Malte Drescher

Gd(III)-PyMTA Label Is Suitable for In-Cell EPR

Distance measurement in the nanometer range by electron paramagnetic resonance spectroscopy (EPR) in combination with site-directed spin labeling is a very powerful tool to monitor the structure and dynamics of biomacromolecules in their natural environment. However, in-cell application is hampered by the short lifetime of the commonly used nitroxide spin labels in the reducing milieu inside a cell. Here, we demonstrate that the Gd(III) based spin label Gd-PyMTA is suitable for in-cell EPR. Gd-PyMTA turned out to be cell compatible and was proven to be inert in in-cell extracts of Xenopus laevis oocytes at 18 °C for more than 24 h. The proline rich peptide H-AP10CP10CP10-NH2 was site-directedly spin labeled with Gd-PyMTA at both cysteine moieties. The resulting peptide, H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2, as well as the model compound Gd-spacer-Gd, which consists of a spacer of well-known stiffness, were microinjected into Xenopus laevis oocytes, and the Gd(III)-Gd(III) distances were determined by double electron-electron resonance (DEER) spectroscopy. To analyze the intracellular peptide conformation, a rotamer library was set up to take the conformational flexibility of the tether between the Gd(III) ion and the Cα of the cysteine moiety into account. The results suggest that the spin labeled peptide H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2 is inserted into cell membranes, coinciding with a conformational change of the oligoproline from a PPII into a PPI helix.

Oxidative and nitrative alpha-synuclein modifications and proteostatic stress: implications for disease mechanisms and interventions in synucleinopathies.

Alpha‐synuclein (ASYN) is a major constituent of the typical protein aggregates observed in several neurodegenerative diseases that are collectively referred to as synucleinopathies. A causal involvement of ASYN in the initiation and progression of neurological diseases is suggested by observations indicating that single‐point (e.g., A30P, A53T) or multiplication mutations of the gene encoding for ASYN cause early onset forms of Parkinsons disease (PD). The relative regional specificity of ASYN pathology is still a riddle that cannot be simply explained by its expression pattern. Also, transgenic over‐expression of ASYN in mice does not recapitulate the typical dopaminergic neuronal death observed in PD. Thus, additional factors must contribute to ASYN‐related toxicity. For instance, synucleinopathies are usually associated with inflammation and elevated levels of oxidative stress in affected brain areas. In turn, these conditions favor oxidative modifications of ASYN. Among these modifications, nitration of tyrosine residues, formation of covalent ASYN dimers, as well as methionine sulfoxidations are prominent examples that are observed in post‐mortem PD brain sections. Oxidative modifications can affect ASYN aggregation, as well as its binding to biological membranes. This would affect neurotransmitter recycling, mitochondrial function and dynamics (fission/fusion), ASYNs degradation within a cell and, possibly, the transfer of modified ASYN to adjacent cells. Here, we propose a model on how covalent modifications of ASYN link energy stress, altered proteostasis, and oxidative stress, three major pathogenic processes involved in PD progression. Moreover, we hypothesize that ASYN may act physiologically as a catalytically regenerated scavenger of oxidants in healthy cells, thus performing an important protective role prior to the onset of disease or during aging.

Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment

Double electron-electron resonance (DEER) is an electron paramagnetic resonance (EPR) technique used to determine distance distributions in the nanometer range between spin labels by measuring their dipole-dipole interactions. Here we describe how in-cell DEER can be applied to spin-labeled DNA sequences to unravel their conformations in living cells by long-range distance measurements in cellula. As EPR detects unpaired electron spins only, diamagnetic molecules provide no background and do not reduce detection sensitivity of the specific signal. Compared with in-cell NMR spectroscopy, low concentrations of spin-labeled molecules can be used owing to the higher sensitivity of EPR per spin. This protocol describes the synthesis of the spin labels, their introduction in DNA strands, the injection of labeled DNA solutions in cells and the performance of in-cell EPR measurements. Completion of the entire protocol takes ∼20 d.

The NOX1/4 Inhibitor GKT136901 as Selective and Direct Scavenger of Peroxynitrite

NADPH oxidases (NOX), catalyzing the reduction of molecular oxygen to form the superoxide radical anion (•O₂⁻) and hydrogen peroxide (H₂O₂), are involved in several pathological conditions, such as stroke, diabetes, atherosclerosis, but also in chronic neurodegenerative diseases such as Parkinsons disease, Alzheimers disease, or multiple sclerosis. GKT136901 is a novel NOX-1/4 inhibitor with potential application in the areas of diabetic nephropathy, stroke, or neurodegeneration. In the present study, we investigated additional pharmacological activities of the compound with respect to direct free radical scavenging. GKT136901 did not interact with nitric oxide (•NO), •O₂⁻, or hydroxyl radicals (•OH), but it acted as selective scavenger of peroxynitrite (PON) already in the submicromolar concentration range. Alpha synuclein (ASYN) is a protein involved in the pathogenesis of Parkinsons disease and a known target for PON-dependent tyrosine nitration. Submicromolar concentrations of GKT136901 prevented tyrosine nitration and di-tyrosine-dependent dimer formation of ASYN by PON as indicated by Western blot and mass spectrometric analysis. GKT136901 itself was degraded when exposed to PON. In a human neuronal cell model, GKT136901 prevented both the depletion of reduced intracellular glutathione, and the degeneration of neurites when present during PON treatment of the cells. When GKT136901 was applied after PON treatment, no protective effect was observed, thus excluding an impact of GKT136901 on cellular death/survival pathways. In summary, selective scavenging of PON is an additional pharmacological property of the NOX-1/4 inhibitor GKT136901, and this may add to the efficiency of the drug in several disease models.

Structural characterization of quadruplex DNA with in-cell EPR approaches

Guanosine-rich DNA sequences have the potential to adopt four-stranded conformations termed quadruplexes. The chromosomes of higher organisms are capped by so-called telomeres that are composed of repeats of the sequence TTAGGG. Up to 200 nucleotides of the G-rich strand form an overhang that is suspected to fold into intramolecular G-quadruplexes. Since induction of quadruplexes at the telomeres results in anti-proliferative effects, the intracellular structure of G-quadruplexes is of high interest as an anti-cancer drug target. Here we give a perspective on the elucidation of DNA sequence folds by electron paramagnetic resonance (EPR) distance measurements. The technique complements X-ray crystallography and NMR spectroscopy, as it can be applied in noncrystalline states, is not intrinsically limited by the size of the bio-macromolecular complex, and is able to analyze flexible structures or coexisting DNA conformation.

A bacterial DNA quadruplex with exceptional K+ selectivity and unique structural polymorphism

The G-rich sequence d[(G4CT)3G4] was recently identified as a potential quadruplex-forming sequence associated with loci involved in antigenic variation in the human pathogen Treponema pallidum. We found this motif to be enriched in eubacterial genomes. Employing a combination of CD spectroscopy, EPR spectroscopy, analytical ultracentrifugation, and EMSA, we demonstrate that d[(G4CT)3G4] displays unique features among the many G-quadruplex-forming sequences studied so far. To our knowledge d[(G4CT)3G4] shows a so far unprecedented selectivity for K+ with even high concentrations of Na+ unable to induce pronounced G-quadruplex formation. A remarkable continuous and complete transition from an anti-parallel, monomolecular structure into a tetrameric, parallel conformation is observed upon increasing K+-concentrations. Furthermore we investigate the effects of cation selectivity, quadruplex loop composition and length as well as G-tract length on quadruplex conversion.

A new building block for DNA network formation by self-assembly and polymerase chain reaction

Summary The predictability of DNA self-assembly is exploited in many nanotechnological approaches. Inspired by naturally existing self-assembled DNA architectures, branched DNA has been developed that allows self-assembly to predesigned architectures with dimensions on the nanometer scale. DNA is an attractive material for generation of nanostructures due to a plethora of enzymes which modify DNA with high accuracy, providing a toolbox for many different manipulations to construct nanometer scaled objects. We present a straightforward synthesis of a rigid DNA branching building block successfully used for the generation of DNA networks by self-assembly and network formation by enzymatic DNA synthesis. The Y-shaped 3-armed DNA construct, bearing 3 primer strands is accepted by Taq DNA polymerase. The enzyme uses each arm as primer strand and incorporates the branched construct into large assemblies during PCR. The networks were investigated by agarose gel electrophoresis, atomic force microscopy, dynamic light scattering, and electron paramagnetic resonance spectroscopy. The findings indicate that rather rigid DNA networks were formed. This presents a new bottom-up approach for DNA material formation and might find applications like in the generation of functional hydrogels.

Triplet state kinetics of anthracene studied by pulsed electron paramagnetic resonance

Photoexcited triplet states of anthracene in a glassy toluene matrix were investigated using pulsed UV–laser photoexcitation and pulsed electron paramagnetic resonance spectroscopy. The triplet lifetime, the triplet relaxation rates between the high-field eigenstates and the populations of the triplet sublevels are quantitatively analysed. High-quality data allows for supporting the former conclusion that excitations into the individual zero-field components proceed via independent channels.