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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]

Double quantum coherence (DQC) ESR is a sensitive method to measure magnetic dipolar interactions between spin labels. However, the DQC experiment on Cu 2+ centers presents a challenge at X-band. The Cu 2+ centers are usually coordinated to histidine residues in proteins. The electron–nuclear interaction between the Cu 2+ ion and the remote nitrogen in the imidazole ring can interfere with the electron–electron dipolar interaction. Herein, we report on a modified DQC experiment that has the advantage of reduced contributions from electron–nuclear interactions, which enhances the resolution of the DQC signal to the electron–electron dipolar modulations. The modified pulse-sequence is verified on Cu 2+ –NO system in a polyalanine-based peptide and on a coupled Cu 2+ system in a polyproline-based peptide. The modified DQC data were compared with the DEER data and good agreement was found. [ABSTRACT FROM AUTHOR]

The use of pulsed electron spin resonance (ESR) to measure interspin distance distributions has advanced biophysical research. The three major techniques that use pulsed ESR are relaxation rate based distance measurements, double quantum coherence (DQC), and double electron electron resonance (DEER). Among these methods, the DEER technique has become particularly popular largely because it is easy to implement on commercial instruments and because programs are available to analyze experimental data. [ABSTRACT FROM AUTHOR]

The article focuses on a research which reveals that cysteine-rich GASA5 Arabidopsis is a redox-active metalloprotein that can suppress plant-specific Gibberellin (GA)-stimulated Transcript (GAST1)-like genes. Topics discussed are the ability pf Arabidopsis to promote GA responses, late flowering due to reduces the ability of GASA5-over expressing seeds to germinate in the presence of the GA-biosynthesis inhibitor paclobutrazol.

Double quantum coherence (DQC) ESRspectroscopy is applied to measurethe Cu2+–Cu2+distance in the EcoRI-DNAcomplex. A simple method is proposed to reduce the contribution ofnuclear hyperfine and quadrupole interactions to such data. The effectsof such interactions between the electron spin of Cu2+andneighboring nuclei on the DQC data make it difficult to measure thenanometer range interspin distance. The DQC data is in good agreementwith results obtained by double electron electron resonance (DEER)spectroscopy. At the same time, the signal-to-noise ratio per shotin DQC is high. Taken together, these results provide impetus forfurther development of paramagnetic metal ion-based DQC techniques. [ABSTRACT FROM AUTHOR]

We studied the structural evolution during the formation of large-pore cubic Ia3¯d silica-based mesoporous materials, synthesized with Pluroinc P123 and butanol as structure directing agents. We used cryogenic high resolution scanning electron microscopy (cryo-HRSEM) and freeze-fracture-replication (FFR) transmission electron microscopy (TEM). Typically a silica precursor is added to an acid-catalyzed solution of Pluronic P123 and butanol. The latter serves as a cosolute, which can be added either at the beginning of the reaction, or after precipitation and the formation of a hexagonal phase. In this study we focused on the structural evolution from the hexagonal phase to the final cubic phase in the two different reactions. The same structural evolution with different kinetics was detected for both reactions. Cryo-HRSEM and FFR-TEM images revealed that from the hexagonal phase a perforated layer (PL) phase is formed, which later evolves into a bicontinuous structure. The final cubic phase forms within the layers, maintaining their orientation. We suggest a formation mechanism involving cylinder merging for the hexagonal to PL transition. Upon additional polymerization of the silica, the PL phase relaxes into the stable Ia3¯d cubic phase. Another minor mechanism detected involves the direct transition between the hexagonal to the final cubic phase through cylinder branching. [ABSTRACT FROM AUTHOR]

The evolution of the solution microstructures during the formation of the hexagonal mesoporous material SBA-15 was studied by direct imaging and freeze-fracture replication cryo-TEM. A reaction mixture was sampled at different times after the addition of tetramethoxyorthosilane (TMOS) to an acidic solution of Pluronic P123 held at 50 °C. Solution microstructures were detected by direct imaging cryo-TEM in the time window of 6.5–40 min after the addition of the TMOS (t = 0). The micrographs revealed that the initial spheroidal micelles evolve into threadlike micelles, which become longer and straighter with time. Then bundles with the dimensions similar to those found in the final material appeared, although there was no sign of a hexagonal arrangement up to 40 min. Due to the appearance of a precipitate at 40 min the sample became too viscous, preventing clear observation of its content. To observe the structures present after 40 min, freeze-fracture replication was carried out as well. Such samples were collected also at 22 min and showed the presence of threadlike micelles in agreement with the direct imaging cryo-TEM micrographs. The 2 h samples showed some areas of hexagonal ordered structures, which become very clear at 2 h 50 min. The cryo-TEM measurements were carried out under the same reaction conditions used in earlier in situ EPR experiments, thus allowing us to correlate molecular level events with the microstructure shape evolutions. This showed that the elongation of the micelles is a consequence of a reduction of the polarity and the water content within the micelles due to silicate adsorption and polymerization. Similar experiments were carried out also on SBA-15 prepared with HCI and TMOS at 35 °C. The appearance of threadlike micelles, followed by clustering of the TLMs, was observed under these conditions as well, but the reaction rate was faster. This suggests that the observed mechanism for the formation of SBA-15 is general. [ABSTRACT FROM AUTHOR]

The article reports on the use of the electron paramagnetic resonance (EPR) spectroscopy in differentiating the properties of the pretreated stones and treated colored diamonds.

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]

This review describes the use of Electron Paramagnetic Resonance (EPR) to measure residue specific dynamics in proteins with a specific focus on Cu(II)‐based spin labels. First, we outline approaches used to measure protein motion by nitroxide‐based spin labels. Here, we describe conceptual details and outline challenges that limit the use of nitroxide spin labels to solvent‐exposed α‐helical sites. The bulk of this review showcases the use of newly developed Cu(II)‐based protein labels. In this approach, the strategic mutation of native residues on a protein to generate two neighboring Histidine residues (i.e. the dHis motif) is exploited to enable a rigid site‐selective binding of a Cu(II) complex. The chelation of the Cu(II) complex to dHis directly anchors the Cu(II) spin label to the protein backbone. The improvement in rigidity expands both the spin‐labeling toolkit as well as the resolution of many EPR measurements. We describe how EPR measurements of the Cu(II) label directly reflect backbone motion and fluctuations. The EPR are complemented by Molecular Dynamics simulations. Finally, the dHis motif provides access to the measurement of site‐specific dynamics at both α‐helices and β‐sheets. The review outlines the limitations of the dHis method and provides an outlook for future developments. [ABSTRACT FROM AUTHOR]

Methionine motifs are methionine-rich metal-binding segments found in many human, yeast, and bacterial proteins involved in the transportation of copper ion to other cellular pathways, and in protecting copper from oxidation. Methionine motifs are found to bind Ag(I) and Cu(I) ions. Proteins or peptides that can bind different metal ions should have the ability to differentiate between them, to be able to shuttle them to various pathways in the cell. This study utilizes electron paramagnetic resonance spectroscopy together with circular dichroism and nuclear magnetic resonance to probe structural changes in the methionine segment upon coordinating Cu(I) and Ag(I) metal ions. The data collected here indicate that methionine segments experience structural changes while coordinating Cu(I) and Ag(I), however, the differences between the coordination of Cu(I) vs. Ag(I) to the methionine segment are mild. Since Cu(I) and Ag(I) metal ions are pretty similar in their nature and charge, the minor structural changes reported here are significant towards the understanding of the differences in the transport mechanism of these two metal ions in prokaryotic and eukaryotic cells. [ABSTRACT FROM AUTHOR]

Copper is an essential metal whose localization within the cells must be carefully controlled to avoid copper dependent redox cycling. Although most of the key proteins involved in cellular copper transfer have been identified, fundamental questions regarding the copper transfer mechanism have yet to be resolved. One of the blood carrier proteins believed to be involved in copper transfer to the cell is human serum albumin (HSA). However, direct evidence for close interaction between HSA and the extracellular domain of the copper transporter Ctr1 has not yet been found. By utilizing EPR spectroscopy, we show here that HSA closely interacts with the first 14 amino acids of the Ctr1, even without the presence of copper ions. [ABSTRACT FROM AUTHOR]

The essentiality and toxicity of copper in human, yeast, and bacteria cells requires precise mechanisms for acquisition, intimately linked to controlled distribution, which have yet to be fully understood. Herein, we utilise continuous wave and pulsed electron paramagnetic resonance (EPR) spectroscopy to explore one aspect in the controlled copper transportation mechanism. This was achieved by probing structural changes that occur in the c-terminal domain of the human copper transporter, CTR1, upon interacting with a methionine segment. The copper transporter CTR1 transports Cu(I) and Ag(I) ions to various intracellular pathways. Methionine motifs are methionine-rich metal binding segments found in many proteins involved in the transportation of copper ions to other cellular pathways. They are also found to bind Ag(I) with an affinity comparable to Cu(I). This study indicates that the methionine motif experiences conformational changes in the presence of the CTR1 c-terminal domain. These structural changes are dependent on the nature of the metal ion, Cu(I) vs. Ag(I). In addition, the data collected in this study emphasise how important the cysteine residue of the CTR1 c-terminal domain is to a correct conformational state of the target metal binding site. [ABSTRACT FROM PUBLISHER]

• The paper discusses the significant challenge in analytical chemistry of determining chirality in solid materials using electron paramagnetic resonance (EPR) spectroscopy. • The paper introduces a novel approach using electron paramagnetic resonance (EPR) spectroscopy on chiral crystals doped with Cu2+ ions to determine chirality of crystals. • The method is demonstrated using l - and DL-Histidine crystals doped with Cu2+, showing promising results in accurately determining chirality and enantiomeric composition. • This work provides an initial example of this technique and envisions its potential application to various other chiral organic crystal systems in the future. One of the most challenging tasks in analytical chemistry is the determination of the chirality (identification of an enantiomeric composition) in solids mainly because of the strict requirements of the pharmaceutical industry for enantiomerically pure drugs. Although there are a few methods available to accomplish enantio‑differentiation in solids, for example: X-ray diffraction (XRD), Thermogravimetric analysis (TGA), CD spectroscopy, and low-frequency (LF) Raman spectroscopy, this is still very challenging. In this work, we have developed a new method to measure the chirality of crystals, based on electron paramagnetic resonance (EPR) spectroscopy of chiral crystals doped with Cu2+ as the EPR active ion. Here, we demonstrate our approach using a model system of l - and DL-Histidine crystals doped with Cu2+. We show that EPR measurements of the Cu2+-doped Histidine crystals can accurately determine the chirality and enantiomeric composition of the crystals. We present a very preliminary example of this technique, and we hope that in the future it will be possible to refine and develop this method for many other chiral organic crystal systems. [Display omitted] [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]

Thirty-five thousand people die as a result of more than 2.8 million antibiotic-resistant infections in the United States of America per year. Pseudomonas aeruginosa (P. aeruginosa) is classified a serious threat, the second-highest threat category of the U.S. Department of Health and Human Services. Among others, the World Health Organization (WHO) encourages the discovery and development of novel antibiotic classes with new targets and mechanisms of action without cross-resistance to existing classes. To find potential new target sites in pathogenic bacteria, such as P. aeruginosa, it is inevitable to fully understand the molecular mechanism of homeostasis, metabolism, regulation, growth, and resistances thereof. P. aeruginosa maintains a sophisticated copper defense cascade comprising three stages, resembling those of public safety organizations. These stages include copper scavenging, first responder, and second responder. Similar mechanisms are found in numerous pathogens. Here we compare the copper-dependent transcription regulators cueR and copRS of Escherichia coli (E. coli) and P. aeruginosa. Further, phylogenetic analysis and structural modelling of mexPQ-opmE reveal that this efflux pump is unlikely to be involved in the copper export of P. aeruginosa. Altogether, we present current understandings of the copper homeostasis in P. aeruginosa and potential new target sites for antimicrobial agents or a combinatorial drug regimen in the fight against multidrug resistant pathogens. [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]

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]

Metallo-corroles have shown to have unique catalytic properties for oxygen reduction when adsorbed on a high surface area carbon such as BP2000. The nature of the interaction between carbon surfaces and metallo-corroles has yet to be identified. This work suggests that the catalytic activity lays not in a bond between the carbon surface and the corrole molecule, but rather in the arrangement of corrole molecules within the porous structure of the carbon surface. This hypothesis was studied here using Electron Paramagnetic Resonance (EPR), X-ray diffraction (XRD), UV-VIS spectroscopy, elemental analysis, N 2 adsorption and several electrochemical methods. BP2000 carbon was compared to four other carbons with different properties (oxygen surface groups, surface area, porous structure and crystallinity). All of these show that the Fe-corrole that was incorporated in BP2000 tends to adsorb in pores of 20–25 nm in diameter, which allow synergistic effects to take place, and enhance the electrocatalysis of oxygen reduction reaction. Image 1 [ABSTRACT FROM AUTHOR]

• The main copper transporter, Ctr1, can transfer Cu(I) in the cell, through two different intracellular domains. • Conformational flexibility of the copper metallochaperone Atox1 controls copper transfer mechanism in the cell. • Each metal binding domain in ATP7B has a specific role. Copper plays a vital role in fundamental cellular functions, and its concentration in the cell must be tightly regulated, as dysfunction of copper homeostasis is linked to severe neurological diseases and cancer. This review provides a compendium of current knowledge regarding the mechanism of copper transfer from the blood system to the Golgi apparatus; this mechanism involves the copper transporter hCtr1, the metallochaperone Atox1, and the ATPases ATP7A/B. We discuss key insights regarding the structural and functional properties of the hCtr1-Atox1-ATP7B cycle, obtained from diverse studies relying on distinct yet complementary biophysical, biochemical, and computational methods. We further address the mechanistic aspects of the cycle that continue to remain elusive. These knowledge gaps must be filled in order to be able to harness our understanding of copper transfer to develop therapeutic approaches with the capacity to modulate copper metabolism. [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 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]

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. [ABSTRACT FROM AUTHOR]

Abstract We propose herein a bio-mimetic catalytic structure as a new approach to increase the oxygen reduction catalytic selectivity of transition metal complexes. As in biological systems, where there is a series of mediators between the active site and the surface, that lowers the activation energy of the overall reaction and increases its selectivity, we use here a structure where a molecular catalyst Fe(III)tetrakis(1-methyl-4-pyridyl) porphyrin is bonded to a surface via a benzimidazole mediator. Using electron paramagnetic resonance (EPR), UV–vis spectroscopy and electrochemical measurements, we compared the ORR activity of the molecular catalyst with and without benzimidazole and show that this ligand strongly influences the selectivity of the reaction and shifts it from the 2 to the 2+2-electron reduction. Graphical abstract Image 1 [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]

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]

Understanding the human copper cycle is essential to understand the role of metals in promoting neurological diseases and disorders. One of the cycles controlling the cellular concentration and distribution of copper involves the copper transporter, Ctr1; the metallochaperone, Atox1; and the ATP7B transporter. It has been shown that the C-terminus of Ctr1, specifically the last three amino acids, HCH, is involved in both copper coordination and the transfer mechanism to Atox1. In contrast, the role of the intracellular loop of Ctr1, which is an additional intracellular segment of Ctr1, in facilitating the copper transfer mechanism has not been investigated yet. Here, we combine various biophysical methods to explore the interaction between this Ctr1 segment and metallochaperone Atox1 and clearly demonstrate that the Ctr1 intracellular loop (1) can coordinate Cu(I) via interactions with the side chains of one histidine and two methionine residues and (2) closely interacts with the Atox1 metallochaperone. Our findings are another important step in elucidating the mechanistic details of the eukaryotic copper cycle. [ABSTRACT FROM AUTHOR]

The oxidation level and properties of reduced graphene oxides (rGOs) were fine-tuned using temperature-programmed reductive annealing. rGOs were annealed at different temperatures (from 500 to 1000 °C) in hydrogen to modulate their oxidation levels. The surface of the rGOs was fully characterized using electron paramagnetic resonance backed by Raman, X-ray diffraction, and chemical analysis measurements. These experiments were used to study the changes in the surface of the rGO, its surface functionalities, and its defects as a function of the reduction temperature. In addition, electrochemical measurements to quantify the oxidation level of the rGOs offer a simple tool to correlate the properties of rGOs with their structure. Finally, we explored the effect of different levels of reduction on conductivity, capacitance, and surface reactivity. This research offers simple methodological techniques and routes to control and characterize the oxidation level of bulk quantities of rGO. [ABSTRACT FROM AUTHOR]

Metal organic frameworks (MOFs) have unique properties that make them excellent candidates for many high-tech applications. Nevertheless, their nonconducting character is an obstacle to their practical utilization in electronic and energy systems. Using the familiar HKUST-1 MOF as a model, we present a new method of imparting electrical conductivity to otherwise nonconducting MOFs by preparing MOF nanoparticles within the conducting matrix of mesoporous activated carbon (AC). This composite material was studied by X-ray diffraction (XRD), scanning electron microscopy (SEM), gas adsorption measurements, and electron paramagnetic resonance (EPR) spectroscopy. We show that MOF nanoparticles grown within the carbon matrix maintain their crystalline characteristics and their surface area. Surprisingly, as a result of the composition process, EPR measurements revealed a copper signal that had not yet been achieved. For the first time, we could analyze the complex EPR response of HKUST-1. We demonstrate the high conductivity of the MOF composite and discuss various factors that are responsible for these results. Finally, we present an optional application for using the conductive MOF composite as a high-performance electrode for pseudocapacitors. [ABSTRACT FROM AUTHOR]

Manganese dissolution from positive electrodes significantly reduces the durability of lithium-ion batteries. Knowledge of dissolution rates and oxidation states of manganese ions is essential for designing effective mitigation measures for this problem. We show that electron paramagnetic resonance (EPR) combined with atomic absorption spectroscopy (AAS) or inductively coupled plasma (ICP) can determine both manganese dissolution rates and relative Mn3+ amounts, by comparing the correlation between EPR and AAS/ICP data for Mn2+ standards with that for samples containing manganese cations dissolved from active materials (LiMn2O4 (LMO) and LiNi0.5Mn1.5O4 (LNMO)) into the same electrolyte solution. We show that Mn3+, and not Mn2+, is the dominant species dissolved from LMO, while Mn2+ is predominant for LNMO. Although the dissolution rate of LMO varies significantly for the two investigated materials, due to particle morphology and the presence of Cr in one of them, the Mn speciation appears independent of such details. Thus, the relative abundance of dissolved manganese ions in various oxidation states depends mainly on the overall chemical identity of the active material (LMO vs LNMO). We demonstrate the relevance of our methodology for practical batteries with data for graphite-LMO cells after high-temperature cycling or stand at 4.2 V. [ABSTRACT FROM AUTHOR]

C. elegans is a heme auxotroph that requires environmental heme for sustenance. As such, worms utilize HRG-3, a small heme-trafficking protein, to traffic heme from the intestine to extra-intestinal tissues and embryos. However, how HRG-3 binds and delivers heme remains unknown. In this study, we utilized electron paramagnetic resonance spectroscopy together with site-directed spin labeling, absorption spectroscopy, circular dichroism, and mutagenesis to gain structural and molecular insights into HRG-3. We showed that HRG-3 is a dimer, whereas H9 and H10 are significant residues that preserve a specific conformational state in the HRG-3 dimer. In the absence of H9 and H10, HRG-3 can still bind heme, although with a different affinity. Furthermore, the heme-binding site is closer to the N-termini than to the C-termini. Taken together, our results lay the groundwork for future mechanistic and structural studies of HRG-3 and inter-tissue heme trafficking in metazoans. [ABSTRACT FROM AUTHOR]

The search for novel anticancer therapeutic agents is an urgent and important issue in medicinal chemistry. Here, we report on the biological activity of the copper-based bioinorganic complex Cu (2,4-di- tert-butyl-6-(1H-imidazo- [1, 10] phenanthrolin-2-yl)phenol)]·10 CHCN ( 2), which was tested in rat L6 myotubes, mouse NSC-34 motor neurone-like cells, and HepG-2 human liver carcinoma. Upon 96 h incubation, 2 exhibited a significant cytotoxic effect on all three types of cells via activation of two cell death mechanisms (apoptosis and necrosis). Complex 2 exhibited better potency and efficacy than the canonical cytotoxic drug cisplatin. Moreover, during shorter incubations, complex 2 demonstrated a significant SOD mimetic activity, and it was more effective and more potent than the well-known SOD mimetic TEMPOL. In addition, complex 2 was able to interact with DNA and, cleave DNA in the presence of sodium ascorbate. This study shows the potential of using polynuclear redox active compounds for developing novel anticancer drugs through SOD-mimetic redox pathways. [ABSTRACT FROM AUTHOR]

Boththe essentiality and the toxicity of copper in human, yeast, and bacteriacells require precise mechanisms for acquisition, intimately linkedto controlled distribution, which have yet to be fully understood.This work explores one aspect in the copper cycle, by probing theinteraction between the human copper chaperone Atox1 and the c-terminaldomain of the copper transporter, CTR1, using electron paramagneticresonance (EPR) spectroscopy and circular dichroism (CD). The datacollected here shows that the Atox1 keeps its dimer nature also inthe presence of the CTR1 c-terminal domain; however, two geometricalstates are assumed by the Atox1. One is similar to the geometricalstate reported by the crystal structure, while the latter has notyet been constructed. In the presence of the CTR1 c-terminal domain,both states are assumed; however, the structure of Atox1 is more restrictedin the presence of the CTR1 c-terminal domain. This study also showsthat the last three amino acids of the CTR1 c-terminal domain, HCH,are important for maintaining the crystal structure of the Atox1,allowing less structural flexibility and improved thermal stabilityof Atox1. [ABSTRACT FROM AUTHOR]

Diamonds contain carbon paramagnetic centers (stable carbon radicals) in small concentrations (at the level of ~1 × 1012 spins/mg) that can help in elucidating the structure of the nitrogen atoms' contaminants in the diamond crystal. All diamonds that undergo polishing are exposed to high temperatures, owing to the friction force during the polishing process, which may affect the carbon-centered radicals' concentration and structure. The temperature is increased appreciably; consequently, the black body radiation in the visible range turns orange. During polishing, diamonds emit an orange light (at a wavelength of about 600 nm) that is typical of a black body temperature of 900 °C or higher. Other processes in which color-enhanced diamonds are exposed to high temperatures are thermal treatments or the high-pressure, high-temperature (HPHT) process in which the brown color (resulting from plastic deformation) is bleached. The aim of the study was to examine how thermal treatment and polishing influence the paramagnetic centers in the diamond. For this purpose, four rough diamonds were studied: two underwent a polishing process, and the other two were thermally treated at 650 °C and 1000 °C. The diamonds were analyzed pre- and post-treatment by EPR (Electron Paramagnetic resonance), FTIR (Fourier transform infrared, fluorescence, and their visual appearance. The results indicate that the polishing process results in much more than just thermal heating the paramagnetic centers. [ABSTRACT FROM AUTHOR]

Copper's essentiality and toxicity mean it requires a sophisticated regulation system for its acquisition, cellular distribution and excretion, which until now has remained elusive. Herein, we applied continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy in solution to resolve the copper trafficking mechanism in humans, by considering the route travelled by Cu(I) from the metallochaperone Atox1 to the metal binding domains of ATP7B. Our study revealed that Cu(I) is most likely mediated by the binding of the Atox1 monomer to metal binding domain 1 (MBD1) and MBD4 of ATP7B in the final part of its extraction pathway, while the other MBDs mediate this interaction and participate in copper transfer between the various MBDs to the ATP7B membrane domain. This research also proposes that MBD1-3 and MBD4-6 act as two independent units. [ABSTRACT FROM AUTHOR]

Five out of six people receive at least one antibiotic prescription per year. However, the ever-expanding use of antibiotics in medicine, agriculture, and food production has accelerated the evolution of antibiotic-resistant bacteria, which, in turn, made the development of novel antibiotics based on new molecular targets a priority in medicinal chemistry. One way of possibly combatting resistant bacterial infections is by inhibiting the copper transporters in prokaryotic cells. Copper is a key element within all living cells, but it can be toxic in excess. Both eukaryotic and prokaryotic cells have developed distinct copper regulation systems to prevent its toxicity. Therefore, selectively targeting the prokaryotic copper regulation system might be an initial step in developing next-generation antibiotics. One such system is the Gram-negative bacterial CusCFBA efflux system. CusB is a key protein in this system and was previously reported to play an important role in opening the channel for efflux via significant structural changes upon copper binding while also controlling the assembly and disassembly process of the entire channel. In this study, we aimed to develop novel peptide copper channel blockers, designed by in silico calculations based on the structure of CusB. Using a combination of magnetic resonance spectroscopy and various biochemical methods, we found a lead peptide that promotes copper-induced cell toxicity. Targeting copper transport in bacteria has not yet been pursued as an antibiotic mechanism of action. Thus, our study lays the foundation for discovering novel antibiotics. [ABSTRACT FROM AUTHOR]

Appropriate maintenance of Cu(I) homeostasis is an essential requirement for proper cell function because its misregulation induces the onset of major human diseases and mortality. For this reason, several research efforts have been devoted to dissecting the inner working mechanism of Cu(I)-binding proteins and transporters. A commonly adopted strategy relies on mutations of cysteine residues, for which Cu(I) has an exquisite complementarity, to serines. Nevertheless, in spite of the similarity between these two amino acids, the structural and functional impact of serine mutations on Cu(I)-binding biomolecules remains unclear. Here, we applied various biochemical and biophysical methods, together with all-atom simulations, to investigate the effect of these mutations on the stability, structure, and aggregation propensity of Cu(I)-binding proteins, as well as their interaction with specific partner proteins. Among Cu(I)-binding biomolecules, we focused on the eukaryotic Atox1-ATP7B system, and the prokaryotic CueR metalloregulator. Our results reveal that proteins containing cysteine-to-serine mutations can still bind Cu(I) ions; however, this alters their stability and aggregation propensity. These results contribute to deciphering the critical biological principles underlying the regulatory mechanism of the in-cell Cu(I) concentration, and provide a basis for interpreting future studies that will take advantage of cysteine-to-serine mutations in Cu(I)-binding systems. [ABSTRACT FROM AUTHOR]

CuII‐‐NTA spin labeling of the transcription regulator CueR, an E. coli copper‐sensing protein, enabled the high‐resolution detection of its various conformational states, as described by S. Ruthstein and co‐workers in their Communication on page 3053 ff. Two of the conformations, activators 1 and 2, represent CueR as it binds DNA and CuI ions. The computed structures suggest that one is more compressed on the DNA than the other and the transition between those states depends on CuI concentration in the bacterial cell. [ABSTRACT FROM AUTHOR]

CuII‐NTA‐Spinmarkierung des Transkriptionsregulators CueR, eines Kupfer erkennenden Proteins aus E. coli, ermöglichte die hochaufgelöste Detektion seiner verschiedenen Konformationszustände, wie S. Ruthstein et al. in ihrer Zuschrift auf S. 3085 demonstrieren. Zwei der Zustände, Aktivator 1 und 2, repräsentieren CueR in seiner Bindung an DNA und CuI‐Ionen. Aus den berechneten Strukturen lässt sich schließen, dass einer der beiden Zustände an der DNA stärker komprimiert ist und der Übergang zwischen den beiden Zuständen von der CuI‐Konzentration in der Bakterienzelle abhängt. [ABSTRACT FROM AUTHOR]

Summary CueR (Cu export regulator) is a metalloregulator protein that “senses” Cu(I) ions with very high affinity, thereby stimulating DNA binding and the transcription activation of two other metalloregulator proteins. The crystal structures of CueR when unbound or bound to DNA and a metal ion are very similar to each other, and the role of CueR and Cu(I) in initiating the transcription has not been fully understood yet. Using double electron-electron resonance (DEER) measurements and structure modeling, we investigate the conformational changes that CueR undergoes upon binding Cu(I) and DNA in solution. We observe three distinct conformations, corresponding to apo-CueR, DNA-bound CueR in the absence of Cu(I) (the “repression” state), and CueR-Cu(I)-DNA (the “activation” state). We propose a detailed structural mechanism underlying CueR's regulation of the transcription process. The mechanism explicitly shows the dependence of CueR activity on copper, thereby revealing the important negative feedback mechanism essential for regulating the intracellular copper concentration. [ABSTRACT FROM AUTHOR]