Robustness and sensitivity of Gd(III)-Gd(III) double electron electron resonance (DEER) measurements: comparative study of high-frequency EPR spectrometer designs and spin label variants

Elena Mihaela Mocanu; Yasmin Ben-Ishay; Lydia Topping; Stuart Ronan Fisher; Robert Iain Hunter; Xun-Cheng Su; Stephen Butler; Graham Murray Smith; Daniella Goldfarb; Janet Eleanor Lovett

doi:10.1007/s00723-024-01741-0

Robustness and sensitivity of Gd(III)-Gd(III) double electron electron resonance (DEER) measurements: comparative study of high-frequency EPR spectrometer designs and spin label variants

Elena Mihaela Mocanu, Yasmin Ben-Ishay, Lydia Topping, Stuart Ronan Fisher, Robert Iain Hunter, Xun-Cheng Su, Stephen Butler, Graham Murray Smith, Daniella Goldfarb, Janet Eleanor Lovett^*

^*Corresponding author for this work

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Abstract

In this paper we explore the robustness and sensitivity of Gd(III)-Gd(III) double electron-electron resonance (DEER) distance measurements in proteins for different spectrometer designs and a three spin labels. To do this a protein was labelled at the same two positions with Gd(III) spin labels and measurements were performed on two home-built high-frequency (W-band, ~95 GHz) EPR spectrometers with different design approaches, and a commercial Q-band spectrometer. The first W-band measurement approach uses a conventional, narrow band single mode cavity, while the second approach uses a broad band non-resonant induction mode sample holder. Both systems incorporate advanced arbitrary waveform generators (AWGs) that give flexibility over excitation bandwidth. We use three DOTA-like Gd(III) spin labels, Gd.C12, Gd.DO3A and the new Gd.L¹, conjugated to the model protein calmodulin. We compare measurements taken by including or excluding the excitation of the Gd(III) central transition. The advantages and disadvantages of the EPR spectrometers for the measurement of Gd(III)-Gd(III) DEER are discussed in terms of the robustness of the resulting distance distribution width, absolute and concentration sensitivity, sample handling, ease of use, and flexibility of measurement.

Original language	English
Pages (from-to)	591-611
Number of pages	21
Journal	Applied Magnetic Resonance
Volume	56
Issue number	5
Early online date	3 Jan 2025
DOIs	https://doi.org/10.1007/s00723-024-01741-0
Publication status	Published - 1 May 2025

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Mocanu, E. M., Ben-Ishay, Y., Topping, L., Fisher, S. R., Hunter, R. I., Su, X.-C., Butler, S., Smith, G. M., Goldfarb, D., & Lovett, J. E. (2025). Robustness and sensitivity of Gd(III)-Gd(III) double electron electron resonance (DEER) measurements: comparative study of high-frequency EPR spectrometer designs and spin label variants. Applied Magnetic Resonance, 56(5), 591-611. https://doi.org/10.1007/s00723-024-01741-0

@article{e76fe3ea695e4d9995e1e354c28396c9,

title = "Robustness and sensitivity of Gd(III)-Gd(III) double electron electron resonance (DEER) measurements: comparative study of high-frequency EPR spectrometer designs and spin label variants",

abstract = "In this paper we explore the robustness and sensitivity of Gd(III)-Gd(III) double electron-electron resonance (DEER) distance measurements in proteins for different spectrometer designs and a three spin labels. To do this a protein was labelled at the same two positions with Gd(III) spin labels and measurements were performed on two home-built high-frequency (W-band, \textasciitilde{}95 GHz) EPR spectrometers with different design approaches, and a commercial Q-band spectrometer. The first W-band measurement approach uses a conventional, narrow band single mode cavity, while the second approach uses a broad band non-resonant induction mode sample holder. Both systems incorporate advanced arbitrary waveform generators (AWGs) that give flexibility over excitation bandwidth. We use three DOTA-like Gd(III) spin labels, Gd.C12, Gd.DO3A and the new Gd.L1, conjugated to the model protein calmodulin. We compare measurements taken by including or excluding the excitation of the Gd(III) central transition. The advantages and disadvantages of the EPR spectrometers for the measurement of Gd(III)-Gd(III) DEER are discussed in terms of the robustness of the resulting distance distribution width, absolute and concentration sensitivity, sample handling, ease of use, and flexibility of measurement. ",

author = "Mocanu, \{Elena Mihaela\} and Yasmin Ben-Ishay and Lydia Topping and Fisher, \{Stuart Ronan\} and Hunter, \{Robert Iain\} and Xun-Cheng Su and Stephen Butler and Smith, \{Graham Murray\} and Daniella Goldfarb and Lovett, \{Janet Eleanor\}",

note = "Funding: This work was supported financially by the Bluston Making Connections (UK-WIS) scheme, The Royal Society (International Exchanges) and the Carnegie Trust. The Q-band spectrometer high power Q-band with cryogen-free upgrade was provided by the Wellcome Trust (099149/Z/12/Z) and the BBSRC (BB/T017740/1) respectively. The authors thank EPSRC (EP/N509759/1) and the School of Physics and Astronomy, St Andrews for studentships for SRF and EMM respectively.",

year = "2025",

month = may,

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doi = "10.1007/s00723-024-01741-0",

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Mocanu, EM, Ben-Ishay, Y, Topping, L, Fisher, SR, Hunter, RI, Su, X-C, Butler, S, Smith, GM, Goldfarb, D & Lovett, JE 2025, 'Robustness and sensitivity of Gd(III)-Gd(III) double electron electron resonance (DEER) measurements: comparative study of high-frequency EPR spectrometer designs and spin label variants', Applied Magnetic Resonance, vol. 56, no. 5, pp. 591-611. https://doi.org/10.1007/s00723-024-01741-0

Robustness and sensitivity of Gd(III)-Gd(III) double electron electron resonance (DEER) measurements: comparative study of high-frequency EPR spectrometer designs and spin label variants. / Mocanu, Elena Mihaela; Ben-Ishay, Yasmin; Topping, Lydia et al.
In: Applied Magnetic Resonance, Vol. 56, No. 5, 01.05.2025, p. 591-611.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Robustness and sensitivity of Gd(III)-Gd(III) double electron electron resonance (DEER) measurements

T2 - comparative study of high-frequency EPR spectrometer designs and spin label variants

AU - Mocanu, Elena Mihaela

AU - Ben-Ishay, Yasmin

AU - Topping, Lydia

AU - Fisher, Stuart Ronan

AU - Hunter, Robert Iain

AU - Su, Xun-Cheng

AU - Butler, Stephen

AU - Smith, Graham Murray

AU - Goldfarb, Daniella

AU - Lovett, Janet Eleanor

N1 - Funding: This work was supported financially by the Bluston Making Connections (UK-WIS) scheme, The Royal Society (International Exchanges) and the Carnegie Trust. The Q-band spectrometer high power Q-band with cryogen-free upgrade was provided by the Wellcome Trust (099149/Z/12/Z) and the BBSRC (BB/T017740/1) respectively. The authors thank EPSRC (EP/N509759/1) and the School of Physics and Astronomy, St Andrews for studentships for SRF and EMM respectively.

PY - 2025/5/1

Y1 - 2025/5/1

N2 - In this paper we explore the robustness and sensitivity of Gd(III)-Gd(III) double electron-electron resonance (DEER) distance measurements in proteins for different spectrometer designs and a three spin labels. To do this a protein was labelled at the same two positions with Gd(III) spin labels and measurements were performed on two home-built high-frequency (W-band, ~95 GHz) EPR spectrometers with different design approaches, and a commercial Q-band spectrometer. The first W-band measurement approach uses a conventional, narrow band single mode cavity, while the second approach uses a broad band non-resonant induction mode sample holder. Both systems incorporate advanced arbitrary waveform generators (AWGs) that give flexibility over excitation bandwidth. We use three DOTA-like Gd(III) spin labels, Gd.C12, Gd.DO3A and the new Gd.L1, conjugated to the model protein calmodulin. We compare measurements taken by including or excluding the excitation of the Gd(III) central transition. The advantages and disadvantages of the EPR spectrometers for the measurement of Gd(III)-Gd(III) DEER are discussed in terms of the robustness of the resulting distance distribution width, absolute and concentration sensitivity, sample handling, ease of use, and flexibility of measurement.

AB - In this paper we explore the robustness and sensitivity of Gd(III)-Gd(III) double electron-electron resonance (DEER) distance measurements in proteins for different spectrometer designs and a three spin labels. To do this a protein was labelled at the same two positions with Gd(III) spin labels and measurements were performed on two home-built high-frequency (W-band, ~95 GHz) EPR spectrometers with different design approaches, and a commercial Q-band spectrometer. The first W-band measurement approach uses a conventional, narrow band single mode cavity, while the second approach uses a broad band non-resonant induction mode sample holder. Both systems incorporate advanced arbitrary waveform generators (AWGs) that give flexibility over excitation bandwidth. We use three DOTA-like Gd(III) spin labels, Gd.C12, Gd.DO3A and the new Gd.L1, conjugated to the model protein calmodulin. We compare measurements taken by including or excluding the excitation of the Gd(III) central transition. The advantages and disadvantages of the EPR spectrometers for the measurement of Gd(III)-Gd(III) DEER are discussed in terms of the robustness of the resulting distance distribution width, absolute and concentration sensitivity, sample handling, ease of use, and flexibility of measurement.

UR - https://www.scopus.com/pages/publications/85213950618

U2 - 10.1007/s00723-024-01741-0

DO - 10.1007/s00723-024-01741-0

M3 - Article

SN - 0937-9347

VL - 56

SP - 591

EP - 611

JO - Applied Magnetic Resonance

JF - Applied Magnetic Resonance

IS - 5

ER -

Robustness and sensitivity of Gd(III)-Gd(III) double electron electron resonance (DEER) measurements: comparative study of high-frequency EPR spectrometer designs and spin label variants

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Enabling Shaped Pulse Capability: Enabling Shaped Pulse Capability for Superior Biological Structural Determination Using EPR Spectroscopy.

State of the art pulse EPR instrumentati: State of the art pulse EPR instrumentation for long range distance measurements in biomacromolecules

Data underpinning: Robustness and sensitivity of Gd(III)-Gd(III) double electron electron resonance (DEER) measurements: Comparative study of high-frequency EPR spectrometer designs and spin label variants (dataset)

DEER measurements of calmodulin: novel spin labels and structural insights

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Robustness and sensitivity of Gd(III)-Gd(III) double electron electron resonance (DEER) measurements: comparative study of high-frequency EPR spectrometer designs and spin label variants

Abstract

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Enabling Shaped Pulse Capability: Enabling Shaped Pulse Capability for Superior Biological Structural Determination Using EPR Spectroscopy.

State of the art pulse EPR instrumentati: State of the art pulse EPR instrumentation for long range distance measurements in biomacromolecules

Datasets

Data underpinning: Robustness and sensitivity of Gd(III)-Gd(III) double electron electron resonance (DEER) measurements: Comparative study of high-frequency EPR spectrometer designs and spin label variants (dataset)

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DEER measurements of calmodulin: novel spin labels and structural insights

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