Towards liquid EPR dosimetry using nitroxides in aqueous solution.

Objective. Water-equivalent dosimeters are desirable for dosimetry in radiotherapy. The present work investigates basic characteristics of novel aqueous detector materials and presents a signal loss approach for electron paramagnetic resonance (EPR) dosimetry.Approach. The proposed principle is based on the radiation dose dependent annihilation of EPR active nitroxides (NO·) in aqueous solutions. Stable nitroxide radicals (3-Maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (MmP), 3-Carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (CmP)) in aqueous solutions containing dimethyl sulfoxide (DMSO) as an additive were filled in glass capillaries for irradiation and EPR readout. Radiation doses ranging from 1 to 64 Gy were applied with a clinical 6 MV flattening filter free photon beam. EPR readout was then performed with a X-band benchtop spectrometer. The dose response, temporal stability and reproducibility of the samples' EPR signal amplitudes as well as the influence of the nitroxide concentration between 10 and 160µM on the absolute signal loss were investigated using MmP. CmP was used to examine the dependence of the dose response on DMSO concentration between 0 and 10 vol%. An indirect effect model was fitted to the experimental data assuming irradiation induced radical reactions as the underlying mechanism.Main results. For an initial MmP concentration of 20µM, absolute EPR signal loss is linear up to a dose of 16 Gy with a yield G(-NO·) of approximately 0.4µmol J-1. Within five weeks upon sample irradiation to doses between 0 and 32 Gy relative EPR signal fluctuations were on average (126 readouts) below 1% (1σ). For c(MmP) ≥ 20µM, absolute signal loss is only weakly dependent on c(MmP), whereas it increases strongly with increasing c(DMSO) in the range 0-5 vol%. An indirect effect model is applicable to describe the reaction mechanism resulting in the observed dose response curve.Significance. Liquids consisting of nitroxides in aqueous solution and small amounts of DMSO (2 vol%) show promising basic characteristics for application as water-equivalent EPR dosimeter materials in radiotherapy. The EPR signal loss is based on an indirect effect mediated by diffusing radicals originating from the radiolysis of the water/DMSO mixture.

Evaluation of Copper(II) Transfer between Amyloid-beta Peptides by Relaxation-Induced Dipolar Modulation Enhancement (RIDME).

In the brains of Alzheimer's disease patients, fibrillar aggregates containing amyloid-beta (Aß) peptides are found, along with elevated concentrations of Cu(II) ions. The aggregation pathways of Aß peptides can be modulated by Cu(II) ions and is determined by the formation and nature of the Cu(II)-Aß complex. If spin-labeled, the Cu(II)-Aß complex contains two dipolar coupled paramagnetic centers, the spin label and the Cu(II) ion. Measurement of the dipolar coupling between these paramagnetic centers by relaxation-induced dipolar modulation enhancement (RIDME) allows to monitor the complex formation and thus opens a way to follow the Cu(II) transfer between peptides if a mixture of wild-type and spin-labeled ones is used. We evaluate this approach for a specific Cu(II)-Aß complex, the aggregation-inert Component II. The kinetics of the Cu(II) transfer can be resolved by performing RIDME in a time-dependent manner. A temporal resolution of seconds has been achieved, with the potential to reach milliseconds, using a rapid-freeze quench device to stop the Cu(II) transfer in solution after defined incubation times.

Adapting a practical EPR dosimetry protocol to measure output factors in small fields with alanine.

PURPOSE: Modern radiotherapy techniques often deliver small radiation fields. In this work, a practical Electron Paramagnetic Resonance (EPR) dosimetry protocol is adapted and applied to measure output factors (OF) in small fields of a 6 MV radiotherapy system. Correction factors and uncertainties are presented and OFs are compared to the values obtained by following TRS-483 using an ionization chamber (IC). METHODS: Irradiations were performed at 10 cm depth inside a water phantom positioned at 90 cm source to surface distance with a 6 MV flattening filter free photon beam of a Halcyon radiotherapy system. OFs for different nominal field sizes (1 × 1, 2 × 2, 3 × 3, 4 × 4, normalized to 10 × 10 cm2 ) were determined with a PinPoint 3D (PTW 31022) IC following TRS-483 as well as with alanine pellets with a diameter of 4 mm and a height of 2.4 mm. EPR readout was performed with a benchtop X-band spectrometer. Correction factors due to volume averaging and due to positional uncertainties were derived from 2D film measurements. RESULTS: OFs obtained from both dosimeter types agreed within 0.7% after applying corrections for the volume averaging effect. For the used alanine pellets, volume averaging correction factors of 1.030(2) for the 1 × 1 cm2 field and <1.002 for the larger field sizes were determined. The correction factor for positional uncertainties of 1 mm was in the order of 1.018 for the 1 × 1 cm2 field. Combined relative standard uncertainties uc for the OFs resulting from alanine measurements were estimated to be below 1.5% for all field sizes. For IC measurements, uc was estimated to be below 1.0%. CONCLUSIONS: A practical EPR dosimetry protocol is adaptable for precisely measuring OFs in small fields down to 1 × 1 cm2 . It is recommended to consider the effect of positional uncertainties for field sizes <2 × 2 cm2 .

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

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.

Titin UN2A Acts as a Stable, Non-Polymorphic Scaffold in its Binding to CARP.

The N2A segment of titin functions as a pivotal hub for signal transduction and interacts with various proteins involved in structural support, chaperone activities, and transcriptional regulation. Notably, the "unique N2A" (UN2A) subdomain has been shown to interact with the stress-regulated cardiac ankyrin repeat protein (CARP), which contributes to the regulation of sarcomeric stiffness. Previously, the UN2A domain's three-dimensional structure was modelled based on its secondary structure content identified by NMR spectroscopy, considering the domain in isolation. In this study, we report experimental long-range distance distributions by electron paramagnetic resonance (EPR) spectroscopy between the three helixes within the UN2A domain linked to the immunoglobulin domain I81 in the presence and absence of CARP. The data confirm the central three-helix bundle fold of UN2A and show that this adopts a compact and stable conformation in absence of CARP. After binding to CARP, no significant conformational change was observed, suggesting that the UN2A domain retains its structure upon binding to CARP thereby, mediating the interaction approximately as a rigid-body.

Insights into Enzymatic Catalysis from Binding and Hydrolysis of Diadenosine Tetraphosphate by E. coli Adenylate Kinase.

Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how adenylate kinase (AdK) from Escherichia coli interacts with diadenosine tetraphosphate (AP4A), a putative alarmone associated with transcriptional regulation, stress, and DNA damage response. From a combination of EPR and NMR spectroscopy together with X-ray crystallography, we found that AdK interacts with AP4A with two distinct modes that occur on disparate time scales. First, AdK dynamically interconverts between open and closed states with equal weights in the presence of AP4A. On a much slower time scale, AdK hydrolyses AP4A, and we suggest that the dynamically accessed substrate-bound open AdK conformation enables this hydrolytic activity. The partitioning of the enzyme into open and closed states is discussed in relation to a recently proposed linkage between active site dynamics and collective conformational dynamics.

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

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.

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

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.

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

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.

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

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.

Tracking protein domain movements by EPR distance determination and multilateration.

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.

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

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.

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

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.

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

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.

Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules.

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.

Prospective superficial EPR in-vivo dosimetry study during hypofractionated radiotherapy of breast cancer patients treated with helical tomotherapy.

BACKGROUND: In-vivo dosimetry (IVD) is a patient specific measure of quality control and safety during radiotherapy. With regard to current reporting thresholds for significant occurrences in radiotherapy defined by German regulatory authorities, the present study examines the clinical feasibility of superficial electron paramagnetic resonance (EPR) IVD of cumulative total doses applied to breast cancer patients treated with helical intensity-modulated radiotherapy (tomotherapy). METHODS: In total, 10 female patients with left- or right-sided breast cancer were enrolled in this prospective IVD study. Each patient received a hypofractionated whole breast irradiation. A total median dose of 42.4 Gy in 16 fractions (5 fractions per week) was prescribed to the planning target volume. The treatments were completely delivered using helical tomotherapy and daily image guidance via megavoltage CT (MVCT). For each patient, three EPR dosimeters were prepared and placed at distinct locations on the patient's skin during the delivery of all fractions. Two dosimeters were placed next to the ipsilateral and contralateral mammilla and one dosimeter was placed ventrally to the thyroid (out-of-primary-beam). The total doses delivered to the dosimeters were readout after all fractions had been administered. The measured total dose values were compared to the planned dose values derived from the treatment planning system (TPS). Daily positional variations (displacement vectors) of the ipsilateral mammilla and of the respective dosimeter were analyzed with respect to the planned positions using the daily registered MVCT image. RESULTS: Averaged over all patients, the mean absolute dose differences between measured and planned total dose values (± standard deviation (SD)) were: 0.49 ± 0.85 Gy for the ipsilateral dosimeter, 0.17 ± 0.49 Gy for the contralateral dosimeter and -0.12 ± 0.30 Gy for the thyroid dosimeter. The mean lengths of the ipsilateral displacement vectors (± SD) averaged over all patients and fractions were: 10 ± 7 mm for the dosimeter and 8 ± 4 mm for the mammilla. CONCLUSION: Superficial EPR IVD is suitable as additional safeguard for dose delivery during helical tomotherapy of breast cancer. Despite positional uncertainties in clinical routine, the observed dose deviations at the ipsilateral breast were on average small compared to national reporting thresholds for total dose deviations (i.e. 10%/4 Gy). EPR IVD may allow for the detection of critical dose errors during whole breast irradiations.

Suitability of superficial electron paramagnetic resonance dosimetry for in vivo measurement and verification of cumulative total doses during IMRT: A proof of principle.

PURPOSE: The present study investigates superficial in vivo dosimetry (IVD) by means of a previously proposed electron paramagnetic resonance (EPR) dosimetry system aiming at measuring and verifying total doses delivered by complex radiotherapy treatments. In view of novel regulatory requirements in Germany, differences between measured and planned total doses to the EPR dosimeters are analyzed and compared to reporting thresholds for significant occurrences. METHODS: EPR dosimeters, each consisting of one lithium formate monohydrate (LFM) and one polycrystalline l-alanine (ALA) pellet, were attached to the surface of an anthropomorphic head phantom. Three head and neck treatments with total target doses ranging from 30 to 64Gy were fully delivered to the phantom by helical tomotherapy. During each treatment, eight EPR dosimeters were placed at distinct spots: (i) within or next to the planning target volume (PTV), (ii) near to organs at risk including the parotids and the lenses, (iii) at the thyroid lying out-of-field. EPR read out was always performed after all fractions were delivered. EPR results were compared to thermoluminescence dosimeter (TLD) measurements and to the planned total doses derived from the treatment planning system (TPS). Planned total doses to the EPR dosimeters ranged from about 2 to 64Gy. RESULTS: By taking uncertainties into account, the measured and planned doses were in good agreement. Exceptions occurred mainly at the thyroid (out-of-field) and lenses (extreme sparing). The maximum total dose difference between EPR results and corresponding planned doses was 1.3Gy occurring at the lenses. Remarkably, each LFM and ALA pellet placed within or next to the PTV provided dose values that were within ±4% of the planned dose. Dose deviations from planned dose values were comparable for EPR and TLD measurements. CONCLUSION: The results of this proof of principle study suggests that superficial EPR-IVD is applicable in a wide dose range and in various irradiation conditions - being a valuable tool for monitoring cumulative total doses delivered by complex IMRT treatments. EPR-IVD in combination with helical tomotherapy is suitable to reliably detect local dose deviations at superficial dosimeter spots in the order of current national reporting thresholds for significant occurrences (i.e. 10%/4Gy).

Deciphering molecular details of the RAC-ribosome interaction by EPR spectroscopy.

The eukaryotic ribosome-associated complex (RAC) plays a significant role in de novo protein folding. Its unique interaction with the ribosome, comprising contacts to both ribosomal subunits, suggests a RAC-mediated coordination between translation elongation and co-translational protein folding. Here, we apply electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling (SDSL) to gain deeper insights into a RAC-ribosome contact affecting translational accuracy. We identified a local contact point of RAC to the ribosome. The data provide the first experimental evidence for the existence of a four-helix bundle as well as a long α-helix in full-length RAC, in solution as well as on the ribosome. Additionally, we complemented the structural picture of the region mediating this functionally important contact on the 40S ribosomal subunit. In sum, this study constitutes the first application of SDSL-EPR spectroscopy to elucidate the molecular details of the interaction between the 3.3 MDa translation machinery and a chaperone complex.

Tailoring Valence Tautomerism by Using Redox Potentials: Studies on Ferrocene-Based Triarylmethylium Dyes with Electron-Poor Fluorenylium and Thioxanthylium Acceptors.

Three new electrochromic ferrocenyl triarylmethylium dyes with fluorenylium (1 a+ , 1 b+ ) or thioxanthylium (1 c+ ) residues were selected in order to keep the intrinsic differences of redox potentials for ferrocene oxidation and triarylmethylium reduction small and to trigger valence tautomerism (VT). UV/Vis/NIR and quantitative EPR spectroscopy identified paramagnetic diradical isomers 1 a..+ -1 c..+ alongside diamagnetic forms 1 a+ -1 c+ , which renders these complexes magnetochemical switches. The diradical forms 1 a..+ -1 c..+ as well as the one-electron-reduced triarylmethyl forms of the complexes were found to dimerize in solution. For radical 1 a. , dimerization occurs on the timescale of cyclic voltammetry; this allowed us to determine the kinetics and equilibrium constant for this process by digital simulation. Mößbauer spectroscopy indicated that 1 a+ and 1 b+ retain VT even in the solid state. UV/Vis/NIR spectro-electrochemistry revealed the poly-electrochromic behaviour of these complexes by establishing the distinctly different electronic absorption profiles of the corresponding oxidized and reduced forms.

Intracellular Protein-Lipid Interactions Studied by Rapid-Scan Electron Paramagnetic Resonance Spectroscopy.

Protein-membrane interactions play key roles in essential cellular processes; studying these interactions in the cell is a challenging task of modern biophysical chemistry. A prominent example is the interaction of human α-synuclein (αS) with negatively charged membranes. It has been well-studied in vitro, but in spite of the huge amount of lipid membranes in the crowded environment of biological cells, to date, no interactions have been detected in cells. Here, we use rapid-scan (RS) electron paramagnetic resonance (EPR) spectroscopy to study αS interactions with negatively charged vesicles in vitro and upon transfection of the protein and lipid vesicles into model cells, i.e., oocytes of Xenopus laevis. We show that protein-vesicle interactions are reflected in RS spectra in vitro and in cells, which enables time-resolved monitoring of protein-membrane interaction upon transfection into cells. Our data suggest binding of a small fraction of αS to endogenous membranes.