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  1. 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. 
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    Pulsed electron paramagnetic resonance (EPR) based distance measurements using the recently developed Cu 2+ -DPA label present a promising strategy for measuring DNA backbone distance constraints. Herein we develop force field parameters for Cu 2+ -DPA in order to understand the features of this label at an atomic level. We perform molecular dynamics (MD) simulations using the force field parameters of Cu 2+ -DPA on four different DNA duplexes. The distance between the Cu 2+ centers, extracted from the 2 μs MD trajectories, agrees well with the experimental distance for all the duplexes. Further analyses of the trajectory provide insight into the orientation of the Cu 2+ -DPA inside the duplex that leads to such agreement with experiments. The MD results also illustrate the ability of the Cu 2+ -DPA to report on the DNA backbone distance constraints. Furthermore, measurement of fluctuations of individual residues showed that the flexibility of Cu 2+ -DPA in a DNA depends on the position of the label in the duplex, and a 2 μs MD simulation is not sufficient to fully capture the experimental distribution in some cases. Finally, the MD trajectories were utilized to understand the key aspects of the double electron electron resonance (DEER) results. The lack of orientational selectivity effects of the Cu 2+ -DPA at Q-band frequency is rationalized in terms of fluctuations in the Cu 2+ coordination environment and rotameric fluctuations of the label linker. Overall, a combination of EPR and MD simulations based on the Cu 2+ -DPA labelling strategy can contribute towards understanding changes in DNA backbone conformations during protein–DNA interactions. 
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  4. Abstract Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2′dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules. 
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  5. Spin labels attached to two residues of a protein chain have less conformational flexibility than those attached to a single residue and thus lead to a narrower spatialdistribution of the unpaired electron. The case of Cu(II) labels based on the double-histidine (dHis) motif is of particular interest, as it combines the advantage of precise localization of the unpaired electron with a labelling scheme orthogonal to the more common cysteine-based labelling. Here, we introduce an approach for in silico spin labelling of a protein by dHis motifs and Cu(II) complexes of iminodiacetic acid or nitrilotriacetic acid. We discuss a computerized scan for native histidine pairs that might be prone to bind such Cu(II) complexes and spin-labelling site pair scans that can identify suitable double mutants for labelling. Predicted distance distributions between two Cu(II) labels are compared to experimental distance distributions. We also test the hypothesis that elastic network modelling of conformational transitions with Cu2(II)- dHis labels can provide more accurate structural models than with nitroxide labels. 
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  6. Abstract

    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 anEscherichia colicopper‐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 CuIIspin labeling for the identification of various conformational states of CueR during transcription is shown.

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