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Bacteria use specialized proteins, like transcription factors, to rapidly control metal ion balance. CueR is a Gram‐negative bacterial copper regulator. The structure ofE. coliCueR complexed with Cu(I) and DNA was published, since then many studies have shed light on its function. However,P. aeruginosaCueR, which shows high sequence similarity toE. coliCueR, has been less studied. Here, we applied room‐temperature electron paramagnetic resonance (EPR) measurements to explore changes in dynamics ofP. aeruginosaCueR in dependency of copper concentrations and interaction with two different DNA promoter regions. We showed thatP. aeruginosaCueR is less dynamic than theE. coliCueR protein and exhibits much higher sensitivity to DNA binding as compared to itsE. coliCueR homolog. Moreover, a difference in dynamical behavior was observed whenP. aeruginosaCueR binds to thecopZ2DNA promoter sequence compared to themexPQ‐opmEpromoter sequence. Such dynamical differences may affect the expression levels of CopZ2 and MexPQ‐OpmE proteins inP. 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. 

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. 

Abstract 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.