Pulsed electron–electron double resonance: beyond nanometre distance measurements on biomacromolecules

2011 ◽  
Vol 434 (3) ◽  
pp. 353-363 ◽  
Author(s):  
Gunnar W. Reginsson ◽  
Olav Schiemann
Keyword(s):  
Protein Complexes ◽  
Double Resonance ◽  
Resonance Method ◽  
Spin Labels ◽  
Coupling Mechanism ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Distance Distributions ◽  
Nitroxide Spin Labels ◽  
Amino Acid Radicals

PELDOR (or DEER; pulsed electron–electron double resonance) is an EPR (electron paramagnetic resonance) method that measures via the dipolar electron–electron coupling distances in the nanometre range, currently 1.5–8 nm, with high precision and reliability. Depending on the quality of the data, the error can be as small as 0.1 nm. Beyond mere mean distances, PELDOR yields distance distributions, which provide access to conformational distributions and dynamics. It can also be used to count the number of monomers in a complex and allows determination of the orientations of spin centres with respect to each other. If, in addition to the dipolar through-space coupling, a through-bond exchange coupling mechanism contributes to the overall coupling both mechanisms can be separated and quantified. Over the last 10 years PELDOR has emerged as a powerful new biophysical method without size restriction to the biomolecule to be studied, and has been applied to a large variety of nucleic acids as well as proteins and protein complexes in solution or within membranes. Small nitroxide spin labels, paramagnetic metal ions, amino acid radicals or intrinsic clusters and cofactor radicals have been used as spin centres.

Long-range distance determinations in biomacromolecules by EPR spectroscopy

2007 ◽  
Vol 40 (1) ◽  
pp. 1-53 ◽  
Author(s):  
Olav Schiemann ◽  
Thomas F. Prisner
Keyword(s):  
Long Range ◽  
Epr Spectroscopy ◽  
Structural Changes ◽  
Continuous Wave ◽  
Double Resonance ◽  
Theoretical Background ◽  
Spin Labels ◽  
Paramagnetic Resonance ◽  
Pulsed Epr ◽  
Amino Acid Radicals

AbstractElectron paramagnetic resonance (EPR) spectroscopy provides a variety of tools to study structures and structural changes of large biomolecules or complexes thereof. In order to unravel secondary structure elements, domain arrangements or complex formation, continuous wave and pulsed EPR methods capable of measuring the magnetic dipole coupling between two unpaired electrons can be used to obtain long-range distance constraints on the nanometer scale. Such methods yield reliably and precisely distances of up to 80 Å, can be applied to biomolecules in aqueous buffer solutions or membranes, and are not size limited. They can be applied either at cryogenic or physiological temperatures and down to amounts of a few nanomoles. Spin centers may be metal ions, metal clusters, cofactor radicals, amino acid radicals, or spin labels. In this review, we discuss the advantages and limitations of the different EPR spectroscopic methods, briefly describe their theoretical background, and summarize important biological applications. The main focus of this article will be on pulsed EPR methods like pulsed electron–electron double resonance (PELDOR) and their applications to spin-labeled biosystems.

scholarly journals Studies of transmembrane peptides by pulse dipolar spectroscopy with semi-rigid TOPP spin labels

2021 ◽  
Author(s):  
Igor Tkach ◽  
Ulf Diederichsen ◽  
Marina Bennati
Keyword(s):  
Molecular Level ◽  
Dipolar Interaction ◽  
Spin Labels ◽  
Paramagnetic Centers ◽  
Structural Adaptation ◽  
Paramagnetic Resonance ◽  
Transmembrane Peptides ◽  
Distance Distributions ◽  
Lipid Environment ◽  
Electron Paramagnetic

AbstractElectron paramagnetic resonance (EPR)-based pulsed dipolar spectroscopy measures the dipolar interaction between paramagnetic centers that are separated by distances in the range of about 1.5–10 nm. Its application to transmembrane (TM) peptides in combination with modern spin labelling techniques provides a valuable tool to study peptide-to-lipid interactions at a molecular level, which permits access to key parameters characterizing the structural adaptation of model peptides incorporated in natural membranes. In this mini-review, we summarize our approach for distance and orientation measurements in lipid environment using novel semi-rigid TOPP [4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-L-phenylglycine] labels specifically designed for incorporation in TM peptides. TOPP labels can report single peak distance distributions with sub-angstrom resolution, thus offering new capabilities for a variety of TM peptide investigations, such as monitoring of various helix conformations or measuring of tilt angles in membranes. Graphical Abstract

scholarly journals Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution

2020 ◽  
Vol 48 (9) ◽  
pp. e49-e49 ◽  
Author(s):  
Shreya Ghosh ◽  
Matthew J Lawless ◽  
Hanna J Brubaker ◽  
Kevin Singewald ◽  
Michael R Kurpiewski ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Conformational Changes ◽  
Continuous Wave ◽  
Md Simulations ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Pulsed Epr ◽  
Distance Distributions ◽  
Dna Backbone ◽  
Double Electron Electron Resonance

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.

scholarly journals Darunavir-Resistant HIV-1 Protease Constructs Uphold a Conformational Selection Hypothesis for Drug Resistance

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1275
Author(s):  
Zhanglong Liu ◽  
Trang T. Tran ◽  
Linh Pham ◽  
Lingna Hu ◽  
Kyle Bentz ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Highly Active Antiretroviral Therapy ◽  
Double Resonance ◽  
Resonance Spectroscopy ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Highly Active ◽  
Open Conformation ◽  
Potent Protease Inhibitor ◽  
Hiv 1

Multidrug resistance continues to be a barrier to the effectiveness of highly active antiretroviral therapy in the treatment of human immunodeficiency virus 1 (HIV-1) infection. Darunavir (DRV) is a highly potent protease inhibitor (PI) that is oftentimes effective when drug resistance has emerged against first-generation inhibitors. Resistance to darunavir does evolve and requires 10–20 amino acid substitutions. The conformational landscapes of six highly characterized HIV-1 protease (PR) constructs that harbor up to 19 DRV-associated mutations were characterized by distance measurements with pulsed electron double resonance (PELDOR) paramagnetic resonance spectroscopy, namely double electron–electron resonance (DEER). The results show that the accumulated substitutions alter the conformational landscape compared to PI-naïve protease where the semi-open conformation is destabilized as the dominant population with open-like states becoming prevalent in many cases. A linear correlation is found between values of the DRV inhibition parameter Ki and the open-like to closed-state population ratio determined from DEER. The nearly 50% decrease in occupancy of the semi-open conformation is associated with reduced enzymatic activity, characterized previously in the literature.

scholarly journals Pulsed ELDOR Measurement of the Distance Between a Spin-Label and Copper (II) Centre in the Copper Loaded R48C Mutant of N. gonorrhoeae Ferric Binding Protein

2018 ◽  
Author(s):  
Matt Bawn ◽  
Justin Bradley ◽  
Fraser MacMillan
Keyword(s):  
Spin Label ◽  
Binding Protein ◽  
Double Resonance ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Distance Determination ◽  
Pulsed Epr ◽  
Ferric Binding Protein ◽  
Relaxation Experiments ◽  
Electron Paramagnetic

AbstractDistance determination in proteins and biomolecules using pulsed EPR (electron paramagnetic resonance) techniques is becoming an increasingly popular and accessible technique. PELDOR (pulsed electron-electron double resonance), is a technique designed for distance determination over a nanoscopic scale. Here, ferric binding protein (Fbp) is used to demonstrate the practicability of this technique to Cu (II) Metalloproteins. PELDOR is usually applied to bi-radicals or endogenous radicals, and distance determination using pulsed EPR of metal containing centres in biomolecules has been restricted to relaxation experiments. PELDOR distance measurements between a Cu (II) ion and a nitroxide have previously only been reported for model compounds [1, 2].Fbp as the name suggests usually, contains a Fe (III) ion centre. For the purposes of this investigation the Fe (III) ion was removed and replaced by a Cu (II) ion, after a nitroxide spin-label was added to the Fbp using of site directed spin-labelling (SDSL). PELDOR was then applied to measure the distance between the two centres.Simulation methods were then employed to fully investigate these data and allow a quantitative interpretation of the copper nitroxide PELDOR data. The observed PELDOR time traces were analysed using DEER analysis[3].

scholarly journals A General Model to Optimise Copper(II) Labelling Efficiency of Double-Histidine Motifs for Pulse Dipolar EPR Applications

2020 ◽  
Author(s):  
Joshua L. Wort ◽  
Katrin Ackermann ◽  
David G. Norman ◽  
Bela E. Bode
Keyword(s):  
Conformational Changes ◽  
Dissociation Constants ◽  
Modulation Depth ◽  
Nitrilotriacetic Acid ◽  
Geometric Constraints ◽  
Spin Labels ◽  
Labelling Efficiency ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Model Protein

<div> <p>Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to studies of biomolecules underpinning health and disease by providing highly accurate and precise geometric constraints. Combining double-histidine (dH) motifs with Cu<sup>II</sup> spin labels shows promise for further increasing the precision of distance measurements, and for investigating subtle conformational changes. However, non-covalent coordination-based spin labelling is vulnerable to low binding affinity. Dissociation constants of dH motifs for Cu<sup>II</sup>-nitrilotriacetic acid were previously investigated <i>via </i>relaxation induced dipolar modulation enhancement (RIDME), and demonstrated the feasibility of exploiting the double histidine motif for EPR applications at sub-μM protein concentrations. Herein, the feasibility of using modulation depth quantitation in Cu<sup>II</sup>-Cu<sup>II </sup>RIDME to simultaneously estimate a pair of non-identical independent <i>K<sub>D</sub></i> values in such a tetra-histidine model protein is addressed. Furthermore, we develop a general speciation model to optimise Cu<sup>II </sup>labelling efficiency, in dependence of pairs of identical or disparate <i>K<sub>D</sub></i> values and total Cu<sup>II</sup> label concentration. We find the dissociation constant estimates are in excellent agreement with previously determined values, and empirical modulation depths support the proposed model. </p> </div> <br>

scholarly journals DNA complexes with human apurinic/apyrimidinic endonuclease 1: structural insights revealed by pulsed dipolar EPR with orthogonal spin labeling

2019 ◽  
Vol 47 (15) ◽  
pp. 7767-7780 ◽  
Author(s):  
Olesya A Krumkacheva ◽  
Georgiy Yu Shevelev ◽  
Alexander A Lomzov ◽  
Nadezhda S Dyrkheeva ◽  
Andrey A Kuzhelev ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Md Simulations ◽  
Dna Lesions ◽  
Spin Labels ◽  
Paramagnetic Resonance ◽  
Applied Pulse ◽  
Oligonucleotide Duplex ◽  
Nitroxide Spin Labels ◽  
Ap Sites ◽  
Electron Paramagnetic

Abstract A DNA molecule is under continuous influence of endogenous and exogenous damaging factors, which produce a variety of DNA lesions. Apurinic/apyrimidinic sites (abasic or AP sites) are among the most common DNA lesions. In this work, we applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy in combination with molecular dynamics (MD) simulations to investigate in-depth conformational changes in DNA containing an AP site and in a complex of this DNA with AP endonuclease 1 (APE1). For this purpose, triarylmethyl (TAM)-based spin labels were attached to the 5′ ends of an oligonucleotide duplex, and nitroxide spin labels were introduced into APE1. In this way, we created a system that enabled monitoring the conformational changes of the main APE1 substrate by EPR. In addition, we were able to trace substrate-to-product transformation in this system. The use of different (orthogonal) spin labels in the enzyme and in the DNA substrate has a crucial advantage allowing for detailed investigation of local damage and conformational changes in AP-DNA alone and in its complex with APE1.

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