EPR Spectroscopy Detects Various Active State Conformations of the Transcriptional Regulator CueR

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 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.

A general model to optimise CuIIlabelling efficiency of double-histidine motifs for pulse dipolar EPR applications

“…[28] These Cu II -chelate spin-labels also yield exquisite precision in the distance domain owing to the bipedal mode of attachment, and the rigidity of the co-ordinating imidazole moieties with respect to the protein backbone. [50,53] The lack of a flexible linker means dH Cu II -labelling is appealing for structural studies in systems with subtle conformational changes [54] or nuanced conformational equilibria. [55] Furthermore, use of Cu II -NTA in conjunction with the commercially available methanethiosulfonate spin label MTSL, in the 5-pulse dead-time free Relaxation Induced Dipolar Modulation Enhancement (RIDME) experiment [56] yields superb concentration sensitivity, down to hundreds of nM.…”

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site‐Specific Elucidation of Hidden Dynamics in Enzymes

“…[5a] Distance measurements using the dHis motif have been exploited to understand the localization of a native metal binding site, large-amplitude conformational changes, regulation mechanisms, and the relative orientations between two sites in a protein. [12] Here we present the utility of the dHis-Cu II NTA label to measure the role of site-specific protein dynamics in enzyme function.…”

Pulsed Dipolar EPR Spectroscopy and Metal Ions: Methodology and Biological Applications

“…In PDS, the reorganization of a protein can be followed by monitoring the change in the distance between two spin‐labeled protein sites, which are involved this reorganization. The potential of this method was nicely shown in a study by Sameach et al . The object of that study was the homodimer of the copper‐sensing transcription regulator CueR from E. coli (Figure a).…”

“…If a biomolecule does not contain any of these, spin centers can be introduced through exchanging intrinsic diamagnetic metal ions for paramagnetic ones, e. g., Mg 2+ for Mn 2+ , or attaching paramagnetic tags, called spin labels, via site‐directed spin labeling (SDSL) . In the latter case, nitroxides are usually used, but spin labels based on trityls, Gd 3+ , Cu 2+ , and Mn 2+[29,30] are gaining importance, especially, for in‐cell PDS applications and, in the case of trityls, also for PDS at physiological temperatures …”

“…[11][12][13][14] If a biomolecule does not contain any of these, spin centers can be introduced through exchanging intrinsic diamagnetic metal ions for paramagnetic ones, e. g., Mg 2 + for Mn 2 + , [19] or attaching paramagnetic tags, called spin labels, via site-directed spin labeling (SDSL). [20] In the latter case, nitroxides are usually used, [21] but spin labels based on trityls, [22,23] Gd 3 + , [24][25][26] Cu 2 + , [27,28] and Mn 2 + [29,30] are gaining importance, especially, for in-cell PDS applications [23,[31][32][33] and, in the case of trityls, also for PDS at physiological temperatures. [34,35] If a biomolecule contains two or more spin centers, PDS can be applied to measure the dipolar coupling between these centers, which encodes the distance and relative orientation between the spin centers and, therefore, is an important source of structural information.…”