Pulsed EPR spectroscopy distance measurements of DNA internally labelled with Gd<sup>3+</sup>-DOTA

Wojciechowski, Filip; Groß, A.; Holder, Isabelle T.; Knörr, Laura; Drescher, Malte; Hartig, Jörg S.

doi:10.1039/c5cc04234h

Cited by 31 publications

(15 citation statements)

References 43 publications

(95 reference statements)

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“…these metal centers are generally stable within biological environments.G d III ÀGd III PELDOR distance measurements at Q (34 GHz) and W-band (95 GHz) frequencies have been performed in vitro on synthetic model compounds, [29][30][31] proteins, [32][33][34][35] peptides, [36,37] and DNA, [38] and also on proteins, [39] peptides, [40] and DNA [41] inside cells. Mn II centers,w hich have received much less attention, are very promising in abiological contextb ecause Mn II is endogenous in biological environments and present at the active site of numerous enzymes.…”

Section: Introductionmentioning

confidence: 99%

A Bis‐Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

et al. 2016

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High-spin gadolinium(III) and manganese(II) complexes have emerged as alternatives to standard nitroxide radical spin labels for measuring nanometric distances by using pulsed electron-electron double resonance (PELDOR or DEER) at high fields/frequencies. For certain complexes, particularly those with relatively small zero-field splitting (ZFS) and short distances between the two metal centers, the pseudosecular term of the dipolar coupling Hamiltonian is non-negligible. However, in general, the contribution from this term during conventional data analysis is masked by the flexibility of the molecule of interest and/or the long tethers connecting them to the spin labels. The efficient synthesis of a model system consisting of two [Mn(dota)](2-) (MnDOTA; DOTA(4-) =1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) directly connected to the ends of a central rodlike oligo(phenylene-ethynylene) (OPE) spacer is reported. The rigidity of the OPE is confirmed by Q-band PELDOR measurements on a bis-nitroxide analogue. The Mn(II) -Mn(II) distance distribution profile determined by W-band PELDOR is in reasonable agreement with one simulated by using a simple rotamer analysis. The small degree of flexibility arising from the linking MnDOTA arm appears to outweigh the contribution from the pseudosecular term at this interspin distance. This study illustrates the potential of MnDOTA-based spin labels for measuring fairly short nanometer distances, and also presents an interesting candidate for in-depth studies of pulsed dipolar spectroscopy methods on Mn(II) -Mn(II) systems.

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Section: Introductionmentioning

confidence: 99%

A Bis‐Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

et al. 2016

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“…The obtained time trace (Figure a) exhibits a SNR of 23 h −1/2 (2 h −1/2 μ m −1 ), which is considerably higher than previously reported for in‐cell measurements with nitroxide‐ and trityl‐labeled biomolecules. Even in comparison to W‐band PELDOR/trityl and PELDOR/Gd III measurements, the Q‐band DQC/SLIM combination is at least on par . Remarkably, the distance distribution from the in‐cell measurement differs from the in‐vitro‐derived ones (Figures d and S38,S39).…”

Section: Resultsmentioning

confidence: 90%

SLIM: A Short‐Linked, Highly Redox‐Stable Trityl Label for High‐Sensitivity In‐Cell EPR Distance Measurements

et al. 2020

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The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules,p referably acquired under native conditions.I nt his regard, pulsed dipolar EPR spectroscopy( PDS) in conjunction with site-directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However,t he currently available spin labels have diverse deficiencies for in-cell applications,f or example,l ow radical stability or long bioconjugation linkers.Inthis work, asynthesis strategy is introduced for the derivatization of trityl radicals with am aleimide-functionalized methylene group.T he resulting trityl spin label, called SLIM, yields narrowd istance distributions,e nables highly sensitive distance measurements down to concentrations of 90 nm,a nd shows high stability against reduction. Using this label, the guanine-nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O( YopO) is shown to changei ts conformation within eukaryotic cells.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

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“…Gd(III) and Mn(II) tags have been attached to DNA and/or RNA for EPR distance measurements, but these tags are larger than current nitroxide labels. Cu(II)‐Cu(II) distances in DNA have also been reported using Cu(II) binding porphyrins bound to quadraplexes containing multiple G‐quartets (4 guanines that hydrogen bond to each other in a circle via Hoogsteen base pairing) .…”

Section: Paramagnetic Metal Ion‐based Spin Labels For Proteins and Fomentioning

confidence: 99%

EPR Distance Measurements as a Tool to Characterize Protein‐DNA Interactions

Sameach

Ruthstein

2019

Israel Journal of Chemistry

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Over recent decades, electron paramagnetic resonance (EPR) spectroscopy has become an essential tool for exploring complex biological systems. Herein, we discuss the potential of pulsed EPR spectroscopy to shed light on the mechanisms of protein-DNA interactions. To this end, we first provide an overview of pulsed EPR methodology (and specifically, double electron electron resonance; DEER), with a focus on the various spin-labeling methods used today both for protein labeling and for DNA labeling. Next, after briefly discussing recent applications of DEER in protein-DNA studies, we introduce a detailed case study, an example of how EPR spectroscopy has been used to resolve the transcription mechanism of the CueR copper regulator.

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Pulsed EPR spectroscopy distance measurements of DNA internally labelled with Gd³⁺-DOTA

Cited by 31 publications

References 43 publications

A Bis‐Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

A Bis‐Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

SLIM: A Short‐Linked, Highly Redox‐Stable Trityl Label for High‐Sensitivity In‐Cell EPR Distance Measurements

EPR Distance Measurements as a Tool to Characterize Protein‐DNA Interactions

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Pulsed EPR spectroscopy distance measurements of DNA internally labelled with Gd3+-DOTA

Cited by 31 publications

References 43 publications

A Bis‐Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

A Bis‐Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

SLIM: A Short‐Linked, Highly Redox‐Stable Trityl Label for High‐Sensitivity In‐Cell EPR Distance Measurements

EPR Distance Measurements as a Tool to Characterize Protein‐DNA Interactions

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Pulsed EPR spectroscopy distance measurements of DNA internally labelled with Gd³⁺-DOTA