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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 Componentâ 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.
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Péptidos beta-Amiloides , Cobre , Cobre/química , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Cinética , Humanos , Espectroscopía de Resonancia por Spin del ElectrónRESUMEN
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.
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Adenilato Quinasa , Escherichia coli , Escherichia coli/metabolismo , Adenilato Quinasa/química , Hidrólisis , Fosfatos de Dinucleósidos/metabolismo , Catálisis , Dominio CatalíticoRESUMEN
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.
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The serine/threonine kinase Akt1 is part of the PI3â 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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Neoplasias , Proteínas Proto-Oncogénicas c-akt , Humanos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Mutación , Espectroscopía de Resonancia por Spin del ElectrónRESUMEN
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 .
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Alanina , Radiometría , Humanos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Radiometría/métodos , Aceleradores de Partículas , Fantasmas de Imagen , FotonesRESUMEN
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.
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5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , ADN/aislamiento & purificación , Proteína 2 de Unión a Metil-CpG/química , Fragmentos de Péptidos/química , ADN/química , Metilación de ADN , Evolución Molecular Dirigida , Células HEK293 , Humanos , Proteína 2 de Unión a Metil-CpG/genética , Fragmentos de Péptidos/genética , Dominios ProteicosRESUMEN
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.
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Óxidos N-Cíclicos/química , Espectroscopía de Resonancia por Spin del Electrón/normas , Proteínas/química , Marcadores de Spin , Benchmarking , Espectroscopía de Resonancia por Spin del Electrón/métodos , Reproducibilidad de los ResultadosRESUMEN
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.
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The authors wish to make the following two corrections to this paper [...].
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Synthetic polymers are mixtures of chains with different lengths, and their chain length and chain conformation are often experimentally characterized by ensemble averages. We demonstrate that double electron-electron resonance (DEER) spectroscopy can reveal the chain length distribution and the chain conformation and flexibility of the individual n-mers in oligo-(9,9-dioctylfluorene) from controlled Suzuki-Miyaura coupling polymerization. The required spin-labeled chain ends were introduced efficiently via a TEMPO-substituted initiator and chain-terminating agent, respectively, with an in situ catalyst system. Individual precise chain length oligomers as reference materials were obtained by a stepwise approach. Chain length distribution, chain conformation, and flexibility can also be accessed within poly(fluorene) nanoparticles.
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Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool in protein structural research. Nitroxides are highly suitable spin labeling reagents, but suffer from limited stability, particularly in the cellular environment. Herein we present the synthesis of a maleimide- and an azide-modified tetraethyl-shielded isoindoline-based nitroxide (M- and Az-TEIO) for labeling of cysteines or the noncanonical amino acid para-ethynyl-l-phenylalanine (pENF). We demonstrate the high stability of TEIO site-specifically attached to the protein thioredoxin (TRX) against reduction in prokaryotic and eukaryotic environments, and conduct double electron-electron resonance (DEER) measurements. We further generate a rotamer library for the new residue pENF-Az-TEIO that affords a distance distribution that is in agreement with the measured distribution.
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Alquinos/química , Aminoácidos/química , Cisteína/química , Óxidos de Nitrógeno/química , Azidas/química , Espectroscopía de Resonancia por Spin del Electrón , Isoindoles/química , Marcadores de Spin , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMEN
Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E. coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.
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Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/química , Alquinos/química , Azidas/química , Catálisis , Cobre/química , Reacción de Cicloadición , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Óxidos de Nitrógeno/química , Marcadores de SpinRESUMEN
EPR spectroscopy of diamagnetic bio-macromolecules is based on site-directed spin labeling (SDSL). Herein, a novel labeling strategy for proteins is presented. A nitroxide-based spin label has been developed and synthesized that can be ligated to proteins by an inverse-electron-demand Diels-Alder (DAinv ) cycloaddition to genetically encoded noncanonical amino acids. The nitroxide moiety is shielded by a photoremovable protecting group with an attached tetra(ethylene glycol) unit to achieve water solubility. SDSL is demonstrated on two model proteins with the photoactivatable nitroxide for DAinv reaction (PaNDA) label. The strategy features high reaction rates, combined with high selectivity, and the possibility to deprotect the nitroxide in Escherichia coli lysate.
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Aminoácidos/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Óxidos de Nitrógeno/química , Proteínas/química , Marcadores de Spin , Reacción de Cicloadición , HumanosRESUMEN
Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy enables studies of the structure, dynamics, and interactions of proteins in the noncrystalline state. The scope and analytical value of SDSLâ»EPR experiments crucially depends on the employed labeling strategy, with key aspects being labeling chemoselectivity and biocompatibility, as well as stability and spectroscopic properties of the resulting label. The use of genetically encoded noncanonical amino acids (ncAA) is an emerging strategy for SDSL that holds great promise for providing excellent chemoselectivity and potential for experiments in complex biological environments such as living cells. We here give a focused overview of recent advancements in this field and discuss their potentials and challenges for advancing SDSLâ»EPR studies.
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Código Genético , Marcadores de Spin , Aminoácidos/genética , Catálisis , Reacción de CicloadiciónRESUMEN
Norovirus infection is the major cause of nonbacterial gastroenteritis in humans and has been the subject of numerous studies investigating the virus's biophysical properties and biochemical function with the aim of deriving novel and highly potent entry inhibitors to prevent infection. Recently, it has been shown that the protruding P domain dimer (P-dimer) of a GII.10 Norovirus strain exhibits two new binding sites for l-fucose in addition to the canonical binding sites. Thus, these sites provide a novel target for the design of multivalent fucose ligands as entry inhibitors of norovirus infections. In this current study, a first generation of multivalent fucose-functionalized glycomacromolecules was synthesized and applied as model structures to investigate the potential targeting of fucose binding sites in human norovirus P-dimer. Following previously established solid phase polymer synthesis, eight precision glycomacromolecules varying in number and position of fucose ligands along an oligo(amidoamine) backbone were obtained and then used in a series of binding studies applying native MS, NMR, and X-ray crystallography. We observed only one fucose per glycomacromolecule binding to one P-dimer resulting in similar binding affinities for all fucose-functionalized glycomacromolecules, which based on our current findings we attribute to the overall size of macromolecular ligands and possibly to steric hindrance.
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Antivirales/síntesis química , Proteínas de la Cápside/metabolismo , Fucosa/química , Norovirus/efectos de los fármacos , Antivirales/farmacología , Proteínas de la Cápside/química , Ligandos , Simulación del Acoplamiento Molecular , Unión ProteicaRESUMEN
The intrinsically disordered human protein alpha-Synuclein (αS) has a prominent role in Parkinson's disease (PD) pathology. Several familial variants of αS are correlated with inherited PD. Disease mutations have been shown to have an impact on lipid membrane binding. Here, using electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling, we show that familial PD-associated variants are structurally defective in membrane binding and alter the local binding properties of the protein.
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Membrana Dobles de Lípidos/metabolismo , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Mutación , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
The intrinsically disordered protein α-synuclein (αS), a known pathogenic factor for Parkinson's disease, can adopt defined secondary structures when interacting with membranes or during fibrillation. The αS-lipid interaction and the implications of this process for aggregation and damage to membranes are still poorly understood. Therefore, we established a label-free infrared (IR) spectroscopic approach to allow simultaneous monitoring of αS conformation and membrane integrity. IR showed its unique sensitivity for identifying distinct ß-structured aggregates. A comparative study of wild-type αS and the naturally occurring splicing variant αS Δexon3 yielded new insights into the membrane's capability for altering aggregation pathways.
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Membrana Dobles de Lípidos/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , alfa-Sinucleína/metabolismo , Cinética , Membrana Dobles de Lípidos/química , Unión Proteica , Estructura Secundaria de Proteína , Solventes/química , alfa-Sinucleína/químicaRESUMEN
Following the first evidence of the triplet character of the optically excited phosphorescent state of naphthalene by Hutchison and Mangum in 1958, electron paramagnetic resonance (EPR) spectroscopy has become widely used to study and understand the properties and kinetic characteristics of excited triplet states. This minireview gives an overview over EPR techniques based on continuous microwave methods using lock-in or direct detection as well as pulsed EPR methods with respect to their suitability for kinetic studies of excited triplet states. A short historical overview of the experimental and theoretical developments in this field of research as well as of the triplet systems studied, with a final focus on fullerenes, is given. This may help newcomers to the field as a guide to the relevant literature.
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Separation of compounds using liquid chromatography is a process of enormous technological importance. This is true in particular for chiral substances, when one enantiomer has the desired set of properties and the other one may be harmful. The degree of development in liquid chromatography is extremely high, but still there is a lack in understanding based on experimental data how selectivity works on a molecular level directly at the surfaces of a porous host material. We have prepared amino-acid containing organosilica as such host materials. Watching the rotational dynamics of chiral spin probes using electron paramagnetic resonance spectroscopy allows us to differentiate between surface adsorbed and free guest species. Diastereotopic selectivity factors were determined, and the influence of chiral surface group density, chemical character of the surface groups, pore-size, and temperature was investigated. We found higher selectivity values in macroporous solids with a rather rigid organosilica network and at lower temperature, indicating the significant effect of confinement effects. In mesoporous materials features are opposed with regards to the T-dependent behavior. From EPR imaging techniques and the resulting (macroscopic) diffusion coefficients, we could confirm that the correlations found on the microscopic level transform also to the macroscopic behavior. Thus, our study is of value for the development of future chromatography materials by design.
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Human alpha-Synuclein (aS), implicated in Parkinson's disease, adopts a rich variety of different conformations depending on the macromolecular context. In order to unravel its pathophysiological role, monitoring its intracellular conformational state and identifying differences for the disease variants is crucial. Here, we present an intracellular spectroscopy approach based on a systematic spin-labeling site-scan in combination with intracellular electron paramagnetic resonance spectroscopy determining conformations on a molecular scale. A quantitative and model-based data analysis revealed that the vast majority of aS, be it wild-type or disease variants A30P or A53T, exists in the monomeric intrinsically disordered form in the cell.