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EPR in-cell spin-labeling was applied to CueR in E. coli. The methodology employed a Cu(II)-NTA complexed with dHis. High resolved in-cell distance distributions were obtained revealing minor differences between in vitro and in-cell data. This methodology allows study of structural changes of any protein in-cell, independent of size or cellular system. [ABSTRACT FROM AUTHOR]

Copper is an essential element in nature but in excess, it is toxic to the living cell. The human metallochaperone Atox1 participates in copper homeostasis and is responsible for copper transmission. In a previous multiscale simulation study, we noticed a change in the coordination state of the Cu(I) ion, from 4 bound cysteine residues to 3, in agreement with earlier studies. Here, we perform and analyze classical molecular dynamic simulations of various coordination states: 2, 3, and 4. The main observation is an increase in protein flexibility as a result of a decrease in the coordination state. In addition, we identified several populated conformations that correlate well with double electron–electron resonance distance distributions or an X‐ray structure of Cu(I)‐bound Atox1. We suggest that the increased flexibility might benefit the process of ion transmission between interacting proteins. Further experiments can scrutinize this hypothesis and shed additional light on the mechanism of action of Atox1. [ABSTRACT FROM AUTHOR]

Color induction in nitrogen-contaminated diamonds was carried out via various procedures that involve irradiation, thermal treatments (annealing), and more. These treatments affect vacancy defect production and atom orientation centers in the diamond lattice. Natural diamonds underwent color enhancement treatments in order to produce green, blue, and yellow fancy diamonds. The aim of this study was to follow the changes occurring during the treatment, mainly by EPR spectroscopy, which is the main source for the determination of the effect of paramagnetic centers (carbon-centered radicals) on the color centers produced via the treatments, but also via visual assessment, fluorescence, UV-vis, and FTIR spectroscopy. The results indicate that diamonds containing high levels of nitrogen contamination are associated with high carbon-centered radical concentrations. Four paramagnetic center structures (N1, N4, and P2/W21) were generated by the treatment. It is suggested that the N4 structure correlates with the formation of blue color centers, whereas yellow color centers are attributed to the presence of N1 species. While to produce blue and yellow colors, a thermal treatment is needed after irradiation, for treated green diamonds, no thermal treatment is needed (only irradiation). [ABSTRACT FROM AUTHOR]

The modification of nitrogen-contaminated diamonds into color-enhanced diamonds is usually achieved by irradiation and thermal treatment (annealing). These treatments affect nitrogen contamination chemical bonding, vacancy concentration, and atom orientation centers in the diamond lattice. In this study, natural diamonds were subjected to irradiation and thermal annealing color enhancement treatments to produce green, blue, and yellow fancy diamonds. The study followed the changes that occur during treatment relying on visual assessment, fluorescence, UV-vis, FTIR, and EPR spectroscopy to characterize paramagnetic centers. The results indicated that diamonds containing high levels of nitrogen contamination presented a relatively high carbon-centered radical concentration. Two paramagnetic groups with different g-values were found, namely, low g-value centers of 2.0017–2.0027 and high g-value centers of 2.0028–2.0035. It is suggested that the 2.0017–2.0022 centers correlate with blue centers, whereas the 2.0023–2.0027 centers correlate with yellow centers. It was also found that thermal treatment was required to produce blue and yellow fancy diamonds, whereas no such treatment was needed to produce green diamonds. [ABSTRACT FROM AUTHOR]

Metalloregulators bind and respond to metal ions by regulating the transcription of metal homeostasis genes. Copper efflux regulator (CueR) is a copper‐responsive metalloregulator that is found in numerous Gram‐negative bacteria. Upon Cu(I) coordination, CueR initiates transcription by bending the bound DNA promoter regions facilitating interaction with RNA polymerase. The structure of Escherichia coli CueR in presence of DNA and metal ion has been reported using X‐ray crystallography and cryo‐EM, providing information about the mechanism of action. However, the specific role of copper in controlling this transcription mechanism remains elusive. Herein, we use room temperature electron paramagnetic resonance (EPR) experiments to follow allosterically driven dynamical changes in E. coli CueR induced by Cu(I) binding. We suggest that more than one Cu(I) ion binds per CueR monomer, leading to changes in site‐specific dynamics at the Cu(I) binding domain and at the distant DNA binding site. Interestingly, Cu(I) binding leads to an increase in dynamics about 27 Å away at the DNA binding domain. These changes in the dynamics of the DNA binding domain are important for exact coordination with the DNA. Thus, Cu(I) binding is critical to initiate a series of conformational changes that regulate and initiate gene transcription. Broad audience statement: The dynamics of metal transcription factors as a function of metal and DNA binding are complex. In this study, we use EPR spectroscopy to measure dynamical changes of Escherichia coli CueR as a function of copper and DNA binding. We show that copper controls the activation of the transcription processes by initiation a series of dynamical changes over the protein. [ABSTRACT FROM AUTHOR]

Copper is a trace element vital to many cellular functions. Yet its abnormal levels are toxic to cells, provoking a variety of severe diseases. The high affinity copper transporter 1 (CTR1), being the main in-cell copper [Cu(I)] entry route, tightly regulates its cellular uptake via a still elusive mechanism. Here, all-atoms simulations unlock the molecular terms of Cu(I) transport in eukaryotes disclosing that the two methionine (Met) triads, forming the selectivity filter, play an unprecedented dual role both enabling selective Cu(I) transport and regulating its uptake rate thanks to an intimate coupling between the conformational plasticity of their bulky side chains and the number of bound Cu(I) ions. Namely, the Met residues act as a gate reducing the Cu(I) import rate when two ions simultaneously bind to CTR1. This may represent an elegant autoregulatory mechanism through which CTR1 protects the cells from excessively high, and hence toxic, in-cell Cu(I) levels. Overall, our outcomes resolve fundamental questions in CTR1 biology and open new windows of opportunity to tackle diseases associated with an imbalanced copper uptake. [ABSTRACT FROM AUTHOR]

Paramagnetic centers in diamonds are affected by nitrogen atom contamination in the lattice. This study aimed to better understand the nitrogen concentration and anisotropy impact on the EPR (electron paramagnetic resonance) spectrum of natural diamonds (i.e. appropriate for color enhancement treatments). All the diamonds used were natural either colorless or faintly colored (D–M color range), which were selected according to the targeted final color post-treatment: green, blue, pink, orange, and yellow. Their paramagnetic centers were studied using EPR spectroscopy at various microwave power levels and evaluated for nitrogen concentration via FTIR spectroscopy. The results show a correlation between nitrogen concentrations and spin concentrations in all the pretreated diamonds, whereas the most abundant fast-relaxing paramagnetic center is similar to the C-centered radical. In addition, the hyperfine interaction anisotropy of the paramagnetic centers of nearby nitrogen atoms was found to be valid for pretreated green, pink, and orange diamonds. By clarifying the final structure of the treated fancy diamonds, which are visibly affected by adjacent nitrogen atoms, this study contributes to the refinement of colorless natural diamond selection for color enhancement. [ABSTRACT FROM AUTHOR]

The Cu(II)‐diacetyl‐bis (N4‐methylthiosemicarbazone) complex (ATSM−Cu(II)) has been suggested as a promising positron emission tomography (PET) agent for hypoxia imaging. Several in‐vivo studies have shown its potential to detect hypoxic tumors. However, its uptake mechanism and its specificity to various cancer cell lines have been less studied. Herein, we tested ATSM−Cu(II) toxicity, uptake, and reduction, using four different cell types: (1) mouse breast cancer cells (DA‐3), (2) human embryonic kidney cells (HEK‐293), (3) breast cancer cells (MCF‐7), and (4) cervical cancer cells (Hela) under normoxic and hypoxic conditions. We showed that ATSM−Cu(II) is toxic to breast cancer cells under normoxic and hypoxic conditions; however, it is not toxic to normal HEK‐293 non‐cancer cells. We showed that the Cu(I) content in breast cancer cell after treatment with ATSM−Cu(II) under hypoxic conditions is higher than in normal cells, despite that the uptake of ATSM−Cu(II) is a bit higher in normal cells than in breast cancer cells. This study suggests that the redox potential of ATSM−Cu(II) is higher in breast cancer cells than in normal cells; thus, its toxicity to cancer cells is increased. [ABSTRACT FROM AUTHOR]

Many inorganic materials can form crystals, but little is known about their enantioselective crystallization. Herein, we report on the enantioselective crystallization of ϵ‐Zn(OH)2 (Wulfingite) chiral crystals by using amino acids. Crystals of ϵ‐Zn(OH)2 were crystallized from supersaturated sodium hydroxide and zinc nitrate aqueous solutions in the presence of l‐ or d‐arginine. All of the chiral measurements, such as selective chiral adsorption by circular dichroism (CD), chiral chromatography, and polarimetry measurements, clearly show chiral discrimination during the crystallization of ϵ‐Zn(OH)2. In addition, a new method has been developed for identifying chirality in crystals by using electron paramagnetic resonance (EPR). Although the values of chiral induction of the ϵ‐Zn(OH)2 crystals obtained are somewhat low, these values are still significant because they demonstrate that enantioselectivity during the crystallization of chiral inorganic crystals with chiral additives can be achieved. The method can be applied to many chiral inorganic systems. Understanding and controlling the crystallization of chiral inorganic crystals is important for gaining knowledge on the interaction of chiral molecules with inorganic surfaces. This knowledge can lead to an understanding of basic scientific questions such as the evolution of homochirality in biomolecules and the development of chiral inorganic crystals for a variety of purposes such as asymmetric catalysis and optical applications. [ABSTRACT FROM AUTHOR]

Over recent decades, electron paramagnetic resonance (EPR) spectroscopy has become an essential tool for exploring complex biological systems. Herein, we discuss the potential of pulsed EPR spectroscopy to shed light on the mechanisms of protein‐DNA interactions. To this end, we first provide an overview of pulsed EPR methodology (and specifically, double electron electron resonance; DEER), with a focus on the various spin‐labeling methods used today both for protein labeling and for DNA labeling. Next, after briefly discussing recent applications of DEER in protein‐DNA studies, we introduce a detailed case study, an example of how EPR spectroscopy has been used to resolve the transcription mechanism of the CueR copper regulator. [ABSTRACT FROM AUTHOR]

The dissemination of resistant pathogenic microbes has become one of the most challenging problems that modern medicine has faced. Developing novel drugs based on new molecular targets that previously were not targeted, is therefore the highest priority in antibiotics research. One approach that has been recently suggested is to inhibit copper transporters in prokaryotic systems. Copper is required for many biological pathways, but sometimes it can harm the cell. Pathogenic systems have a highly sophisticated copper-regulation network; therefore, a better understanding of how this network operates at the molecular level should assist in developing the next generation of antibiotics. The CusB protein is part of the CusCBA periplasmic Cu(I) efflux system in Gram-negative bacteria, and was recently reported to play a key role in the functioning of the whole CusCBA system, in which conformational changes as well as the assembly/disassembly process control the opening of the transporter. More knowledge of the underlying mechanism is needed to attain a full understanding of CusB functioning, which is associated with targeting specific and crucial residues in CusB. Here, we combine in-vitro structural measurements, which use EPR spectroscopy and UV-Vis measurements, with cell experiments to explore the role of the various methionine residues in CusB. We targeted two methionine residues (M227 and M241) that are essential for the proper functioning of CusB. [ABSTRACT FROM AUTHOR]

The structural evolution during the formation of large-pore cubic lacd silica-based mesoporous materials, prepared with Pluroinic P123 and butanol as structure directing agents, is studied. Cryogenic high resolution scanning electron microscopy (cryo-HRSEM) and freeze-fracture-replication (FFR) transmission electron microscopy (TEM) images have shown that from the hexagonal phase a perforated layer (PL) phase is formed.

The evolution of the solution micellar structures during the formation of the hexagonal mesoporous material SBA-15 was studied by direct imaging and freeze-fracture replication cryo-TEM. Results showed a continuous transformation from spheroidal micelles, into threadlike micelles, which become longer and stiffer, followed by the formation of bundles with the dimensions similar to those found in the final material.

A methodology for measuring the sizes of micelles and their aggregation number by double electron resonance is demonstrated. It is found that the volume occupied by the hydrophobic spin-probe 4HTB in 7.5 wt % F127 and 6 wt % L64 is 88-90% and 74% respectively, of the total volume of the hydrophobic core.

A proposed model for the initial steps of SBA-15 formation (Si/P123 molar ratio of 59) is suggested, based on electron paramagnetic resonance (EPR) and electron spin-echo envelope modulation (ESEEM) experiments of different pluronic spin probes. The reaction was shown to propogate from the core of micelles into the carona/water interface.

The investigation of the formation mechanism of SBA-15 is focused with emphasis on the PEO interactions with the silica and the initiation of the micropores. A partitioning of the L62-NO between the precursors of the mesopores and micropores of the SBA-15 structure takes place at the very early stage of the reaction, and a continuous depletion of water within the corona-core interface was observed.

The interactions between proteins and their specific DNA sequences are the basis of many cellular processes. Hence, developing methods to build an atomic level picture of these interactions helps improve our understanding of key cellular mechanisms. CueR is an Escherichia coli copper‐sensing transcription regulator. The inhibition of copper‐sensing transcription regulators can kill pathogens, without harming the host. Several spectroscopic studies and crystallographic data have suggested that changes in the conformation of both the DNA and the protein control transcription. However, due to the inadequate resolution of these methods, the exact number of active conformations of CueR has not been determined. Resolving the structure of CueR in its active state is highly important for the development of specific inhibitors. Herein, the potential of double‐histidine (dHis)‐based CuII spin labeling for the identification of various conformational states of CueR during transcription is shown. CuII–NTA spin labeling: By using CuII–NTA spin labeling together with DEER spectroscopy, the active state conformations of the metal regulator CueR can be captured. [ABSTRACT FROM AUTHOR]

Protein kinases are among the most important regulators of cellular homeostasis, regulating many aspects of the cell life cycle through phosphorylation. The threonine/serine kinase cAMP-dependent protein kinase (PKA) is a major intracellular effector for a variety of bioactive molecules and hormones that work via a cAMP signaling pathway. In this work we report the discovery of a peptide and its simple peptidomimetic derivatives, which can activate the PKA catalytic unit. The direct binding of these new compounds to PKA and their resulting and activation mode were demonstrated by cellular (cardiomyocytes) and in vitro methods. Activation of PKA in cardiomyocytes after myocardial infarction might be a promising therapeutic strategy to protect the heart from ischemia and reperfusion. In addition, small-molecule PKA activators may prove useful as pharmacological tools for studying transduction mechanisms related to PKA. Several small molecules, which can activate a regulatory PKA unit, were developed. However, no small molecules that can activate a PKA catalytic unit were described in the scientific literature. [ABSTRACT FROM AUTHOR]

Coal exposed to an air atmosphere absorbs the atmospheric oxygen; this involves physical adsorption and chemisorption to form surface oxides (including hydro-peroxides). These weathering processes, which are denoted as LTO (Low Temperature Oxidation), decrease the calorific value of the coal and emit different gases such as carbon oxides (CO, CO2), water vapor, hydrogen (H2), and also some low-molecular-weight organic gases (C1–5). Some of these gases are toxic and flammable. The mechanism by which the molecular oxygen interacts with the coal macromolecule is thought to occur in several steps. The main concept is that a chain of radical reactions takes place; however, the exact underlying mechanism is not yet clear. We succeeded in identifying various carbon-centered radical species, depending on the coal rank and the degree of oxidation and suggested a new scheme for the formation of radicals via the coal oxidation process. [ABSTRACT FROM AUTHOR]

Copper transporter 1 (CTR1) is the main copper transporter in the eukaryotic system. CTR1 has several important roles: It binds Cu(II) ions that are present in the blood; it reduces those Cu(II) ions to Cu(I); and it subsequently transfers Cu(I) to the cytoplasmic domain, where the ion is delivered to various cellular pathways. Here, we seek to identify CTR1 binding sites for Cu(II) and Cu(I) and to shed light on the Cu(II)-to-Cu(I) reduction process. We focus on the first 14 amino acids of CTR1. This N-terminal segment is rich with histidine and methionine residues, which are known to bind Cu(II) and Cu(I), respectively; thus, this region has been suggested to have an important function in recruiting Cu(II) and reducing it to Cu(I). We utilize electron paramagnetic resonance (EPR) spectroscopy together with nuclear magnetic resonance (NMR) and UV-VIS spectroscopy and alanine substitution to reveal Cu(II) and Cu(I) binding sites in the focal 14-amino-acid segment. We show that H5 and H6 directly coordinate to Cu(II), whereas M7, M9, and M12 are involved in Cu(I) binding. This research is another step on the way to a complete understanding of the cellular copper regulation mechanism in humans. [ABSTRACT FROM AUTHOR]