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Herein, we develop an efficient rotamer library-based approach to predict distance distributions from Cu(ii) protein labels.

 

Combining rigid Cu( ii ) labels and pulsed-EPR techniques enables distance constraint measurements that are incisive probes of protein structure and dynamics. However, the labels can lead to a dipolar signal that is biased by the relative orientation of the two spins, which is typically unknown a priori in a bilabeled protein. This effect, dubbed orientational selectivity, becomes a bottleneck in measuring distances. This phenomenon also applies to other pulsed-EPR techniques that probe electron–nucleus interactions. In this work, we dissect orientational selectivity by generating an in silico sample of Cu( ii )-labeled proteins to evaluate pulse excitation in the context of double electron–electron resonance (DEER) at Q-band frequencies. This approach enables the observation of the contribution of each protein orientation to the dipolar signal, which provides direct insights into optimizing acquisition schemes to mitigate orientational effects. Furthermore, we incorporate the excitation profile of realistic pulses to identify the excited spins. With this method, we show that rectangular pulses, despite their imperfect inversion capability, can sample similar spin orientations as other sophisticated pulses with the same bandwidth. Additionally, we reveal that the efficiency of exciting spin-pairs in DEER depends on the frequency offset of two pulses used in the experiment and the relative orientation of the two spins. Therefore, we systematically examine the frequency offset of the two pulses used in this double resonance experiment to determine the optimal frequency offset for optimal distance measurements. This procedure leads to a protocol where two measurements are sufficient to acquire orientational-independent DEER at Q-band. Notably, this procedure is feasible with any commercial pulsed-EPR spectrometer. Furthermore, we experimentally validate the computational results using DEER experiments on two different proteins. Finally, we show that increasing the amplitude of the rectangular pulse can increase the efficiency of DEER experiments by almost threefold. Overall, this work provides an attractive new approach for analyzing pulsed-EPR spectroscopy to obtain microscopic nuances that cannot be easily discerned from analytical or numerical calculations. 

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 as 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. 

Pulsed dipolar EPR spectroscopy (PDS) in combination with site‐directed spin labeling is a powerful tool in structural biology. However, the commonly used spin labels are conjugated to biomolecules via rather long and flexible linkers, which hampers the translation of distance distributions into biomolecular conformations. In contrast, the spin label copper(II)‐nitrilotriacetic acid [Cu²⁺(NTA)] bound to two histidines (dHis) is rigid and yields narrow distance distributions, which can be more easily translated into biomolecular conformations. Here, we use this label on the 71 kDaYersiniaouter protein O (YopO) to decipher whether a previously experimentally observed bimodal distance distribution is due to two conformations of the biomolecule or of the flexible spin labels. Two different PDS experiments, that is, pulsed electron–electron double resonance (PELDOR aka DEER) and relaxation‐induced dipolar modulation enhancement (RIDME), yield unimodal distance distribution with the dHis‐Cu²⁺(NTA) motif; this result suggests that the α‐helical backbone of YopO adopts a single conformation in frozen solution. In addition, we show that the Cu²⁺(NTA) label preferentially binds to the target double histidine (dHis) sites even in the presence of 22 competing native histidine residues. Our results therefore suggest that the generation of a His‐null background is not required for this spin labeling methodology. Together these results highlight the value of the dHis‐Cu²⁺(NTA) motif in PDS experiments.

 

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. 

Site‐specific dynamics in proteins are at the heart of protein function. While electron paramagnetic resonance (EPR) has potential to measure dynamics in large protein complexes, the reliance on flexible nitroxide labels is limitating especially for the accurate measurement of site‐specific β‐sheet dynamics. Here, we employed EPR spectroscopy to measure site‐specific dynamics across the surface of a protein, GB1. Through the use of the double Histidine (dHis) motif, which enables labeling with a Cu(II) – nitrilotriacetic acid (NTA) complex, dynamics information was obtained for both α‐helical and β‐sheet sites. Spectral simulations of the resulting CW‐EPR report unique site‐specific fluctuations across the surface of GB1. Additionally, we performed molecular dynamics (MD) simulations to complement the EPR data. The dynamics observed from MD agree with the EPR results. Furthermore, we observe small changes ing_ǁvalues for different sites, which may be due to small differences in coordination geometry and/or local electrostatics of the site. Taken together, this work expands the utility of Cu(II)NTA‐based EPR measurements to probe information beyond distance constraints.