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1. Orientation and dynamics of Cu ²⁺ based DNA labels from force field parameterized MD elucidates the relationship between EPR distance constraints and DNA backbone distances

   https://doi.org/10.1039/d0cp05016d  

   Ghosh, Shreya ; Casto, Joshua ; Bogetti, Xiaowei ; Arora, Charu ; Wang, Junmei ; Saxena, Sunil ( December 2020 , Physical Chemistry Chemical Physics)

   Pulsed electron paramagnetic resonance (EPR) based distance measurements using the recently developed Cu 2+ -DPA label present a promising strategy for measuring DNA backbone distance constraints. Herein we develop force field parameters for Cu 2+ -DPA in order to understand the features of this label at an atomic level. We perform molecular dynamics (MD) simulations using the force field parameters of Cu 2+ -DPA on four different DNA duplexes. The distance between the Cu 2+ centers, extracted from the 2 μs MD trajectories, agrees well with the experimental distance for all the duplexes. Further analyses of the trajectory provide insight into the orientation of the Cu 2+ -DPA inside the duplex that leads to such agreement with experiments. The MD results also illustrate the ability of the Cu 2+ -DPA to report on the DNA backbone distance constraints. Furthermore, measurement of fluctuations of individual residues showed that the flexibility of Cu 2+ -DPA in a DNA depends on the position of the label in the duplex, and a 2 μs MD simulation is not sufficient to fully capture the experimental distribution in some cases. Finally, the MD trajectories were utilized to understand the key aspects of the double electron electronmore »resonance (DEER) results. The lack of orientational selectivity effects of the Cu 2+ -DPA at Q-band frequency is rationalized in terms of fluctuations in the Cu 2+ coordination environment and rotameric fluctuations of the label linker. Overall, a combination of EPR and MD simulations based on the Cu 2+ -DPA labelling strategy can contribute towards understanding changes in DNA backbone conformations during protein–DNA interactions.« less
2. 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 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
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 conformationsmore »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.« less
4. Cu(II) EPR Reveals Two Distinct Binding Sites and Oligomerization of Innate Immune Protein Calgranulin C

   https://doi.org/https://doi.org/10.1007/s00723-018-1053-7  

   Ghosh, S ; Garcia, V ; Singewald, K ; Damo, S ; Saxena, S. ( January 2018 , Applied magnetic resonance)

   S100A12 or Calgranulin C is a homodimeric antimicrobial protein of the S100 family of EF-hand calcium-modulated proteins. S100A12 is involved in many diseases like inflammation, tumor invasion, cancer and neurological disorders like Alzheimer’s disease. The binding of transition metal ions to the protein is important as the sequestering of the metal ion induces conformational changes in the protein, inhibiting the growth of various pathogenic microorganisms. In this work, we probe the Cu(II) binding properties of Calgranulin C. We demonstrate that the two Cu(II) binding sites in Calgranulin C show different coordination environments in solution. Electron spin resonance (ESR) spectra of Cu(II)-bound protein clearly show two distinct components at higher Cu(II):protein ratios, which is indicative of the two different binding environments for the Cu(II) ions. The g|| and A|| values are also different for the two components, indicating that the number of directly coordinated nitrogens in each site differs. Furthermore, we perform Continuous Wave (CW)-titrations to obtain the binding affinity of the Ca(II)-loaded protein to Cu2+ ions. We observe a positive cooperativity in binding of the two Cu(II) ions. In order to further probe the Cu2+ coordination, we also perform Electron Spin Echo Envelope Modulation (ESEEM) experiment. We perform ESEEM atmore »two different fields where one Cu(II) binding site dominates over the other. At both sites we see distinct signatures of Cu(II)-histidine coordination. However, we clearly see that the ESEEM spectra corresponding to the two Cu2+ binding sites are significantly different. There is clear change in the intensity of the double quantum (DQ) peak with respect to the nuclear quadrupole interaction (NQI) peak at the two different fields. Furthermore, ESEEM along with Hyperfine Sublevel Correlation (HYSCORE) show that only one of the two Cu(II) binding sites has backbone coordination, confirming our previous observation. Finally, we perform Double Electron Electron Resonance (DEER) spectroscopy to probe if the difference in binding environment is due to the Cu(II) binding to different sites in the protein. We obtain a distance distribution with a sharp peak at ~ 3 nm and a broad peak at ~ 4 nm. The shorter distance agrees with the Cu(II)-Cu(II) distance expected for a dimer from the crystal structure. The longer distance is consistent with the Cu(II)-Cu(II) distance when oligomerization occurs.« less
5. Efficient localization of a native metal ion within a protein by Cu ²⁺ -based EPR distance measurements

   https://doi.org/10.1039/c8cp07143h  

   Gamble Jarvi, Austin ; Cunningham, Timothy F. ; Saxena, Sunil ( May 2019 , Physical Chemistry Chemical Physics)

   Electron paramagnetic resonance (EPR) based distance measurements have been exploited to measure protein–protein docking, protein–DNA interactions, substrate binding and metal coordination sites. Here, we use EPR to locate a native paramagnetic metal binding site in a protein with less than 2 Å resolution. We employ a rigid Cu 2+ binding motif, the double histidine (dHis) motif, in conjunction with double electron electron resonance (DEER) spectroscopy. Specifically, we utilize a multilateration approach to elucidate the native Cu 2+ binding site in the immunoglobulin binding domain of protein G. Notably, multilateration performed with the dHis motif required only the minimum number of four distance constraints, whereas comparable studies using flexible nitroxide-based spin labels require many more for similar precision. This methodology demonstrates a significant increase in the efficiency of structural determinations via EPR distance measurements using the dHis motif.