Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules

Schiemann, Olav; Heubach, Caspar A.; Abdullin, Dinar; Ackermann, Katrin; Azarkh, Mykhailo; Bagryanskaya, Elena G.; Drescher, Malte; Endeward, Burkhard; Freed, Jack H.; Galazzo, Laura; Goldfarb, Daniella; Hett, Tobias; Esteban-Hofer, Laura; Ibáñez, Luis Fábregas; Hustedt, Eric J.; Kucher, Svetlana; Kuprov, Ilya; Lovett, Janet E.; Meyer, Andreas; Ruthstein, Sharon; Saxena, Sunil; Stoll, Stefan; Timmel, Christiane R.; Valentin, Marilena Di; Mchaourab, Hassane S.; Prisner, Thomas F.; Bode, Bela E.; Bordignon, Enrica; Bennati, Marina

doi:10.1021/jacs.1c07371

Cited by 174 publications

(212 citation statements)

References 113 publications

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“…One such interaction is the dipolar interaction between two paramagnetic centers, which can report on nanometer distances (usually at a range of 2.0-10.0 nm) between two paramagnetic sites. The most common approaches to acquire nanoscale structural information are double electron electron resonance (DEER), also referred to as pulsed electron double resonance (PELDOR) [51,[71][72][73][74][75], Double Quantum Coherence (DQC) [76][77][78], and relaxation induced dipolar modulation enhancement (RIDME) [79,80]. In all experiments, the Fourier transform (FT) spectra yield characteristic lineshapes and splittings that may be analyzed for distances and distance distributions.…”

Section: Epr Spectroscopymentioning

confidence: 99%

The Advantages of EPR Spectroscopy in Exploring Diamagnetic Metal Ion Binding and Transfer Mechanisms in Biological Systems

2021

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Electron paramagnetic resonance (EPR) spectroscopy has emerged as an ideal biophysical tool to study complex biological processes. EPR spectroscopy can follow minor conformational changes in various proteins as a function of ligand or protein binding or interactions with high resolution and sensitivity. Resolving cellular mechanisms, involving small ligand binding or metal ion transfer, is not trivial and cannot be studied using conventional biophysical tools. In recent years, our group has been using EPR spectroscopy to study the mechanism underlying copper ion transfer in eukaryotic and prokaryotic systems. This mini-review focuses on our achievements following copper metal coordination in the diamagnetic oxidation state, Cu(I), between biomolecules. We discuss the conformational changes induced in proteins upon Cu(I) binding, as well as the conformational changes induced in two proteins involved in Cu(I) transfer. We also consider how EPR spectroscopy, together with other biophysical and computational tools, can identify the Cu(I)-binding sites. This work describes the advantages of EPR spectroscopy for studying biological processes that involve small ligand binding and transfer between intracellular proteins.

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Section: Epr Spectroscopymentioning

confidence: 99%

The Advantages of EPR Spectroscopy in Exploring Diamagnetic Metal Ion Binding and Transfer Mechanisms in Biological Systems

2021

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“…The chosen experimental setup (measuring temperature, length of the dipolar trace, sampling frequency, and length of the pumping pulse) is in line with the recommended conditions to extract the distance distribution function within the range 1.57-2.9 nm [56][57][58].…”

Section: Epr Measurementsmentioning

confidence: 99%

A Peptide-Based Trap for Metal Ions Studied by Electron Paramagnetic Resonance

et al. 2022

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Peptide-based materials provide a versatile platform for sensing and ion sequestration since peptides are endowed with stimuli-responsive properties. The mechanism of molecular sensing is often based on peptide structural changes (or switching), caused by the binding of the target molecule. One scope of sensing applications is the selection of a specific analyte, which may be achieved by adjusting the structure of the peptide binding site. Therefore, exact knowledge of peptide properties and 3D-structure in the ‘switched’ state is desirable for tuning the detection and for further molecular construction. Hence, here we demonstrate the performance of Electron Paramagnetic Resonance (EPR) spectroscopy in the identification of metal ion binding by the antimicrobial peptide trichogin GA IV. Na(I), Ca(II), and Cu(II) ions were probed as analytes to evaluate the impact of coordination number, ionic radii, and charge. Conclusions drawn by EPR are in line with literature data, where other spectroscopic techniques were exploited to study peptide-ion interactions for trichogin GA IV, and the structural switch from an extended helix to a hairpin structure, wrapped around the metal ion upon binding of divalent cations was proposed.

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“…PELDOR data were subjected to the Comparative DEER Analyzer (CDA) within DeerAnalysis2021b [64] for unbiased data processing and analysis according to recent recommendations [65], employing DEERNet [66] neural network processing and Deer-Lab [67] Tikhonov regularisation. Full reports of the CDA analysis are provided in the supplementary information.…”

Section: Pulse Dipolar Epr-sample Preparation and Measurementmentioning

confidence: 99%

A Comparison of Cysteine-Conjugated Nitroxide Spin Labels for Pulse Dipolar EPR Spectroscopy

2021

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The structure-function and materials paradigms drive research on the understanding of structures and structural heterogeneity of molecules and solids from materials science to structural biology. Functional insights into complex architectures are often gained from a suite of complementary physicochemical methods. In the context of biomacromolecular structures, the use of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) has become increasingly popular. The main interest in PDS is providing long-range nanometre distance distributions that allow for identifying macromolecular topologies, validating structural models and conformational transitions as well as docking of quaternary complexes. Most commonly, cysteines are introduced into protein structures by site-directed mutagenesis and modified site-specifically to a spin-labelled side-chain such as a stable nitroxide radical. In this contribution, we investigate labelling by four different commercial labelling agents that react through different sulfur-specific reactions. Further, the distance distributions obtained are between spin-bearing moieties and need to be related to the protein structure via modelling approaches. Here, we compare two different approaches to modelling these distributions for all four side-chains. The results indicate that there are significant differences in the optimum labelling procedure. All four spin-labels show differences in the ease of labelling and purification. Further challenges arise from the different tether lengths and rotamers of spin-labelled side-chains; both influence the modelling and translation into structures. Our comparison indicates that the spin-label with the shortest tether in the spin-labelled side-group, (bis-(2,2,5,5-Tetramethyl-3-imidazoline-1-oxyl-4-yl) disulfide, may be underappreciated and could increase the resolution of structural studies by PDS if labelling conditions are optimised accordingly.

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Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules

Cited by 174 publications

References 113 publications

The Advantages of EPR Spectroscopy in Exploring Diamagnetic Metal Ion Binding and Transfer Mechanisms in Biological Systems

The Advantages of EPR Spectroscopy in Exploring Diamagnetic Metal Ion Binding and Transfer Mechanisms in Biological Systems

A Peptide-Based Trap for Metal Ions Studied by Electron Paramagnetic Resonance

A Comparison of Cysteine-Conjugated Nitroxide Spin Labels for Pulse Dipolar EPR Spectroscopy

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