- NSF-PAR ID:
- 10186788
- Date Published:
- Journal Name:
- The Journal of Physical Chemistry B
- ISSN:
- 1520-6106
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)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 electron 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.more » « less
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An optimal acquisition scheme for Q-band EPR distance measurements using Cu 2+ -based protein labelsRecent 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.more » « less
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Abstract Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2′dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules.more » « less
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Abstract 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 kDa
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