Electron Paramagnetic Resonance Spectroscopy In Combination Research Articles

Crystallographic snapshots of a B12-dependent radical SAM methyltransferase

By catalysing the microbial formation of methane, methyl-coenzyme M reductase has a central role in the global levels of this greenhouse gas1,2. The activity of methyl-coenzyme M reductase is profoundly affected by several unique post-translational modifications3–6, such as a unique C-methylation reaction catalysed by methanogenesis marker protein 10 (Mmp10), a radical S-adenosyl-l-methionine (SAM) enzyme7,8. Here we report the spectroscopic investigation and atomic resolution structure of Mmp10 from Methanosarcina acetivorans, a unique B12 (cobalamin)-dependent radical SAM enzyme9. The structure of Mmp10 reveals a unique enzyme architecture with four metallic centres and critical structural features involved in the control of catalysis. In addition, the structure of the enzyme–substrate complex offers a glimpse into a B12-dependent radical SAM enzyme in a precatalytic state. By combining electron paramagnetic resonance spectroscopy, structural biology and biochemistry, our study illuminates the mechanism by which the emerging superfamily of B12-dependent radical SAM enzymes catalyse chemically challenging alkylation reactions and identifies distinctive active site rearrangements to provide a structural rationale for the dual use of the SAM cofactor for radical and nucleophilic chemistry.

A novel combinatorial technique for simultaneous quantification of oxygen radicals and aggregation reveals unexpected redox patterns in the activation of platelets by different physiopathological stimuli.

The regulation of platelets by oxidants is critical for vascular health and may explain thrombotic complications in diseases such as diabetes and dementia, but remains poorly understood. Here, we describe a novel technique combining electron paramagnetic resonance spectroscopy and turbidimetry, which has been utilized to monitor simultaneously platelet activation and oxygen radical generation. This technique has been used to investigate the redox-dependence of human and mouse platelets. Using selective peptide inhibitors of NADPH oxidases (NOXs) on human platelets and genetically modified mouse platelets (NOX1−/− or NOX2−/−), we discovered that: 1) intracellular but not extracellular superoxide anion generated by NOX is critical for platelet activation by collagen; 2) superoxide dismutation to hydrogen peroxide is required for thrombin-dependent activation; 3) NOX1 is the main source of oxygen radicals in response to collagen, while NOX2 is critical for activation by thrombin; 4) two platelet modulators, namely oxidized low density lipoproteins (oxLDL) and amyloid peptide β (Aβ), require activation of both NOX1 and NOX2 to pre-activate platelets. This study provides new insights into the redox dependence of platelet activation. It suggests the possibility of selectively inhibiting platelet agonists by targeting either NOX1 (for collagen) or NOX2 (for thrombin). Selective inhibition of either NOX1 or NOX2 impairs the potentiatory effect of tested platelet modulators (oxLDL and Aβ), but does not completely abolish platelet hemostatic function. This information offers new opportunities for the development of disease-specific antiplatelet drugs with limited bleeding side effects by selectively targeting one NOX isoenzyme.

Capturing Conformational Changes of the Tau Protein Upon Aggregation

Many neurodegenerative diseases (NDs) are associated with abnormal accumulation of protein aggregates in the brain. The intrinsically disordered tau protein, which does not possess a well-defined 3D structure in normal conditions, is involved in several NDs, including Alzheimer diseases and frontotemporal dementia. In pathological conditions, tau can aggregate into structured aggregates, in so-called neurofibrillarly tangles, which are deadly to neurons. Different fibers supra-structures, referred to as strains, have been shown to be associated with different diseases, thereby suggesting that the arrangement of tau molecules in the fibers is pathologically relevant. Strikingly, tau has recently been termed as a prion-like protein because of the capacity of one tau strains to effectively propagate from neuron to neuron. The ability of a strain to convert naïve disordered monomers into specific structured aggregates is still not understood, but conformational changes of tau is thought to be a critical factor. Here we have characterized tau structure by combining electron paramagnetic resonance spectroscopy and spin-labelling techniques. In particular, double electron-electron resonance spectroscopy (DEER) allowed us to measure intra-molecular distances at different time points along the aggregation process, in particular at early times before the formation of beta-sheets structures. We have used different aggregation triggers including heparin cofactors and fiber seeds extracted from model mouse brains. We will show some key conformational changes occurring during aggregation, and relate them to the seeding capacity of tau misfolded species. A better view of tau disorder-to-structure transition will help understand the aggregation mechanisms, and pave the way to new drug development strategies that aim at limiting propagation of deleterious aggregates in NDs.

Physiological concentrations of albumin favor drug binding.

The ability to track drug binding and release makes electron paramagnetic resonance (EPR) spectroscopy well suited for drug delivery studies. Using the continuous wave (cw) EPR technique to extract information about the dynamics of the spin labeled drugs we can simultaneously determine the bound and unbound drugs. Here, spin labeled salicylic acid (SLSA) binding to and release from bovine serum albumin (BSA) is investigated, as a model for drug-transport protein interaction. We studied SLSA-BSA binding in a wide concentration range and found that the stoichiometry of the drug-protein increases significantly when the physiological range of BSA concentration is reached. Our EPR results explicitly reveal that up to ∼7 SLSA can bind to one albumin at the physiological concentration, whereas at lower BSA concentrations (<0.125 mM) the SLSA-BSA stoichiometry is maximum 2. Moreover, we studied drug release and showed that the ratio of bound to unbound SLSA concentrations remains relatively stable during dialysis. This indicates that the binding equilibrium of SLSA is not altered through the process of dialysis. This study demonstrates that cw EPR spectroscopy in combination with spin labeled drugs is an effective technique for binding and release studies and stoichiometric analysis of drug-protein interactions.

Open and Closed Conformations of the Isolated Transmembrane Domain of Death Receptor 5 Support a New Model of Activation

It has long been presumed that activation of the apoptosis-initiating Death Receptor 5, as well as other structurally homologous members of the TNF-receptor superfamily, relies on ligand-stabilized trimerization of noninteracting receptor monomers. We and others have proposed an alternate model in which the TNF-receptor dimer—sitting at the vertices of a large supramolecular receptor network of ligand-bound receptor trimers—undergoes a closed-to-open transition, propagated through a scissorslike conformational change in a tightly bundled transmembrane (TM) domain dimer. Here we have combined electron paramagnetic resonance spectroscopy and potential-of-mean force calculations on the isolated TM domain of the long isoform of DR5. The experiments and calculations both independently validate that the opening transition is intrinsic to the physical character of the TM domain dimer, with a significant energy barrier separating the open and closed states.

Remodeling of Lipid Vesicles into Cylindrical Micelles by α-Synuclein in an Extended α-Helical Conformation

α-Synuclein (αS) is a protein with multiple conformations and interactions. Natively unfolded in solution, αS accumulates as amyloid in neurological tissue in Parkinson disease and interacts with membranes under both physiological and pathological conditions. Here, we used cryoelectron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the ability of αS to remodel vesicles. At molar ratios of 1:5 to 1:40 for protein/lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), large spherical vesicles are converted into cylindrical micelles ~50 Å in diameter. Other lipids of the same charge (negative) exhibit generally similar behavior, although bilayer tubes of 150-500 Å in width are also produced, depending on the lipid acyl chains. At higher protein/lipid ratios, discoid particles, 70-100 Å across, are formed. EPR data show that, on cylindrical micelles, αS adopts an extended amphipathic α-helical conformation, with its long axis aligned with the tube axis. The observed geometrical relationship between αS and the micelle suggests that the wedging of its long α-helix into the outer leaflet of a membrane may cause curvature and an anisotropic partition of lipids, leading to tube formation.

Probing Protein Secondary Structure using EPR: Investigating a Dynamic Region of Visual Arrestin.

One key application of site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is the determination of sequence-specific secondary structure in proteins. Regular secondary structure leads to a periodic variation in both side chain motion and solvent accessibility, two properties easily monitored by EPR techniques. Specifically, saturation recovery (SR) EPR spectroscopy has proven to be useful for making accessibility measurements for multiple protein structure populations by determining individual accessibilities and is therefore well suited to study the structure of proteins exhibiting multiple conformations in equilibrium. Here we employ both continuous wave and SR EPR spectroscopy in combination to examine the secondary structure of a short sequence showing conformational heterogeneity in visual rod arrestin. The EPR data presented here clearly distinguish between the unstructured loop and the helical structure formed in the crystallographic tetramer of visual arrestin and show that this region is unstructured in solution.

Nucleophile Addition of Reduced Glutathione on 2-Methyl-2-Nitroso Compound: A Combined Electron Paramagnetic Resonance Spectroscopy and Electrospray Tandem Mass Spectrometry Study

Mass spectrometry (MS) was used in conjunction with electron paramagnetic resonance (EPR) to characterize products arising from reactions between reduced glutathione (GSH) and 2-methyl 2-nitroso propane (MNP) in an oxidative medium, to evaluate the reactivity of this tripeptide as a nucleophile toward a nitroso compound. Depending on the experimental conditions, different radical species could be detected by EPR, which allowed some structural assumptions. These samples were then submitted to electrospray ionization, in both positive and negative ion modes, for structural elucidation in tandem mass spectrometry. Although the primary nitroxide products could not be detected in MS, structurally related compounds such as hydroxylamine and O-methyl hydroxylamine could be fully characterized. In the absence of light, a S-adduct was formed via a Forrester-Hepburn reaction, that is, a nucleophile addition of MNP onto the thiol function in reduced glutathione to yield a hydroxylamine intermediate, further oxidized into nitroxide. In contrast, irradiating the reaction medium with visible light could allow an inverted spin trapping reaction to take place, involving the oxidation of both MNP and GSH before the nucleophilic addition of the sulfenic acid function onto the nitrogen of MNP, yielding a so-called O-adduct. It was also found that dilution of the reaction medium with methanol for the purpose of electrospray ionization could allow nitroxides to be indirectly observed either as hydroxylamine or O-methyl hydroxylamine species.

Vitamin E Chemistry. Studies into Initial Oxidation Intermediates of α-Tocopherol: Disproving the Involvement of 5a-C-Centered “Chromanol Methide” Radicals

Contrary to concepts handed down in the literature from the early days of vitamin E research, one-electron oxidation of vitamin E does not involve 5a-C-centered radicals. A combined approach of analytical techniques, in particular electron paramagnetic resonance spectroscopy (EPR), organic synthesis of special derivatives, isotopic labeling, kinetic studies, and computational chemistry was used to re-evaluate the one-electron and two-electron oxidation chemistry of alpha-tocopherol (alpha-toc). EPR in combination with 5a-13C-labeled compounds provided no indication of the involvement of 5a-C-centered radicals. Oxidation of special tocopherol derivatives were used to disprove the occurrence of 5a-C-centered one-electron intermediates. Additionally it was shown that those vitamin E reactions that were commonly evoked to plead for the involvement of C-centered tocopheryl radicals actually proceeded via heterolytic, i.e., non-radical, intermediates. The results will help to clear widely spread misunderstandings about the chemistry of vitamin E and will have mechanistic implications for the synthesis of tocopherol-based supramolecular structures and 5a-substituted alpha-tocopherol derivatives.

Tetracycline-HCl-loaded poly( dl-lactide-co-glycolide) microspheres prepared by a spray drying technique: influence of γ-irradiation on radical formation and polymer degradation

Tetracycline-HCl (TCH)-loaded microspheres were prepared from poly(lactide-co-glycolide) (PLGA) by spray drying. The drug was incorporated in the polymer matrix either in solid state or as w/o emulsion. The spin probe 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPOL) and the spin trap tert-butyl-phenyl-nitrone (PBN) were co-encapsulated into the TCH-loaded and placebo particles. We investigated the effects of γ-irradiation on the formation of free radicals in polymer and drug and the mechanism of chain scission after sterilization. γ-Irradiation was performed at 26.9 and 54.9 kGy using a 60Co source. The microspheres were characterized especially with respect to the formation of radicals and in vitro polymer degradation. Electron paramagnetic resonance (EPR) spectroscopy, gel permeation chromatography (GPC), differential scanning calorimetry (DSC), high-performance liquid chromatography (HPLC), gas chromatography–mass spectroscopy (GC–MS), and scanning electron microscopy (SEM) were used for characterization of the microspheres. Using EPR spectroscopy, we successfully detected γ-irradiation induced free radicals within the TCH-loaded microspheres, while unloaded PLGA did not contain radicals under the same conditions. The relatively low glass transition temperature of the poly( dl-lactide-co-glycolide) (37–39°C) seems to favor subsequent reactions of free radicals due to the high mobility of the polymeric chains. Because of the high melting point of TCH (214°C), the radicals can only be stabilized in drug loaded microspheres. In order to determine the mechanism of polymer degradation after exposure to γ-rays, the spin trap PBN and the spin probe TEMPOL were encapsulated in the microspheres. γ-Irradiation of microspheres containing PBN resulted in the formation of a lipophilic spin adduct, indicating that a polymeric radical was generated by random chain scission. Polymer degradation by an unzipping mechanism would have produced hydrophilic spin adducts of PBN and monomeric radicals of lactic or glycolic acid. These degradation products were not detected by EPR. This result is confirmed by the observation that possible diamagnetic reaction products of low molecular weight, consisting of TEMPOL and lactide or glycolide monomers, could not be detected by GC–MS. While an irradiation dose-dependent decrease in molecular weight of PLGA could be verified in agreement with the literature, TCH content of the microspheres was not affected by the exposure to γ-rays. It can be concluded that EPR spectroscopy in combination with GPC, DSC, and HPLC allows a detailed characterization of the impact of γ-sterilization on biodegradable parenteral drug delivery systems.