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

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.

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Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements

Molecules ◽

10.3390/molecules25184143 ◽

2020 ◽

Vol 25 (18) ◽

pp. 4143

Author(s):

Jeannette M. Laugwitz ◽

Haleh H. Haeri ◽

Anette Kaiser ◽

Ulrike Krug ◽

Dariush Hinderberger ◽

...

Keyword(s):

Continuous Wave ◽

Conformational Dynamics ◽

Spin Labeling ◽

Conformational States ◽

Distance Measurements ◽

Paramagnetic Resonance ◽

Pulsed Epr ◽

Y2 Receptor ◽

Epr Measurements ◽

Double Electron Electron Resonance

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor.

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

Biochemical Journal ◽

10.1042/bj20101871 ◽

2011 ◽

Vol 434 (3) ◽

pp. 353-363 ◽

Cited By ~ 64

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.

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Long-Range Distance Measurements on Nucleic Acids in Cells by Pulsed EPR Spectroscopy

Angewandte Chemie International Edition ◽

10.1002/anie.201100886 ◽

2011 ◽

Vol 50 (22) ◽

pp. 5070-5074 ◽

Cited By ~ 129

Author(s):

Ivan Krstić ◽

Robert Hänsel ◽

Olga Romainczyk ◽

Joachim W. Engels ◽

Volker Dötsch ◽

...

Keyword(s):

Nucleic Acids ◽

Long Range ◽

Epr Spectroscopy ◽

Distance Measurements ◽

Pulsed Epr ◽

In Cells

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High-resolution models of actin-bound myosin from EPR of a bifunctional spin label

10.1101/458257 ◽

2018 ◽

Cited By ~ 2

Author(s):

Benjamin P. Binder ◽

Andrew R. Thompson ◽

David D. Thomas

Keyword(s):

High Resolution ◽

Spin Label ◽

Continuous Wave ◽

Catalytic Domain ◽

Structural Models ◽

Spin Labels ◽

Paramagnetic Resonance ◽

Distance Constraints ◽

Helix Orientation ◽

Double Electron Electron Resonance

AbstractWe have employed two complementary high-resolution electron paramagnetic resonance (EPR) techniques with a bifunctional spin label (BSL) to test and refine protein structural models based on crystal structures and cryo-EM. We demonstrate this approach by investigating the effects of nucleotide binding on the structure of myosin’s catalytic domain (CD), while myosin is in complex with actin. Unlike conventional spin labels attached to single Cys, BSL reacts with a pair of Cys; in this study, we thoroughly characterize BSL’s rigid, highly stereoselective attachment to protein α-helices, which permits accurate measurements of orientation and distance. Distance constraints were obtained from double electron-electron resonance (DEER) on myosin constructs labeled with BSL specifically at two sites. Constraints for orientation of individual helices were obtained previously from continuous-wave EPR (CW-EPR) of myosin labeled at specific sites with BSL in oriented muscle fibers. We have shown previously that CW-EPR of BSL quantifies helix orientation within actin-bound myosin; here we show that the addition of high-resolution distance constraints by DEER alleviates remaining spatial ambiguity, allowing for direct testing and refinement of atomic structural models. This approach is applicable to any orientable complex (e.g., membranes or filaments) in which site-specific di- Cys mutation is feasible.

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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

10.1101/248054 ◽

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].

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Combining Site-Directed Spin Labeling in Vivo and In-Cell EPR Distance Determination

10.26434/chemrxiv.9746222 ◽

2019 ◽

Author(s):

Pia Widder ◽

Julian Schuck ◽

Daniel Summerer ◽

Malte Drescher

Keyword(s):

Spin Labeling ◽

Distance Measurements ◽

Paramagnetic Resonance ◽

Distance Determination ◽

Bioorthogonal Labeling ◽

In Vitro Measurements ◽

Site Directed Spin Labeling ◽

Double Electron Electron Resonance

Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E.coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.

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Combining Site-Directed Spin Labeling in Vivo and In-Cell EPR Distance Determination

10.26434/chemrxiv.9746222.v1 ◽

2019 ◽

Author(s):

Pia Widder ◽

Julian Schuck ◽

Daniel Summerer ◽

Malte Drescher

Keyword(s):

Spin Labeling ◽

Distance Measurements ◽

Paramagnetic Resonance ◽

Distance Determination ◽

Bioorthogonal Labeling ◽

In Vitro Measurements ◽

Site Directed Spin Labeling ◽

Double Electron Electron Resonance

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

10.26434/chemrxiv.13271501.v1 ◽

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> 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 CuII 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 CuII-nitrilotriacetic acid were previously investigated via 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 CuII-CuII RIDME to simultaneously estimate a pair of non-identical independent KD values in such a tetra-histidine model protein is addressed. Furthermore, we develop a general speciation model to optimise CuII labelling efficiency, in dependence of pairs of identical or disparate KD values and total CuII label concentration. We find the dissociation constant estimates are in excellent agreement with previously determined values, and empirical modulation depths support the proposed model. </div>

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

Nucleic Acids Research ◽

10.1093/nar/gkz620 ◽

2019 ◽

Vol 47 (15) ◽

pp. 7767-7780 ◽

Cited By ~ 6

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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The contribution of modern EPR to structural biology

Emerging Topics in Life Sciences ◽

10.1042/etls20170143 ◽

2018 ◽

Vol 2 (1) ◽

pp. 9-18 ◽

Cited By ~ 33

Author(s):

Gunnar Jeschke

Keyword(s):

System Size ◽

Native Conformation ◽

Paramagnetic Resonance ◽

Building Models ◽

Distance Distributions ◽

Double Electron ◽

The Mean ◽

Structural Restraints ◽

Double Electron Electron Resonance ◽

Electron Paramagnetic

Electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labelling is applicable to biomolecules and their complexes irrespective of system size and in a broad range of environments. Neither short-range nor long-range order is required to obtain structural restraints on accessibility of sites to water or oxygen, on secondary structure, and on distances between sites. Many of the experiments characterize a static ensemble obtained by shock-freezing. Compared with characterizing the dynamic ensemble at ambient temperature, analysis is simplified and information loss due to overlapping timescales of measurement and system dynamics is avoided. The necessity for labelling leads to sparse restraint sets that require integration with data from other methodologies for building models. The double electron–electron resonance experiment provides distance distributions in the nanometre range that carry information not only on the mean conformation but also on the width of the native ensemble. The distribution widths are often inconsistent with Anfinsen's concept that a sequence encodes a single native conformation defined at atomic resolution under physiological conditions.

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