An efficient EPR spin-labeling method enables insights of conformational changes in DNA.

Electron Paramagnetic Resonance (EPR) is a powerful tool for elucidating both static and dynamic conformational alterations in macromolecules. However, to effectively utilize EPR for such investigations, the presence of paramagnetic enters, known as spin-labels, is required. The process of spin-labeling, particularly for nucleotides, typically demands intricate organic synthesis techniques. In this study, we introduce a unique addition-elimination reaction method with a simple spin-labeling process, facilitating the monitoring of structural changes within nucleotide sequence. Our investigation focuses on three distinct labeling positions with a DNA sequence, allowing the measurement of distance between two spin-labels. The experimental mean distances obtained agreed with the calculated distances, underscoring the efficacy of this straightforward spin-labelling approach in studying complex biological processes such as transcription mechanism using EPR measurements.

The Dynamic Plasticity of P. aeruginosa CueR Copper Transcription Factor upon Cofactor and DNA Binding.

Bacteria use specialized proteins, like transcription factors, to rapidly control metal ion balance. CueR is a Gram-negative bacterial copper regulator. The structure of E. coli CueR complexed with Cu(I) and DNA was published, since then many studies have shed light on its function. However, P. aeruginosa CueR, which shows high sequence similarity to E. coli CueR, has been less studied. Here, we applied room-temperature electron paramagnetic resonance (EPR) measurements to explore changes in dynamics of P. aeruginosa CueR in dependency of copper concentrations and interaction with two different DNA promoter regions. We showed that P. aeruginosa CueR is less dynamic than the E. coli CueR protein and exhibits much higher sensitivity to DNA binding as compared to its E. coli CueR homologue. Moreover, a difference in dynamical behavior was observed when P. aeruginosa CueR binds to the copZ2 DNA promoter sequence compared to the mexPQ-opmE promoter sequence. Such dynamical differences may affect the expression levels of CopZ2 and MexPQ-OpmE proteins in P. aeruginosa. Overall, such comparative measurements of protein-DNA complexes derived from different bacterial systems reveal insights about how structural and dynamical differences between two highly homologous proteins lead to quite different DNA sequence-recognition and mechanistic properties.

Revealing the DNA Binding Modes of CsoR by EPR Spectroscopy.

In pathogens, a unique class of metalloregulator proteins, called gene regulatory proteins, sense specific metal ions that initiate gene transcription of proteins that export metal ions from the cell, thereby preventing toxicity and cell death. CsoR is a metalloregulator protein found in various bacterial systems that "sense" Cu(I) ions with high affinity. Upon copper binding, CsoR dissociates from the DNA promoter region, resulting in initiation of gene transcription. Crystal structures of CsoR in the presence and absence of Cu(I) from various bacterial systems have been reported, suggesting either a dimeric or tetrameric structure of these helical proteins. However, structural information about the CsoR-DNA complex is missing. Here, we applied electron paramagnetic resonance (EPR) spectroscopy to follow the conformational and dynamical changes that Mycobacterium tuberculosis CsoR undergoes upon DNA binding in solution. We showed that the quaternary structure is predominantly dimeric in solution, and only minor conformational and dynamical changes occur in the DNA bound state. Also, labeling of the unresolved C- terminus revealed no significant change in dynamics upon DNA binding. These observations are unique, since for other bacterial copper metalloregulators, such as the MerR and CopY families, major conformational changes were observed upon DNA binding, indicating a different mode of action for this protein family.

An in-cell spin-labelling methodology provides structural information on cytoplasmic proteins in bacteria.

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.

Conformations and Local Dynamics of the CopY Metal Sensor Revealed by EPR Spectroscopy.

Metal transcription factors regulate metal concentrations in eukaryotic and prokaryotic cells. Copper is a metal ion that is being tightly regulated, owing to its dual nature. Whereas copper is an essential nutrient for bacteria, it is also toxic at high concentrations. CopY is a metal-sensitive transcription factor belonging to the copper-responsive repressor family found in Gram-positive bacteria. CopY represses transcription in the presence of Zn(II) ions and initiates transcription in the presence of Cu(I) ions. The complete crystal structure of CopY has not been reported yet, therefore most of the structural information on this protein is based on its similarity to the well-studied MecI protein. In this study, electron paramagnetic resonance (EPR) spectroscopy was used to characterize structural and local dynamical changes in Streptococcus pneumoniae CopY as a function of Zn(II), Cu(I), and DNA binding. We detected different conformations and changes in local dynamics when CopY bound Zn(II), as opposed to Cu(I) ions. Furthermore, we explored the effects of metal ions and DNA on CopY conformation. Our results revealed the sensitivity and selectivity of CopY towards metal ions and provide new insight into the structural mechanism of the CopY transcription factor.

Copper coordination states affect the flexibility of copper Metallochaperone Atox1: Insights from molecular dynamics simulations.

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.

Disrupting Cu trafficking as a potential therapy for cancer.

Copper ions play a crucial role in various cellular biological processes. However, these copper ions can also lead to toxicity when their concentration is not controlled by a sophisticated copper-trafficking system. Copper dys-homeostasis has been linked to a variety of diseases, including neurodegeneration and cancer. Therefore, manipulating Cu-trafficking to trigger selective cancer cell death may be a viable strategy with therapeutic benefit. By exploiting combined in silico and experimental strategies, we identified small peptides able to bind Atox1 and metal-binding domains 3-4 of ATP7B proteins. We found that these peptides reduced the proliferation of cancer cells owing to increased cellular copper ions concentration. These outcomes support the idea of harming copper trafficking as an opportunity for devising novel anti-cancer therapies.

EPR Spectroscopy Provides New Insights into Complex Biological Reaction Mechanisms.

In the last 20 years, the use of electron paramagnetic resonance (EPR) has made a pronounced and lasting impact in the field of structural biology. The advantage of EPR spectroscopy over other structural techniques is its ability to target even minor conformational changes in any biomolecule or macromolecular complex, independent of its size or complexity, or whether it is in solution or in the cell during a biological or chemical reaction. Here, we focus on the use of EPR spectroscopy to study transmembrane transport and transcription mechanisms. We discuss experimental and analytical concerns when referring to studies of two biological reaction mechanisms, namely, transfer of copper ions by the human copper transporter hCtr1 and the mechanism of action of the Escherichia coli copper-dependent transcription factor CueR. Last, we elaborate on future avenues in the field of EPR structural biology.

Inherent Minor Conformer of Bordetella Effector BteA Directs Chaperone-Mediated Unfolding.

The pathogen Bordetella pertussis uses a type-3 secretion system (T3SS) to inject its cytotoxic effector BteA into the host cell via a designated needle structure. Prior to injection BteA is bound to its cognate chaperone BtcA presumed to assist in effector unfolding en route to needle passage. We utilized NMR and EPR spectroscopy to uncover the molecular mechanism of BtcA-mediated unfolding of BteA. BtcA induces a global structural change in the effector, which adopts a more extended and partially unfolded conformation. EPR distance measurements further show that the structured helical-bundle form of free BteA exists in conformational equilibrium with a lowly populated minor species. The nature of this equilibrium was probed using NMR relaxation dispersion experiments. At 283 K structural effects are most pronounced for a contiguous surface spanning the A- and B-helices of BteA, extending at 303 K to a second surface including the D- and E-helices. Residues perturbed in the minor conformation coincide with those exhibiting a BtcA-induced increase in flexibility, identifying this conformation as the BtcA-bound form of the effector. Our findings hint at a conformational-selectivity mechanism for the chaperone interaction with the effector, a paradigm that may be common to effector-chaperones secretion complexes in this family of pathogens.

The effects of thermal treatment and irradiation on the chemical properties of natural diamonds.

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.

Allostery-driven changes in dynamics regulate the activation of bacterial copper transcription factor.

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.

Cu(ii)-based DNA labeling identifies the structural link between transcriptional activation and termination in a metalloregulator.

Understanding the structural and mechanistic details of protein-DNA interactions that lead to cellular defence against toxic metal ions in pathogenic bacteria can lead to new ways of combating their virulence. Herein, we examine the Copper Efflux Regulator (CueR) protein, a transcription factor which interacts with DNA to generate proteins that ameliorate excess free Cu(i). We exploit site directed Cu(ii) labeling to measure the conformational changes in DNA as a function of protein and Cu(i) concentration. Unexpectedly, the EPR data indicate that the protein can bend the DNA at high protein concentrations even in the Cu(i)-free state. On the other hand, the bent state of the DNA is accessed at a low protein concentration in the presence of Cu(i). Such bending enables the coordination of the DNA with RNA polymerase. Taken together, the results lead to a structural understanding of how transcription is activated in response to Cu(i) stress and how Cu(i)-free CueR can replace Cu(i)-bound CueR in the protein-DNA complex to terminate transcription. This work also highlights the utility of EPR to measure structural data under conditions that are difficult to access in order to shed light on protein function.

Dynamical interplay between the human high-affinity copper transporter hCtr1 and its cognate metal ion.

Abnormal cellular copper levels have been clearly implicated in genetic diseases, cancer, and neurodegeneration. Ctr1, a high-affinity copper transporter, is a homotrimeric integral membrane protein that provides the main route for cellular copper uptake. Together with a sophisticated copper transport system, Ctr1 regulates Cu(I) metabolism in eukaryotes. Despite its pivotal role in normal cell function, the molecular mechanism of copper uptake and transport via Ctr1 remains elusive. In this study, electron paramagnetic resonance (EPR), UV-visible spectroscopy, and all-atom simulations were employed to explore Cu(I) binding to full-length human Ctr1 (hCtr1), thereby elucidating how metal binding at multiple distinct sites affects the hCtr1 conformational dynamics. We demonstrate that each hCtr1 monomer binds up to five Cu(I) ions and that progressive Cu(I) binding triggers a marked structural rearrangement in the hCtr1 C-terminal region. The observed Cu(I)-induced conformational remodeling suggests that the C-terminal region may play a dual role, serving both as a channel gate and as a shuttle mediating the delivery of copper ions from the extracellular hCtr1 selectivity filter to intracellular metallochaperones. Our findings thus contribute to a more complete understanding of the mechanism of hCtr1-mediated Cu(I) uptake and provide a conceptual basis for developing mechanism-based therapeutics for treating pathological conditions linked to de-regulated copper metabolism.

The conformational plasticity of the selectivity filter methionines controls the in-cell Cu(I) uptake through the CTR1 transporter.

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.

Thermal Stability of Carbon-Centered Radicals Involved in Low-Temperature Oxidation of Bituminous and Lignite Coals as a Function of Temperature.

Coal is intensively used worldwide as a main fuel source. However, it may undergo oxidation processes [i.e., low-temperature oxidation (LTO)] when stored under an air atmosphere in piles post-mining at low temperatures ranging from 300 to 425 K, specifically, a surface gas/solid reaction with molecular oxygen. Therefore, it is of major importance to prevent or appreciably slow down such reactions, which result in a loss in the energy content (calorific value) of coal. Previously, we showed that radicals are formed during the LTO process. In this work, the dependence of radical formation on coal rank as a function of heating (temperature) and the presence of oxygen gas were studied using electron paramagnetic resonance spectroscopy. It was shown that lignite coals are more sensitive than bituminous coals to the atmospheric environment (i.e., molecular oxygen and nitrogen content) and to temperature, as reflected by the formation of surface carbon-centered radicals. Moreover, this is the first publication showing the effects of LTO on micro- and macro-pores by assessing how these structures affect O2 diffusion. The LTO process blocks the micro-pores, such that radicals form mainly at the surface of the coal macromolecules, in both bituminous and lignite coals.

Effect of Diamond Polishing and Thermal Treatment on Carbon Paramagnetic Centers' Nature and Structure.

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.

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.

Molecular Dynamics Simulations of the Apo and Holo States of the Copper Binding Protein CueR Reveal Principal Bending and Twisting Motions.

Copper is essential for proper functioning of cells but is dangerous in unregulated concentrations. One of the members in the bacterial system responsible for facilitating copper homeostasis is the copper efflux regulator (CueR) protein. Upon copper binding, CueR induces transcription of additional copper homeostasis proteins via a cascade of events. There are some available crystal structures of CueR, in the holo (copper-bound), active (copper- and DNA-bound), and repressed (only DNA-bound) states, and these structures suggest that transcription initiation involves a distortion in the promoter DNA strand. In this work, we study the dynamic behavior of the protein, using molecular dynamics simulations, and compare with available electron paramagnetic resonance measurements for validation. We develop simple force-field parameters to describe the copper-binding motif, thus enabling the use of simplified, classical physics equations. This enabled us to access reasonable simulation times that illustrate global motions of the protein. Both in the holo and apo states of CueR, we observed large-scale helical bending motions that could be involved in the bending of a bound DNA molecule so that transcription activation can take place. Additionally, copper binding might afford increased rigidification of the active state via helix α6.

Cellular Uptake of the ATSM-Cu(II) Complex under Hypoxic Conditions.

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.

Advances in Understanding of the Copper Homeostasis in Pseudomonas aeruginosa.

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.