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Recent advances in site-directed Cu 2+ labeling of proteins and nucleic acids have added an attractive new methodology to measure the structure-function relationship in biomolecules. Despite the promise, accessing the higher sensitivity of Q-band Double Electron Electron Resonance (DEER) has been challenging for Cu 2+ labels designed for proteins. Q-band DEER experiments on this label typically require many measurements at different magnetic fields, since the pulses can excite only a few orientations at a given magnetic field. Herein, we analyze such orientational effects through simulations and show that three DEER measurements, at strategically selected magnetic fields, are generally sufficient to acquire an orientational-averaged DEER time trace for this spin label at Q-band. The modeling results are experimentally verified on Cu 2+ labeled human glutathione S-transferase (hGSTA1-1). The DEER distance distribution measured at the Q-band shows good agreement with the distance distribution sampled by molecular dynamics (MD) simulations and X-band experiments. The concordance of MD sampled distances and experimentally measured distances adds growing evidence that MD simulations can accurately predict distances for the Cu 2+ labels, which remains a key bottleneck for the commonly used nitroxide label. In all, this minimal collection scheme reduces data collection time by as much asmore »six-fold and is generally applicable to many octahedrally coordinated Cu 2+ systems. Furthermore, the concepts presented here may be applied to other metals and pulsed EPR experiments.« less

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 ordermore »to shed light on protein function.« less

Sensitive in-cell distance measurements in proteins using pulsed-Electron Spin Resonance (ESR) require reduction-resistant and cleavage-resistant spin-labels. Among the reduction-resistant moieties, the hydrophilic trityl core known as OX063 is promising due to its long phase-memory relaxation time (T_m). This property leads to a sufficiently intense ESR signal for reliable distance measurements. Furthermore, the T_m of OX063 remains sufficiently long at higher temperatures, opening the possibility for measurements at temperatures above 50 K. In this work, we synthesized deuterated OX063 with a maleimide linker (mOX063-d24). We show that the combination of the hydrophilicity of the label and the maleimide linker enables high protein labeling that is cleavage-resistant in-cells. Distance measurements performed at 150 K using this label are more sensitive than the measurements at 80 K. The sensitivity gain is due to the significantly short longitudinal relaxation time (T_1) at higher temperatures, which enables more data collection per unit of time. In addition to in vitro experiments, we perform distance measurements in Xenopus laevis oocytes. Interestingly, the T_m of mOX063-d24 is sufficiently long even in the crowded environment of the cell, leading to signals of appreciable intensity. Overall, mOX063-d24 provides highly sensitive distance measurements both in vitro and in-cells.