discussion of the high-field EPR and ENDOR spectra of the tyrosyl radical (Y⋅) in the R2 subunit of class I RNR. Finally, we describe two examples illustrating the use of high-frequency EPR and ENDOR to elucidate the enzymatic mechanism of RNR. EPR and ENDOR have played an important role for these studies since the mechanism involves several different radical intermediates. These intermediates are all present in low concentrations relative to the Y⋅ concentration and they possess similarg-values. Spectral overlap, therefore, has been a problem with X-band EPR. At high frequencies the spectra are resolved to the point that individual powder lineshapes are discernible. In addition, we describe our approach, on the basis of differential relaxation, to suppress the spectrum of the dominant Y⋅. High-frequency EPR and ENDOR therefore has permitted us to determine the structure of several radical intermediates which in turn have contributed to the understanding of the enzymatic mechanism of RNR.
- Published
- 2001
34. Solid effect in the electron spin dressed state: A new approach for dynamic nuclear polarization
- Author
-
Robert G. Griffin, Marina Bennati, V. Weis, and Melanie Rosay
- Subjects
Spin polarization ,Chemistry ,Dynamic nuclear polarisation ,General Physics and Astronomy ,Field strength ,Nuclear Overhauser effect ,Electron ,Radio frequency ,Physical and Theoretical Chemistry ,Atomic physics ,Polarization (waves) ,Microwave - Abstract
We describe a new type of solid effect for dynamic nuclear polarization ~DNP! that is based on simultaneous, near resonant microwave ~mw! and radio frequency ~rf! irradiation of a coupled electron nuclear spin system. The interaction of the electron spin with the mw field is treated as an electron spin dressed state. In contrast to the customary laboratory frame solid effect, it is possible to obtain nuclear polarization with the dressed state solid effect ~DSSE! even in the absence of nonsecular hyperfine coupling. Efficient, selective excitation of dressed state transitions generates nuclear polarization in the nuclear laboratory frame on a time scale of tens of ms, depending on the strength of the electron‐nuclear coupling, the mw and rf offset and field strength. The experiment employs both pulsed mw and rf irradiation at a repetition rate comparable to T 1e 21 , where T 1e is the electronic spin lattice relaxation time. The DSSE is demonstrated on a perdeuterated BDPA radical in a protonated matrix of polystyrene. © 2000 American Institute of Physics. @S0021-9606~00!01340-4#
- Published
- 2000
35. Organometallic Synthesis and Spectroscopic Characterization of Manganese-Doped CdSe Nanocrystals
- Author
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Robert G. Griffin, D. A. Hall, F. V. Mikulec, Masaru Kuno, Moungi G. Bawendi, and Marina Bennati
- Subjects
Dopant ,Condensed Matter::Other ,Doping ,General Chemistry ,Condensed Matter::Mesoscopic Systems and Quantum Hall Effect ,Biochemistry ,Catalysis ,law.invention ,NMR spectra database ,Condensed Matter::Materials Science ,chemistry.chemical_compound ,Paramagnetism ,Colloid and Surface Chemistry ,chemistry ,Quantum dot ,law ,Magic angle spinning ,Physical chemistry ,Condensed Matter::Strongly Correlated Electrons ,Electron paramagnetic resonance ,Trioctylphosphine oxide - Abstract
The synthesis of II−VI semiconductor nanocrystals doped with transition metals has proved to be particularly difficult. In the case of CdSe quantum dots (QDs) produced via high-temperature pyrolysis in trioctylphosphine oxide (TOPO), specially designed precursors used in this study appear to be necessary to successfully incorporate low levels of Mn. A simple etching experiment and electron paramagnetic resonance (EPR) measurements reveal that most of the dopant atoms reside in the surface layers of the inorganic lattice. The dopant dramatically affects 113Cd magic angle spinning (MAS) nuclear magnetic resonance NMR spectra; the observed paramagnetic shift and decreased longitudinal relaxation time are consistent with Mn incorporated in the QDs. Paramagnetic atoms in QDs generate large effective magnetic fields, which implies that magnetooptical experiments can be performed simply by doping. Results from fluorescence line narrowing (FLN) studies on Mn-doped CdSe QDs mirror previous findings on undoped QDs ...
- Published
- 2000
36. Nitroxide Side-Chain Dynamics in a Spin-Labeled Helix-Forming Peptide Revealed by High-Frequency (139.5-GHz) EPR Spectroscopy
- Author
-
Marina Bennati, Gary V. Martinez, Gary J. Gerfen, Robert G. Griffin, David J. Singel, and Glenn L. Millhauser
- Subjects
Quantitative Biology::Biomolecules ,Nuclear and High Energy Physics ,Nitroxide mediated radical polymerization ,Protein Conformation ,Chemistry ,Electron Spin Resonance Spectroscopy ,Temperature ,Biophysics ,Site-directed spin labeling ,Condensed Matter Physics ,Biochemistry ,law.invention ,Crystallography ,Nuclear magnetic resonance ,law ,Helix ,Side chain ,Anisotropy ,Spin Labels ,Amino Acid Sequence ,Peptides ,Spectroscopy ,Electron paramagnetic resonance ,Spin (physics) ,Spin label - Abstract
High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy tau( perpendicular)/tau( parallel) ( identical with N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.
- Published
- 1999
37. Pulsed-EPR on the photoexcited triplet state of C60
- Author
-
Michael Mehring, Marina Bennati, and A. Grupp
- Subjects
Condensed matter physics ,Pulsed EPR ,Chemistry ,Mechanical Engineering ,Jahn–Teller effect ,Metals and Alloys ,Electronic structure ,Zero field splitting ,Condensed Matter Physics ,Molecular physics ,Spectral line ,Electronic, Optical and Magnetic Materials ,law.invention ,Mechanics of Materials ,law ,Materials Chemistry ,Molecule ,Triplet state ,Electron paramagnetic resonance - Abstract
We report on the electronic structure of photoexcited triplet states of C60 molecules in dilute solutions. The analysis of the temperature dependence and kinetics of EPR spectra clearly indicates the existence of energetically close lying triplet states with different symmetry. Zero field splitting parameters |D| between 50 G and 123.5 G are observed. The local spin density distribution on the molecule is estimated from 13C hyperfine coupling constants.
- Published
- 1997
38. Cross-polarisation edited ENDOR
- Author
-
Ilia Kaminker, Roberto Rizzato, Marina Bennati, and Shimon Vega
- Subjects
Chemistry ,Biophysics ,Resonance ,Pulse sequence ,Condensed Matter Physics ,law.invention ,Paramagnetism ,Nuclear magnetic resonance ,law ,Physical and Theoretical Chemistry ,Spectral resolution ,Atomic physics ,Spectroscopy ,Electron paramagnetic resonance ,Molecular Biology ,Hyperfine structure ,Microwave - Abstract
Electron-nuclear double resonance (ENDOR) is a fundamental technique in electron paramagnetic resonance (EPR) spectroscopy that directly detects hyperfine transitions of nuclei coupled to a paramagnetic centre. Despite its wide use, spin-sensitivity and restricted spectral resolution in powder samples pose limitations of this technique in modern application fields of EPR. In this contribution, we examine the performance of an ENDOR pulse sequence that utilises a preparation scheme different from conventional Davies ENDOR. The scheme is based on electron-nuclear cross-polarisation (eNCP), which requires concomitant microwave (MW) and radio-frequency (RF) irradiation satisfying specific matching conditions between the MW and RF offsets and the hyperfine coupling. Changes in nuclear polarisation generated during eNCP can be detected via a conventional ENDOR read-out sequence consisting of an RF π-pulse followed by EPR-spin echo detection. Using 1H-BDPA as a standard sample, we first examine the CP matching conditions by monitoring the depolarisation of the electron spin magnetisation. Subsequently, so-called CP-edited ENDOR spectra for different matching conditions are reported and analysed based on the provided theoretical description of the time evolution of the spin density matrix during the experiment. The results demonstrate that CP-edited ENDOR provides additional information with respect to the sign of the hyperfine couplings. Furthermore, the sequence is less sensitive to nuclear saturation effects encountered in conventional ENDOR.
- Published
- 2013
39. A structural model of PpoA derived from SAXS-analysis-Implications for substrate conversion
- Author
-
Kirstin Feussner, Florian Brodhun, Oliver Valerius, Giancarlo Tria, Marina Bennati, Christian Koch, Alistair J. Fielding, Ivo Feussner, Gerhard H. Braus, and Dmitri I. Svergun
- Subjects
Stereochemistry ,Electrons ,Aspergillus nidulans ,Catalysis ,Dioxygenases ,Substrate Specificity ,03 medical and health sciences ,Cytochrome P-450 Enzyme System ,Tandem Mass Spectrometry ,Dioxygenase ,Scattering, Small Angle ,Phosphofructokinase 2 ,Protein Structure, Quaternary ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,Small-angle X-ray scattering ,030302 biochemistry & molecular biology ,Substrate (chemistry) ,Cytochrome P450 ,Cell Biology ,Oxylipin ,biology.organism_classification ,Enzyme ,chemistry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Mutagenesis, Site-Directed ,biology.protein ,Protein Binding - Abstract
In plants and mammals, oxylipins may be synthesized via multi step processes that consist of dioxygenation and isomerization of the intermediately formed hydroperoxy fatty acid. These processes are typically catalyzed by two distinct enzyme classes: dioxygenases and cytochrome P450 enzymes. In ascomycetes biosynthesis of oxylipins may proceed by a similar two-step pathway. An important difference, however, is that both enzymatic activities may be combined in a single bifunctional enzyme. These types of enzymes are named Psi-factor producing oxygenases (Ppo). Here, the spatial organization of the two domains of PpoA from Aspergillus nidulans was analyzed by small-angle X-ray scattering and the obtained data show that the enzyme exhibits a relatively flat trimeric shape. Atomic structures of the single domains were obtained by template-based structure prediction and docked into the enzyme envelope of the low resolution structure obtained by SAXS. EPR-based distance measurements between the tyrosyl radicals formed in the activated dioxygenase domain of the enzyme supported the trimeric structure obtained from SAXS and the previous assignment of Tyr374 as radical-site in PpoA. Furthermore, two phenylalanine residues in the cytochrome P450 domain were shown to modulate the specificity of hydroperoxy fatty acid rearrangement.
- Published
- 2013
40. A rapid freeze-quench setup for multi-frequency EPR spectroscopy of enzymatic reactions
- Author
-
Ivo Feussner, Marina Bennati, Roberta Pievo, Brigitta Angerstein, Christian Koch, and Alistair J. Fielding
- Subjects
Time Factors ,Analytical chemistry ,010402 general chemistry ,7. Clean energy ,01 natural sciences ,Spectral line ,Enzyme catalysis ,law.invention ,Substrate Specificity ,03 medical and health sciences ,law ,Freezing ,Physical and Theoretical Chemistry ,Spectral resolution ,Spectroscopy ,Electron paramagnetic resonance ,030304 developmental biology ,0303 health sciences ,Millisecond ,Chemistry ,Electron Spin Resonance Spectroscopy ,Substrate (chemistry) ,Atomic and Molecular Physics, and Optics ,0104 chemical sciences ,Enzymes ,Kinetics ,Reagent ,Oxygenases - Abstract
Electron paramagnetic resonance (EPR) spectroscopy in combination with the rapid freeze-quench (RFQ) technique is a well-established method to trap and characterize intermediates in chemical or enzymatic reactions at the millisecond or even shorter time scales. The method is particularly powerful for mechanistic studies of enzymatic reactions when combined with high-frequency EPR (ν≥90 GHz), which permits the identification of substrate or protein radical intermediates by their electronic g values. In this work, we describe a new custom-designed micro-mix rapid freeze-quench apparatus, for which reagent volumes for biological samples as small as 20 μL are required. The apparatus was implemented with homemade sample collectors appropriate for 9, 34, and 94 GHz EPR capillaries (4, 2, and 0.87 mm outer diameter, respectively) and the performance was evaluated. We demonstrate the application potential of the RFQ apparatus by following the enzymatic reaction of PpoA, a fungal dioxygenase producing hydro(pero)xylated fatty acids. The larger spectral resolution at 94 GHz allows the discernment of structural changes in the EPR spectra, which are not detectable in the same samples at the standard 9 GHz frequency.
- Published
- 2013
41. Orientation selection in distance measurements between nitroxide spin labels at 94 GHz EPR with variable dual frequency irradiation
- Author
-
Falk Wachowius, Giuseppe Sicoli, Marina Bennati, Igor Tkach, Claudia Höbartner, Snorri Th. Sigurdsson, Tatiana Y. Baranova, Soraya Pornsuwan, and Ulf Diederichsen
- Subjects
Nitroxide mediated radical polymerization ,Oligonucleotides ,General Physics and Astronomy ,Duplex (telecommunications) ,010402 general chemistry ,01 natural sciences ,Molecular physics ,law.invention ,Optics ,law ,Dual frequency ,Irradiation ,Physical and Theoretical Chemistry ,Pulsed electron–electron double resonance ,nitroxide spin labels ,Electron paramagnetic resonance ,chemistry.chemical_classification ,Quantitative Biology::Biomolecules ,Molecular Structure ,010405 organic chemistry ,Chemistry ,business.industry ,Biomolecule ,Dual mode ,0104 chemical sciences ,Dipole ,Nitrogen Oxides ,Spin Labels ,business ,Peptides - Abstract
Pulsed electron-electron double resonance (PELDOR, also known as DEER) has become a method of choice to measure distances in biomolecules. In this work we show how the performance of the method can be improved at high EPR frequencies (94 GHz) using variable dual frequency irradiation in a dual mode cavity in order to obtain enhanced resolution toward orientation selection. Dipolar evolution traces of a representative RNA duplex and an α-helical peptide were analysed in terms of possible bi-radical structures by considering the inherent ambiguity of symmetry-related solutions. peerReviewed
- Published
- 2013
42. Zero‐field‐splitting and π‐electron spin densities in the lowest excited triplet state of oligothiophenes
- Author
-
Péter R. Surján, Marina Bennati, Michael Mehring, and Károly Németh
- Subjects
Condensed matter physics ,General Physics and Astronomy ,Electronic structure ,Zero field splitting ,Bond order ,Molecular physics ,law.invention ,Bond length ,chemistry.chemical_compound ,chemistry ,law ,Excited state ,Thiophene ,Physical and Theoretical Chemistry ,Triplet state ,Electron paramagnetic resonance - Abstract
The electronic properties of thiophene oligomers (nT, n=2–8) have been investigated in the lowest excited triplet state. Theoretical calculations of the zero field splitting parameters and of the π‐electron spin density have been performed and compared with previous experimental EPR results. The calculations are based on a simple π‐electron (one‐electron‐per‐site) model including electron–electron interaction at the extended Hubbard level. Optimized bond lengths result from making them self‐consistent to the corresponding bond orders via Coulson’s relationship. The calculated D values decrease from D=0.0959 cm−1 for n=2 to D=0.0597 cm−1 for n=8, in agreement with EPR data. The measured as well as the calculated E values are rather small. Furthermore, we found that ZFS parameters are affected by the torsion angles between the thiophene rings. The chain length dependence of D can be rationalized comparing π‐electron spin density calculations and computed bond length distortions. These clearly indicate that the triplet excitation reaches a finite extension over about four thiophene rings.
- Published
- 1996
43. Zero-field-splitting in the lowest triplet state of C60
- Author
-
Marina Bennati, Michael Mehring, Péter R. Surján, A. Grupp, and Károly Németh
- Subjects
Fullerene ,Chemistry ,General Physics and Astronomy ,Absolute value (algebra) ,Physical and Theoretical Chemistry ,Triplet state ,Atomic physics ,Zero field splitting ,Symmetry (physics) ,Sign (mathematics) - Abstract
Zero-field-splitting parameters D and E for the fullerene C 60 in its Jahn-Teller distorted triplet state were calculated and compared to recent experimental results. The E value vanishes due to symmetry, while we obtained a non-zero D value of D = −0.009 cm −1 in the D 5d distorted lowest triplet state. The absolute value of D is in good agreement with experimental data, which suggests that the sign of D is negative.
- Published
- 1996
44. Pulsed EPR Spectroscopy of the Photoexcited Triplet States of Thiophene Oligomers in Frozen Solution
- Author
-
Marina Bennati, Michael Mehring, and Arthur Grupp, and Peter Bäuerle
- Subjects
education.field_of_study ,Pulsed EPR ,Population ,General Engineering ,Photochemistry ,Spectral line ,law.invention ,Photoexcitation ,chemistry.chemical_compound ,Chain length ,Crystallography ,chemistry ,law ,Thiophene ,Physical and Theoretical Chemistry ,education ,Electron paramagnetic resonance ,Spectroscopy - Abstract
We present a triplet EPR study on photoexcitations in two different series of well defined thiophene oligomers. The first series consists of the so-called ‘end-capped' oligothiophenes with chain length n = 2−6. In the second series dodecyl chains are linked to α-oligothiophenes with n = 3, 6, and 8. Measurements of the triplet EPR spectra were performed using pulsed EPR spectroscopy. The characteristic lineshape of the EPR spectra provides evidence that photoexcitation leads to molecular triplet states in all compounds. We determined the fine structure parameters D and E and the population of the triplet eigenfunctions Px, Py, Pz performing spectra simulations. The chain length dependence and the conformation dependence are discussed.
- Published
- 1996
45. Triplet-EPR spectroscopy on the Diels-Alder adduct of C60 and 4,5-dimethoxy-o-quinodimethane in frozen solution
- Author
-
Michael Mehring, Klaus Müllen, A. Grupp, Andreas Gügel, Pavel Belik, and Marina Bennati
- Subjects
Chemistry ,General Physics and Astronomy ,Spectral line ,law.invention ,Adduct ,Condensed Matter::Materials Science ,symbols.namesake ,law ,Computational chemistry ,Physics::Atomic and Molecular Clusters ,symbols ,Physical chemistry ,Molecule ,Physical and Theoretical Chemistry ,Triplet state ,Hamiltonian (quantum mechanics) ,Electron paramagnetic resonance ,Anisotropy ,Spectroscopy - Abstract
Pulsed-EPR spectroscopy was used to study a modified C60 molecule (1) in its photoexcited triplet state. The analysis of the triplet EPR lineshape shows a break of the high symmetry of the lowest populated triplet state of C60 due to the modification of the C60 unity. The reported temperature dependence of the spectra is influenced by relaxation effects. Lineshape simulations based on a triplet Hamiltonian including anisotropic T1/T2 relaxation were performed. The results are discussed in relation to the behaviour of pure 3C60.
- Published
- 1995
46. Electron paramagnetic resonance lineshape analysis of the photoexcited triplet state of C60 in frozen solution. Exchange narrowing and dynamic Jahn–Teller effect
- Author
-
A. Grupp, Marina Bennati, and Michael Mehring
- Subjects
Pulsed EPR ,Chemistry ,Jahn–Teller effect ,General Physics and Astronomy ,Resonance ,Zero field splitting ,law.invention ,law ,Excited state ,Physical and Theoretical Chemistry ,Triplet state ,Atomic physics ,Spectroscopy ,Electron paramagnetic resonance - Abstract
The EPR lineshape of the photoinduced triplet state of C60 in frozen toluene solution was studied by pulsed EPR spectroscopy. Lineshape calculations of the triplet spectra were performed including dynamical exchange effects. The observed spectra in a glassy matrix are compatible with zero field splitting parameters ‖D1‖ = 0.0114 cm−1 (12.2 mT) and ‖E1‖ = 0.0005 cm−1 (0.5 mT). The temperature dependence of the powder lineshape was simulated using a dynamical exchange model, where the triplet principal axis jumps between all equivalent sites allowed by the D5d symmetry for the lowest excited triplet state. The determined exchange rate turned out to be only weakly temperature dependent and suggests that the dynamic process is due to tunneling between Jahn–Teller distorted states rather than to real molecular jumps. In addition we have observed a different triplet state with zero field splitting parameters ‖D2‖ = 0.0100 cm−1 (10.6 mT), ‖E2‖ = 0.0015 cm−1 (1.6 mT) after annealing of the matrix. We attribute th...
- Published
- 1995
47. W-band orientation selective DEER measurements on a Gd3+/nitroxide mixed-labeled protein dimer with a dual mode cavity
- Author
-
Nurit Manukovsky, Daniella Goldfarb, Gottfried Otting, Marina Bennati, Hiromasa Yagi, Thomas Huber, Ilia Kaminker, and Igor Tkach
- Subjects
Nuclear and High Energy Physics ,Nitroxide mediated radical polymerization ,Biophysics ,Dual mode ,Electron Spin Resonance Spectroscopy ,Protein dimer ,Resonance ,Proteins ,Gadolinium ,Condensed Matter Physics ,Biochemistry ,Signal ,chemistry.chemical_compound ,Nuclear magnetic resonance ,chemistry ,W band ,Orientation (geometry) ,Nitrogen Oxides ,Spin (physics) ,Dimerization ,Algorithms - Abstract
Double electron-electron resonance (DEER) at W-band (95 GHz) was applied to measure the distance between a pair of nitroxide and Gd(3+) chelate spin labels, about 6 nm apart, in a homodimer of the protein ERp29. While high-field DEER measurements on systems with such mixed labels can be highly attractive in terms of sensitivity and the potential to access long distances, a major difficulty arises from the large frequency spacing (about 700 MHz) between the narrow, intense signal of the Gd(3+) central transition and the nitroxide signal. This is particularly problematic when using standard single-mode cavities. Here we show that a novel dual-mode cavity that matches this large frequency separation dramatically increases the sensitivity of DEER measurements, allowing evolution times as long as 12 μs in a protein. This opens the possibility of accessing distances of 8 nm and longer. In addition, orientation selection can be resolved and analyzed, thus providing additional structural information. In the case of W-band DEER on a Gd(3+)-nitroxide pair, only two angles and their distributions have to be determined, which is a much simpler problem to solve than the five angles and their distributions associated with two nitroxide spin labels.
- Published
- 2012
48. ENDOR spectroscopy and DFT calculations: evidence for the hydrogen bond network within α2 in the PCET of E. coli ribonucleotide reductase
- Author
-
Christoph Riplinger, Marina Bennati, JoAnne Stubbe, Tomislav Argirević, and Frank Neese
- Subjects
Models, Molecular ,Stereochemistry ,Electrons ,Photochemistry ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Cofactor ,Article ,Turn (biochemistry) ,Electron transfer ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Ribonucleotide Reductases ,Escherichia coli ,Nucleotide ,chemistry.chemical_classification ,biology ,Hydrogen bond ,Electron Spin Resonance Spectroscopy ,Cytidine ,Hydrogen Bonding ,General Chemistry ,Resonance (chemistry) ,Ribonucleotide reductase ,chemistry ,biology.protein ,Quantum Theory ,Protons - Abstract
Escherichia coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: α2 and β2. β2 contains a stable di-iron tyrosyl radical (Y(122)(•)) cofactor required to generate a thiyl radical (C(439)(•)) in α2 over a distance of 35 Å, which in turn initiates the chemistry of the reduction process. The radical transfer process is proposed to occur by proton-coupled electron transfer (PCET) via a specific pathway: Y(122) ⇆ W(48)[?] ⇆ Y(356) in β2, across the subunit interface to Y(731) ⇆ Y(730) ⇆ C(439) in α2. Within α2 a colinear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH(2)Y) replaced Y(730) in α2, and this mutant was incubated with β2, cytidine 5'-diphosphate, and adenosine 5'-triphosphate to generate a NH(2)Y(730)(•) in D(2)O. [(2)H]-Electron-nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained, and together with DFT models of α2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C(439) and Y(731). A third proton was assigned to a water molecule in close proximity (2.2 Å O-H···O distance) to residue 730. The calculations also suggest that the unusual g-values measured for NH(2)Y(730)(•) are consistent with the combined effect of the hydrogen bonds to Cys(439) and Tyr(731), both nearly perpendicular to the ring plane of NH(2)Y(730.) The results provide the first experimental evidence for the hydrogen-bond network between the pathway residues in α2 of the active RNR complex, for which no structural data are available.
- Published
- 2012
49. Overhauser DNP with 15N labelled Frémy's salt at 0.35 Tesla
- Author
-
Giacomo Parigi, Marina Bennati, Maria-Teresa Türke, and Claudio Luchinat
- Subjects
Nitroxide mediated radical polymerization ,Proton ,Nitrogen Isotopes ,Chemistry ,Radical ,Analytical chemistry ,Electron Spin Resonance Spectroscopy ,General Physics and Astronomy ,Water ,Models, Theoretical ,law.invention ,chemistry.chemical_compound ,law ,Proton NMR ,Frémy's salt ,Physical and Theoretical Chemistry ,Protons ,Electron paramagnetic resonance ,Saturation (chemistry) ,Hyperfine structure ,Nitroso Compounds - Abstract
The effectiveness of dynamic nuclear polarization (DNP) as a tool to enhance the sensitivity of liquid state NMR critically depends on the choice of the optimal polarizer molecule. In this study the performance of (15)N labelled Frémy's salt as a polarizing agent in Overhauser DNP is investigated in detail at X-band (0.35 T, 9.7 GHz EPR, 15 MHz (1)H NMR) and compared to that of TEMPONE-D,(15)N employed in previous studies. Both radicals provide similar maximum enhancements of the solvent water protons under similar conditions but a different saturation behaviour. The factors determining the enhancement and effective saturation were measured independently by EPR, ELDOR and NMRD and are shown to fulfil the Overhauser equation. In particular, following the theory of EPR saturation we provide analytical solutions for the dependence of the enhancement on the microwave field strength in terms of saturation transfer between two coupled hyperfine lines undergoing spin exchange. The negative charge of the radical in Frémy's salt solutions can explain the peculiar properties of this polarizing agent and indicates different suitable application areas for the two types of nitroxide radicals.
- Published
- 2012
50. Evaluation of a shuttle DNP spectrometer by calculating the coupling and global enhancement factors of l-tryptophan
- Author
-
Frank Engelke, Alexander Krahn, Marina Bennati, Peter Höfer, Philip Lottmann, Thorsten Marquardsen, Andreas Tavernier, and Christian Griesinger
- Subjects
Spectrometer ,Proton ,Solid-state physics ,Chemistry ,Analytical chemistry ,Polarizer ,Polarization (waves) ,Molecular physics ,Atomic and Molecular Physics, and Optics ,law.invention ,law ,Proton spin crisis ,Molecule ,Leakage (electronics) - Abstract
A liquid state shuttle dynamic nuclear polarization (DNP) spectrometer is presented, featuring several technical modifications that increase stability and improve reproducibility. For the protons of l-tryptophan, the signal enhancement and the DNP spin properties, such as relaxation, were measured and compared with each other. The calculated coupling factors suggest that the proton accessibility for the polarizer molecule has an important influence on the DNP enhancement. In general, short proton spin longitudinal relaxation times without radical reduce the detectable enhancement by decreasing the leakage factor and increasing the relaxation losses during the course of the sample transfer. The usage of a global enhancement factor gives a more complete overview of the capabilities for the described experimental setup. Global enhancements of up to −4.2 for l-tryptophan protons are found compared to pure Boltzmann enhancements of up to −2.4.
- Published
- 2012
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