Drescher, Malte

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The effect of the zero-field splitting in light-induced pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy

2023-02-08, Scherer, Andreas, Yildirim, Berk, Drescher, Malte

Laser-induced magnetic dipole (LaserIMD) spectroscopy and light-induced double electron–electron resonance (LiDEER) spectroscopy are important techniques in the emerging field of light-induced pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy (light-induced PDS). These techniques use the photoexcitation of a chromophore to the triplet state and measure its dipolar coupling to a neighboring electron spin, which allows the determination of distance restraints. To date, LaserIMD and LiDEER have been analyzed with software tools that were developed for a pair of two S=1/2 spins and that neglected the zero-field splitting (ZFS) of the excited triplet. Here, we explore the limits of this assumption and show that the ZFS can have a significant effect on the shape of the dipolar trace. For a detailed understanding of the effect of the ZFS, a theoretical description for LaserIMD and LiDEER is derived, taking into account the non-secular terms of the ZFS. Simulations based on this model show that the effect of the ZFS is not that pronounced in LiDEER for experimentally relevant conditions. However, the ZFS leads to an additional decay in the dipolar trace in LaserIMD. This decay is not that pronounced in Q-band but can be quite noticeable for lower magnetic field strengths in X-band. Experimentally recorded LiDEER and LaserIMD data confirm these findings. It is shown that ignoring the ZFS in the data analysis of LaserIMD traces can lead to errors in the obtained modulation depths and background decays. In X-band, it is additionally possible that the obtained distance distribution is plagued by long distance artifacts.

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Insights into the Conformational Plasticity of the Protein Kinase Akt1 by Multi-Lateral Dipolar Spectroscopy

2023, Stehle, Juliane, Weisner, Jörn, Eichhorn, Leanne, Rauh, Daniel, Drescher, Malte

The serine/threonine kinase Akt1 is part of the PI3 K/Akt pathway and plays a key role in the regulation of various cellular processes such as cell growth, proliferation, and apoptosis. Here, we analyzed the elasticity between the two domains of the kinase Akt1, connected by a flexible linker, recording a wide variety of distance restraints by electron paramagnetic resonance (EPR) spectroscopy. We studied full length Akt1 and the influence of the cancer-associated mutation E17K. The conformational landscape in the presence of different modulators, like different types of inhibitors and membranes was presented, revealing a tuned flexibility between the two domains, dependent on the bound molecule.

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Tracking protein domain movements by EPR distance determination and multilateration

2022, Stehle, Juliane, Drescher, Malte

Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful approach to supplement the large toolbox of methods for protein structure determination. The strength of EPR spectroscopy is the ability to map flexibility of protein domains and conformational ensembles. EPR distance determination in the nanometer range and subsequent multilateration enables the three-dimensional visualization of the localization of a spin label in a protein domain. Therefore, multilateration can not only be used to represent the degree of flexibility of protein structural elements, but also track movements of whole domains. We report a detailed protocol for all needed steps, beginning with the choice of the labeling sites, the spin labeling reaction, the EPR distance measurement by double electron-electron resonance (DEER) and finally the multilateration exploiting the EPR distance restraints.

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In situ EPR spectroscopy of a bacterial membrane transporter using an expanded genetic code

2021-11-18, Kugele, Anandi, Ketter, Sophie, Silkenath, Bjarne, Wittmann, Valentin, Joseph, Benesh, Drescher, Malte

The membrane transporter BtuB is site-directedly spin labelled on the surface of living Escherichia coli via Diels–Alder click chemistry of the genetically encoded amino acid SCO-L-lysine. The previously introduced photoactivatable nitroxide PaNDA prevents off-target labelling, is used for distance measurements, and the temporally shifted activation of the nitroxide allows for advanced experimental setups. This study describes significant evolution of Diels–Alder-mediated spin labelling on cellular surfaces and opens up new vistas for the the study of membrane proteins.

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Rapid Scan Electron Paramagnetic Resonance Spectroscopy Is a Suitable Tool to Study Intermolecular Interactions of Intrinsically Disordered Protein

2023-01-03, Dröden, Jessica, Drescher, Malte

Intrinsically disordered proteins (IDPs) are involved in most crucial cellular processes. However, they lack a well-defined fold hampering the investigation of their structural ensemble and interactions. Suitable biophysical methods able to manage their inherent flexibility and broad conformational ensemble are scarce. Here, we used rapid scan (RS) electron paramagnetic resonance (EPR) spectroscopy to study the intermolecular interactions of the IDP α-synuclein (aS). aS aggregation and fibril deposition is the hallmark of Parkinson’s disease, and specific point mutations, among them A30P and A53T, were linked to the early onset of the disease. To understand the pathological processes, research intensively investigates aS aggregation kinetics, which was reported to be accelerated in the presence of ethanol. Conventional techniques fail to capture these fast processes due to their limited time resolution and, thus, lose kinetic information. We have demonstrated that RS EPR spectroscopy is suitable for studying aS aggregation by resolving underlying kinetics and highlighting differences in fibrillization behavior. RS EPR spectroscopy outperforms traditional EPR methods in terms of sensitivity by a factor of 5 in our case while significantly reducing data acquisition time. Thus, we were able to sample short time intervals capturing single events taking place during the aggregation process. Further studies will therefore be able to shed light on biological processes proceeding on fast time scales.

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Increasing the Modulation Depth of GdIII-Based Pulsed Dipolar EPR Spectroscopy (PDS) with Porphyrin–GdIII Laser-Induced Magnetic Dipole Spectroscopy

2022-12-01, Scherer, Andreas, Yao, Xuemei, Qi, Mian, Wiedmaier, Max, Godt, Adelheid, Drescher, Malte

Distance determination with pulsed EPR has become an important technique for the structural investigation of biomacromolecules, with double electron–electron resonance spectroscopy (DEER) as the most important method. GdIII-based spin labels are one of the most frequently used spin labels for DEER owing to their stability against reduction, high magnetic moment, and absence of orientation selection. A disadvantage of GdIII–GdIII DEER is the low modulation depth due to the broad EPR spectrum of GdIII. Here, we introduce laser-induced magnetic dipole spectroscopy (LaserIMD) with a spin pair consisting of GdIII(PymiMTA) and a photoexcited porphyrin as an alternative technique. We show that the excited state of the porphyrin is not disturbed by the presence of the GdIII complex and that herewith modulation depths of almost 40% are possible. This is significantly higher than the value of 7.2% that was achieved with GdIII–GdIII DEER.

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Oxygen vacancies in oxidized and reduced vertically aligned α-MoO3 nanoblades

2022, Noby, Sohaila Z., Fakharuddin, Azhar, Schupp, Stefan, Sultan, Muhammad, Krumova, Marina, Drescher, Malte, Azarkh, Mykhailo, Boldt, Klaus, Schmidt-Mende, Lukas

Functionalized materials are highly desired for technological advancements spanning physics, chemistry, materials science, and biology due to their unique electronic properties. One such example is molybdenum trioxide (MoO3), a metal oxide with multiple oxidation states. Manipulating these oxidation states can alter the electronic properties, for instance, defects and electrical conductivity, by several orders of magnitude. In this work, oxygen vacancy-mediated intrinsic defects in vertically aligned a-MoO3 crystals are systematically tuned via thermal treatment under different reducing and oxidizing atmospheres. The positions and the concentration of the oxygen vacancies and restitution of the oxygen ions have been experimentally demonstrated via a range of techniques including electron paramagnetic resonance, X-ray diffraction, and high-resolution electron microscopy. The calculated concentration of the oxygen vacancies in the a-MoO3-x via EPR measurements is in the range of x = 0.004–0.049. The mechanism of the formation of oxygen vacancies in the a-MoO3-x crystal is understood via color center formation and polaron migration models. These oxygen vacancies show no influence on the optical band gap. However, they significantly impact the electrical conductivity on the order of 102 Sm-1 by altering the MoO3 properties from semi-insulating to conducting.

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Dance with spins : site-directed spin labeling coupled to electron paramagnetic resonance spectroscopy directly inside cells

2023, Pierro, Annalisa, Drescher, Malte

Depicting how biomolecules move and interact within their physiological environment is one of the hottest topics of structural biology. This Feature Article gives an overview of the most recent advances in Site-directed Spin Labeling coupled to Electron Paramagnetic Resonance spectroscopy (SDSL–EPR) to study biomolecules in living cells. The high sensitivity, the virtual absence of background, and the versatility of spin-labeling strategies make this approach one of the most promising techniques for the study of biomolecules in physiologically relevant environments. After presenting the milestones achieved in this field, we present a summary of the future goals and ambitions of this community.

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Mapping the helix arrangement of the reconstituted ETR1 ethylene receptor transmembrane domain by EPR spectroscopy

2022-03-01, Kugele, Anandi, Uzun, Buket, Müller, Lena, Schott-Verdugo, Stephan, Gohlke, Holger, Groth, Georg, Drescher, Malte

The plant ethylene receptor ETR1 is a key player in the perception of the phytohormone and subsequent downstream ethylene signal transmission, crucial for processes such as ripening, senescence and abscission. However, to date, there is sparse structural knowledge about the transmembrane sensor domain (TMD) of ETR1 that is responsible for the binding of the plant hormone and initiates the downstream signal transmission. Sequence information and ab initio modelling suggest that the TMD consists of three transmembrane helices. Here, we combined site-directed spin labelling with electron paramagnetic resonance spectroscopy and obtained distance restraints for liposome-reconstituted ETR1_TMD on the orientation and arrangement of the transmembrane helices. We used these data to scrutinize different computational structure predictions of the TMD.

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Evolved DNA Duplex Readers for Strand-Asymmetrically Modified 5-Hydroxymethylcytosine/5-Methylcytosine CpG Dyads

2022, Buchmuller, Benjamin C., Dröden, Jessica, Singh, Himanshu, Palei, Shubhendu, Drescher, Malte, Linser, Rasmus, Summerer, Daniel

5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), the two main epigenetic modifications of mammalian DNA, exist in symmetric and asymmetric combinations in the two strands of CpG dyads. However, revealing such combinations in single DNA duplexes is a significant challenge. Here, we evolve methyl-CpG-binding domains (MBDs) derived from MeCP2 by bacterial cell surface display, resulting in the first affinity probes for hmC/mC CpGs. One mutant has low nanomolar affinity for a single hmC/mC CpG, discriminates against all 14 other modified CpG dyads, and rivals the selectivity of wild-type MeCP2. Structural studies indicate that this protein has a conserved scaffold and recognizes hmC and mC with two dedicated sets of residues. The mutant allows us to selectively address and enrich hmC/mC-containing DNA fragments from genomic DNA backgrounds. We anticipate that this novel probe will be a versatile tool to unravel the function of hmC/mC marks in diverse aspects of chromatin biology.