This content will become publicly available on July 1, 2024
- Award ID(s):
- 2006154
- NSF-PAR ID:
- 10411117
- Date Published:
- Journal Name:
- Physical Chemistry Chemical Physics
- ISSN:
- 1463-9076
- 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 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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Electron paramagnetic resonance (EPR) is a powerful tool for research in chemistry, biology, physics and materials science, which can benefit significantly from moving to frequencies above 100 GHz. In pulsed EPR spectrometers driven by powerful sub-THz oscillators, such as the free electron laser (FEL)-powered EPR spectrometer at UCSB, control of the duration, power and relative phases of the pulses in a sequence must be performed at the frequency and power level of the oscillator. Here we report on the implementation of an all-quasioptical four-step phase cycling procedure carried out directly at the kW power level of the 240 GHz pulses used in the FEL-powered EPR spectrometer. Phase shifts are introduced by modifying the optical path length of a 240 GHz pulse with precision-machined dielectric plates in a procedure we call phase cycling with optomechanical phase shifters (POPS), while numerical receiver phase cycling is implemented in post-processing. The POPS scheme was successfully used to reduce experimental dead times, enabling pulsed EPR of fast-relaxing spin systems such as gadolinium complexes at temperatures above 190 K. Coherence transfer pathway selection with POPS was used to perform spin echo relaxation experiments to measure the phase memory time of P1 centers in diamond in the presence of a strong unwanted FID signal in the background. The large excitation bandwidth of FEL-EPR, together with phase cycling, enabled the quantitative measurement of instantaneous electron spectral diffusion, from which the P1 center concentration was estimated to within 10%. Finally, phase cycling enabled saturation-recovery measurements of T 1 in a trityl-water solution at room temperature – the first FEL-EPR measurement of electron T 1 .more » « less
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