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

Ghosh, Shreya; Casto, Joshua; Bogetti, Xiaowei; Arora, Charu; Wang, Junmei; Saxena, Sunil

doi:10.1039/d0cp05016d

Cited by 11 publications

(13 citation statements)

References 96 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…11 The labeling of DNA with Cu(II) is described in ESI. † 11,14 Electrophoretic mobility shi assays (EMSA) show that CueR binds to the DPA-DNA duplex (Fig. S1 †).…”

Section: Resultsmentioning

confidence: 99%

“…Prior work has shown that the Cu(II)-Cu(II) distance in DNA can be directly related to the backbone C 0 -C 0 distance. 14 A more careful model analysis that accounts for the positioning of the Cu(II) atoms with respect to the DNA backbone, yields a 5 0 endcenter -3 0 end angle of 55 for the duplex with a 3.6 nm Cu(II)-Cu(II) distance (Fig. S8 †).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Cu(ii)-based DNA labeling identifies the structural link between transcriptional activation and termination in a metalloregulator

et al. 2022

Self Cite

View full text Add to dashboard Cite

show abstract

“…11 The labeling of DNA with Cu(II) is described in ESI. † 11,14 Electrophoretic mobility shi assays (EMSA) show that CueR binds to the DPA-DNA duplex (Fig. S1 †).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Cu(ii)-based DNA labeling identifies the structural link between transcriptional activation and termination in a metalloregulator

et al. 2022

Self Cite

View full text Add to dashboard Cite

show abstract

“…The experimental most probable distance is consistent with the prediction of C′–C′ and Cu 2+ –Cu 2+ distances from a 2.0 μs Molecular Dynamics (MD) simulation. Note that previous work has shown that a MD trajectory of such a length is sufficient to reasonably estimate the most probable distance but may not be sufficient to capture the full distribution width due to slow mobility of the DPA side chains …”

mentioning

confidence: 99%

“…Note that previous work has shown that a MD trajectory of such a length is sufficient to reasonably estimate the most probable distance but may not be sufficient to capture the full distribution width due to slow mobility of the DPA side chains. 43 In summary, we present a systematic examination of factors that enhance the phase memory times for Cu 2+ labels. In particular, we show that deuteration of the solvent and cryoprotectant can provide improvements that allow for the rapid measurement of distances up to 6.7 nm.…”

mentioning

confidence: 99%

dHis-troying Barriers: Deuteration Provides a Pathway to Increase Sensitivity and Accessible Distances for Cu²⁺ Labels

Casto

Mandato

Saxena

2021

J. Phys. Chem. Lett.

Self Cite

View full text Add to dashboard Cite

Recently, site-directed Cu2+ labeling has emerged as an incisive biophysical tool to directly report on distance constraints that pertain to the structure, conformational transitions, and dynamics of proteins and nucleic acids. However, short phase memory times inherent to the Cu2+ labels limit measurable distances to 4–5 nm. In this work we systematically examine different methods to dampen electron–nuclear and electron–electron coupled interactions to decrease rapid relaxation. We show that using Cu2+ spin concentrations up to ca. 800 μM has an invariant effect on relaxation and that increasing the cryoprotectant concentration reduces contributions of solvent protons to relaxation. On the other hand, the deuteration of protein and solvent dramatically increases the duration of the dipolar modulated signal by over 6-fold to 32 μs. Based on this increase in signal longevity, distances up to 9 nm and beyond can potentially be measured with Cu2+ labels.

show abstract

“…1,76 This concept was further developed and validated through EPR experimentation combined with MD simulations enabled by force field parametrization of the Cu 2+ −DPA motif. 80 These MD simulations revealed that the DPA residue resides within the helix of a DNA, at an angle roughly perpendicular to the DNA backbone. This orientation, shown in Figure 7A, is comparable to natural DNA bases.…”

Section: For Cu 2+ Labeling Of Dnamentioning

confidence: 99%

Going the dHis-tance: Site-Directed Cu²⁺ Labeling of Proteins and Nucleic Acids

et al. 2021

Self Cite

View full text Add to dashboard Cite

Conspectus In this Account, we showcase site-directed Cu2+ labeling in proteins and DNA, which has opened new avenues for the measurement of the structure and dynamics of biomolecules using electron paramagnetic resonance (EPR) spectroscopy. In proteins, the spin label is assembled in situ from natural amino acid residues and a metal complex and requires no post-expression synthetic modification or purification procedures. The labeling scheme exploits a double histidine (dHis) motif, which utilizes endogenous or site-specifically mutated histidine residues to coordinate a Cu2+ complex. Pulsed EPR measurements on such Cu2+-labeled proteins potentially yield distance distributions that are up to 5 times narrower than the common protein spin labelthe approach, thus, overcomes the inherent limitation of the current technology, which relies on a spin label with a highly flexible side chain. This labeling scheme provides a straightforward method that elucidates biophysical information that is costly, complicated, or simply inaccessible by traditional EPR labels. Examples include the direct measurement of protein backbone dynamics at β-sheet sites, which are largely inaccessible through traditional spin labels, and rigid Cu2+–Cu2+ distance measurements that enable higher precision in the analysis of protein conformations, conformational changes, interactions with other biomolecules, and the relative orientations of two labeled protein subunits. Likewise, a Cu2+ label has been developed for use in DNA, which is small, is nucleotide independent, and is positioned within the DNA helix. The placement of the Cu2+ label directly reports on the biologically relevant backbone distance. Additionally, for both of these labeling techniques, we have developed models for interpretation of the EPR distance information, primarily utilizing molecular dynamics (MD) simulations. Initial results using force fields developed for both protein and DNA labels have agreed with experimental results, which has been a major bottleneck for traditional spin labels. Looking ahead, we anticipate new combinations of MD and EPR to further our understanding of protein and DNA conformational changes, as well as working synergistically to investigate protein–DNA interactions.

show abstract

Orientation and dynamics of Cu²⁺based DNA labels from force field parameterized MD elucidates the relationship between EPR distance constraints and DNA backbone distances

Abstract: Pulsed electron paramagnetic resonance (EPR) based distance measurements using the recently developed Cu(II)-DPA label present a promising strategy for measuring DNA backbone distance constraints. Herein we develop force field parameters...

Cited by 11 publications

References 96 publications

Cu(ii)-based DNA labeling identifies the structural link between transcriptional activation and termination in a metalloregulator

Cu(ii)-based DNA labeling identifies the structural link between transcriptional activation and termination in a metalloregulator

dHis-troying Barriers: Deuteration Provides a Pathway to Increase Sensitivity and Accessible Distances for Cu²⁺ Labels

Going the dHis-tance: Site-Directed Cu²⁺ Labeling of Proteins and Nucleic Acids

Contact Info

Product

Resources

About

Orientation and dynamics of Cu2+based DNA labels from force field parameterized MD elucidates the relationship between EPR distance constraints and DNA backbone distances

Abstract: Pulsed electron paramagnetic resonance (EPR) based distance measurements using the recently developed Cu(II)-DPA label present a promising strategy for measuring DNA backbone distance constraints. Herein we develop force field parameters...

Cited by 11 publications

References 96 publications

Cu(ii)-based DNA labeling identifies the structural link between transcriptional activation and termination in a metalloregulator

Cu(ii)-based DNA labeling identifies the structural link between transcriptional activation and termination in a metalloregulator

dHis-troying Barriers: Deuteration Provides a Pathway to Increase Sensitivity and Accessible Distances for Cu2+ Labels

Going the dHis-tance: Site-Directed Cu2+ Labeling of Proteins and Nucleic Acids

Contact Info

Product

Resources

About

Orientation and dynamics of Cu²⁺based DNA labels from force field parameterized MD elucidates the relationship between EPR distance constraints and DNA backbone distances

dHis-troying Barriers: Deuteration Provides a Pathway to Increase Sensitivity and Accessible Distances for Cu²⁺ Labels

Going the dHis-tance: Site-Directed Cu²⁺ Labeling of Proteins and Nucleic Acids