scholarly journals Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins Using Commercial Labels and Hardware

2020 ◽  
Author(s):  
Katrin Ackermann ◽  
Joshua Wort ◽  
Bela Bode
Keyword(s):  
Protein Concentration ◽  
Geometric Constraints ◽  
Spin Labels ◽  
Paramagnetic Resonance ◽  
Physiological Conditions ◽  
Model Protein ◽  
Concentration Sensitivity ◽  
Health And Disease ◽  
Electron Paramagnetic ◽  
Dipolar Modulation

The study of complex biomolecular assemblies implicated in human health and disease is increasingly performed under native conditions. Pulse Dipolar Electron paramagnetic resonance (PDEPR) spectroscopy is a powerful tool that provides highly precise geometric constraints in frozen solution, however the drive towards <i>in cellulo</i> EPR is limited by the currently achievable concentration sensitivity in the low μM regime. Achieving PDEPR at physiologically relevant sub-μM concentrations is currently very challenging. Recently, relaxation induced dipolar modulation enhancement (RIDME) measurements using a combination of nitroxide and double-histidine Cu<sup>II</sup> based spin labels allowed measuring 500 nM concentration of a model protein. Herein, we demonstrate Cu<sup>II</sup>-Cu<sup>II </sup>RIDME and nitroxide-nitroxide PELDOR measurements down to 500 and 100 nM protein concentration, respectively. This is possible using commercial instrumentation and spin labels. These results herald a transition towards routine sub-μM PDEPR measurements at short to intermediate distances (~1.5-3.5 nm), without the necessity of specialized instrumentation or spin-labelling protocols, particularly relevant for applications in near physiological conditions.

scholarly journals Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins Using Commercial Labels and Hardware

2020 ◽  
Author(s):  
Katrin Ackermann ◽  
Joshua Wort ◽  
Bela Bode
Keyword(s):  
Protein Concentration ◽  
Geometric Constraints ◽  
Spin Labels ◽  
Paramagnetic Resonance ◽  
Physiological Conditions ◽  
Model Protein ◽  
Concentration Sensitivity ◽  
Health And Disease ◽  
Electron Paramagnetic ◽  
Dipolar Modulation

The study of complex biomolecular assemblies implicated in human health and disease is increasingly performed under native conditions. Pulse Dipolar Electron paramagnetic resonance (PDEPR) spectroscopy is a powerful tool that provides highly precise geometric constraints in frozen solution, however the drive towards <i>in cellulo</i> EPR is limited by the currently achievable concentration sensitivity in the low μM regime. Achieving PDEPR at physiologically relevant sub-μM concentrations is currently very challenging. Recently, relaxation induced dipolar modulation enhancement (RIDME) measurements using a combination of nitroxide and double-histidine Cu<sup>II</sup> based spin labels allowed measuring 500 nM concentration of a model protein. Herein, we demonstrate Cu<sup>II</sup>-Cu<sup>II </sup>RIDME and nitroxide-nitroxide PELDOR measurements down to 500 and 100 nM protein concentration, respectively. This is possible using commercial instrumentation and spin labels. These results herald a transition towards routine sub-μM PDEPR measurements at short to intermediate distances (~1.5-3.5 nm), without the necessity of specialized instrumentation or spin-labelling protocols, particularly relevant for applications in near physiological conditions.

scholarly journals Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins; the Copper(II)-Copper(II) and Nitroxide-Nitroxide Cases

2021 ◽  
Author(s):  
Katrin Ackermann ◽  
Joshua Wort ◽  
Bela Bode
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Geometric Constraints ◽  
Spin Labels ◽  
Paramagnetic Resonance ◽  
Frozen Solution ◽  
Model Protein ◽  
Concentration Sensitivity ◽  
Order Of Magnitude ◽  
Health And Disease ◽  
Electron Paramagnetic

The study of ever more complex biomolecular assemblies implicated in human health and disease is facilitated by a suite of complementary biophysical methods. Pulse Dipolar Electron Paramagnetic Resonance (PDEPR) spectroscopy is a powerful tool that provides highly precise geometric constraints in frozen solution, however the drive towards PDEPR at physiologically relevant sub-μM concentrations is limited by the currently achievable concentration sensitivity. Recently, PDEPR using a combination of nitroxide and Cu<sup>II</sup> based spin labels allowed measuring 500 nM concentration of a model protein. Using commercial instrumentation and spin labels we demonstrate Cu<sup>II</sup>-Cu<sup>II</sup> and nitroxide-nitroxide PDEPR measurements at protein concentrations more than an order of magnitude below previous examples reaching 500 and 100 nM, respectively. These results demonstrate the general feasibility of sub-μM PDEPR measurements at short to intermediate distances (~1.5 - 3.5 nm), and are of particular relevance for applications where the achievable concentration is limiting.

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

2021 ◽  
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 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 2′-Alkynyl spin-labelling is a minimally perturbing tool for DNA structural analysis

2020 ◽  
Vol 48 (6) ◽  
pp. 2830-2840 ◽  
Author(s):  
Jack S Hardwick ◽  
Marius M Haugland ◽  
Afaf H El-Sagheer ◽  
Denis Ptchelkine ◽  
Frank R Beierlein ◽  
...  
Keyword(s):  
Epr Spectroscopy ◽  
Spin Labels ◽  
Resonance Spectroscopy ◽  
Conformational Ensemble ◽  
Dna Duplexes ◽  
Paramagnetic Resonance ◽  
Dynamics Simulations ◽  
Electron Paramagnetic ◽  
Spin Labelling

Abstract The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2–10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2′ position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2′-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation.

scholarly journals Triarylmethyl Radicals: An EPR Study of 13C Hyperfine Coupling Constants

2017 ◽  
Vol 231 (4) ◽  
Author(s):  
Andrey A. Kuzhelev ◽  
Victor M. Tormyshev ◽  
Olga Yu. Rogozhnikova ◽  
Dmitry V. Trukhin ◽  
Tatiana I. Troitskaya ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Epr Spectroscopy ◽  
Hyperfine Coupling ◽  
Coupling Constants ◽  
Spin Labels ◽  
Spin Probes ◽  
Paramagnetic Resonance ◽  
Hyperfine Coupling Constants ◽  
Electron Paramagnetic ◽  
Triarylmethyl Radicals

AbstractTriarylmethyl (TAM) radicals are widely used in electron paramagnetic resonance (EPR) spectroscopy as spin labels and in EPR imaging as spin probes for

Sign in / Sign up

Export Citation Format

Share Document