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

10.26434/chemrxiv.13271501.v2 ◽

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

10.26434/chemrxiv.13271501 ◽

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

10.26434/chemrxiv.13370924 ◽

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 in cellulo 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 CuII based spin labels allowed measuring 500 nM concentration of a model protein. Herein, we demonstrate CuII-CuII 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.

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

10.26434/chemrxiv.13370924.v2 ◽

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 CuII based spin labels allowed measuring 500 nM concentration of a model protein. Using commercial instrumentation and spin labels we demonstrate CuII-CuII 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.

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

10.26434/chemrxiv.13370924.v1 ◽

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 in cellulo 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 CuII based spin labels allowed measuring 500 nM concentration of a model protein. Herein, we demonstrate CuII-CuII 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.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa133 ◽

2020 ◽

Vol 48 (9) ◽

pp. e49-e49 ◽

Cited By ~ 6

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.

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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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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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Two closed ATP- and ADP-dependent conformations in yeast Hsp90 chaperone detected by Mn(II) EPR spectroscopic techniques

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1916030116 ◽

2019 ◽

Vol 117 (1) ◽

pp. 395-404 ◽

Cited By ~ 7

Author(s):

Angeliki Giannoulis ◽

Akiva Feintuch ◽

Yoav Barak ◽

Hisham Mazal ◽

Shira Albeck ◽

...

Keyword(s):

Conformational Changes ◽

Close Contact ◽

Energy State ◽

Bound State ◽

High Energy ◽

Distance Distribution ◽

Spin Labels ◽

Atp Binding ◽

Spectroscopic Techniques ◽

Distance Measurements

Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of a large variety of clients. It is a homo-dimer, which functions via hydrolysis of ATP-coupled to conformational changes. Hsp90’s conformational cycle in the absence of cochaperones is currently postulated as apo-Hsp90 being an ensemble of “open”/“closed” conformations. Upon ATP binding, Hsp90 adopts an active ATP-bound closed conformation where the N-terminal domains, which comprise the ATP binding site, are in close contact. However, there is no consensus regarding the conformation of the ADP-bound Hsp90, which is considered important for client release. In this work, we tracked the conformational states of yeast Hsp90 at various stages of ATP hydrolysis in frozen solutions employing electron paramagnetic resonance (EPR) techniques, particularly double electron–electron resonance (DEER) distance measurements. Using rigid Gd(III) spin labels, we found the C domains to be dimerized with same distance distribution at all hydrolysis states. Then, we substituted the ATPase Mg(II) cofactor with paramagnetic Mn(II) and followed the hydrolysis state using hyperfine spectroscopy and measured the inter–N-domain distance distributions via Mn(II)–Mn(II) DEER. The point character of the Mn(II) spin label allowed us resolve 2 different closed states: The ATP-bound (prehydrolysis) characterized by a distance distribution having a maximum of 4.3 nm, which broadened and shortened, shifting the mean to 3.8 nm at the ADP-bound state (posthydrolysis). This provides experimental evidence to a second closed conformational state of Hsp90 in solution, referred to as “compact.” Finally, the so-called high-energy state, trapped by addition of vanadate, was found structurally similar to the posthydrolysis state.

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