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1. Differentiating between Label and Protein Conformers in Pulsed Dipolar EPR Spectroscopy with the dHis‐Cu2+(NTA) Motif

   https://doi.org/10.1002/chem.202302541  

   Heubach, Caspar A.; Hasanbasri, Zikri; Abdullin, Dinar; Reuter, Arne; Korzekwa, Benedict; Saxena, Sunil; Schiemann, Olav (September 2023, Chemistry – A European Journal)

   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 flexible linkers, which hampers the translation of distance distributions into biomolecular conformations. In contrast, the spin label (nitrilotriacetic acid)copper(II) [Cu2+(NTA)] bound to two histidines (dHis) is rigid and can be more easily translated into biomolecular conformations. Here, we use this label on the 71 kDa Yersinia outer 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, i.e., 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, suggesting 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. This suggests 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. 

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2. Caught in the Middle: A Rigid DNA Label That Provides an Incisive Picture of DNA Conformational Flexibility in Protein–DNA Complexes

   https://doi.org/10.1021/jacs.5c01823  

   Casto, Joshua; Palit, Shramana; Little, Anthony; Hasanbasri, Zikri; Jen-Jacobson, Linda; Saxena, Sunil (June 2025, Journal of the American Chemical Society)

   Measurement of the conformation of DNA in protein-DNA complexes is important to decipher the role of DNA conformation and dynamics in protein recognition and function. In this work we report a rigid nucleotide-independent spin label that places paramagnetic Cu(II) within the DNA helix. The labeling strategy exploits the chelation of Cu(II) to two 8-aminoquinoline moieties, one in each strand. Because the rigidity of the probe avoids potentially confounding motions of the label itself, EPR signals can resolve 2-3 Å changes in interspin distance; the breadth of the distance distribution reports dy-namic fluctuations from the most probable conformation. Continuous wave and pulsed electron paramagnetic resonance spectroscopy (EPR) show that the Cu(II) coordinates properly to the labeling sites. Measurements of the interspin dipolar interaction on DNA oligonucleotides with two labels placed at various distances demonstrate that the label provides accurate and narrow distance distributions sensitive to DNA conformation and flexibility. Molecular dynamics simulations support these interpretations. We utilize this label to measure the conformations of DNA when type II restriction endonuclease EcoRV binds to its specific recognition sequence. The results provide in-solution evidence that EcoRV endonuclease induces axial DNA bending in the absence of metal ions, contrary to long-standing belief. Furthermore, the distance distribution nar-rows upon protein binding and even further on subsequent metal binding, implying that bound protein constrains the bend-ing dynamics of DNA. This method provides a novel and accurate approach to assess DNA conformation and dynamics in solution. 

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3. Development of Cu ²⁺ -Based Distance Methods and Force Field Parameters for the Determination of PNA Conformations and Dynamics by EPR and MD Simulations

   https://doi.org/10.1021/acs.jpcb.0c05509  

   Gamble Jarvi, Austin; Sargun, Artur; Bogetti, Xiaowei; Wang, Junmei; Achim, Catalina; Saxena, Sunil (August 2020, The Journal of Physical Chemistry B)

   Peptide nucleic acids (PNAs) are a promising group of synthetic analogues of DNA and RNA that offer several distinct advantages over the naturally occurring nucleic acids for applications in biosensing, drug delivery, and nanoelectronics. Because of its structural differences from DNA/RNA, methods to analyze and assess the structure, conformations, and dynamics are needed. In this work, we develop synergistic techniques for the study of the PNA conformation. We use CuQ2, a Cu(II) complex with 8-hydroxyquinoline (HQ), as an alternative base pair and as a spin label in electron paramagnetic resonance (EPR) distance methods. We use molecular dynamics (MD) simulations with newly developed force field parameters for the spin labels to interpret the distance constraints determined by EPR. We complement these methods by UV–vis and circular dichroism measurements and assess the efficacy of the Cu(II) label on a PNA duplex whose backbone is based on aminoethylglycine and a duplex with a hydroxymethyl backbone modification. We show that the Cu(II) label functions efficiently within the standard PNA and the hydroxymethyl-modified PNA and that the MD parameters may be used to accurately reproduce our EPR findings. Through the combination of EPR and MD, we gain new insights into the PNA structure and conformations as well as into the mechanism of orientational selectivity in Cu(II) EPR at X-band. These results present for the first time a rigid Cu(II) spin label used for EPR distance measurements in PNA and the accompanying MD force fields for the spin label. Our studies also reveal that the spin labels have a low impact on the structure of the PNA duplexes. The combined MD and EPR approach represents an important new tool for the characterization of the PNA duplex structure and provides valuable information to aid in the rational application of PNA at large. 

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4. An optimal acquisition scheme for Q-band EPR distance measurements using Cu ²⁺ -based protein labels

   https://doi.org/10.1039/d2cp01032a  

   Bogetti, Xiaowei; Hasanbasri, Zikri; Hunter, Hannah R.; Saxena, Sunil (January 2022, Physical Chemistry Chemical Physics)

   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. 

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5. Cleavage-Resistant Protein Labeling With Hydrophilic Trityl Enables Distance Measurements In-Cell

   https://doi.org/10.1021/acs.jpcb.1c02371  

   Hasanbasri, Zikri; Singewald, Kevin; Gluth, Teresa D.; Driesschaert, Benoit; Saxena, Sunil (April 2021, The Journal of Physical Chemistry B)

   null (Ed.)

   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. 

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