Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII‐labelling of Double‐Histidine Motifs with Lower Temperature

Joshua L Wort; Katrin Ackermann; Angeliki Giannoulis; Alan J Stewart; David G Norman; Bela E Bode

doi:10.1002/anie.201904848

Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII‐labelling of Double‐Histidine Motifs with Lower Temperature

Joshua L Wort
, Katrin Ackermann
, Angeliki Giannoulis
, Alan J Stewart
, David G Norman
, Bela E Bode

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

74 Citations (Scopus)

167 Downloads (Pure)

Abstract

Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double-histidine motifs with CuII spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol-specific labelling. However, the non-covalent CuII coordination approach is vulnerable to low binding-affinity. Herein, dissociation constants (KD ) are investigated directly from the modulation depths of relaxation-induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low- to sub-μm CuII KD s under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double-histidine motif for EPR applications even at sub-μm protein concentrations in orthogonally labelled CuII -nitroxide systems using a commercial Q-band EPR instrument.

Original language	English
Pages (from-to)	11681-11685
Number of pages	5
Journal	Angewandte Chemie International Edition
Volume	58
Issue number	34
Early online date	19 Jun 2019
DOIs	https://doi.org/10.1002/anie.201904848
Publication status	Published - 9 Aug 2019

Keywords

dissociation constant
double-histidine motif
EPR spectroscopy
non-covalent interactions
RIDME

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10.1002/anie.201904848Licence: CC BY

Final Published VersionFinal published version, 1.47 MBLicence: CC BY

Data underpinning: Sub-micromolar pulse dipolar EPR spectroscopy reveals increasing Cu(II)-labelling of double-histidine motifs with lower temperature
Wort, J. (Creator), Ackermann, K. (Creator), Giannoulis, A. (Creator), Stewart, A. J. (Creator), Norman, D. (Creator) & Bode, B. E. (Creator), University of St Andrews, 4 Jul 2019
DOI: 10.17630/ced80b64-fa40-4d15-8f17-78bb4f06562f
Dataset

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@article{eb4789c264b74a32b1e47b2b7c035b49,

title = "Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII‐labelling of Double‐Histidine Motifs with Lower Temperature",

abstract = "Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double-histidine motifs with CuII spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol-specific labelling. However, the non-covalent CuII coordination approach is vulnerable to low binding-affinity. Herein, dissociation constants (KD ) are investigated directly from the modulation depths of relaxation-induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low- to sub-μm CuII KD s under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double-histidine motif for EPR applications even at sub-μm protein concentrations in orthogonally labelled CuII -nitroxide systems using a commercial Q-band EPR instrument.",

keywords = "dissociation constant, double-histidine motif, EPR spectroscopy, non-covalent interactions, RIDME",

author = "Wort, \{Joshua L\} and Katrin Ackermann and Angeliki Giannoulis and Stewart, \{Alan J\} and Norman, \{David G\} and Bode, \{Bela E\}",

note = "{\textcopyright} 2019 The Authors. Published by Wiley-VCH Verlag GmbH \& Co. KGaA.",

year = "2019",

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T1 - Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII‐labelling of Double‐Histidine Motifs with Lower Temperature

AU - Wort, Joshua L

AU - Ackermann, Katrin

AU - Giannoulis, Angeliki

AU - Stewart, Alan J

AU - Norman, David G

AU - Bode, Bela E

PY - 2019/8/9

Y1 - 2019/8/9

N2 - Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double-histidine motifs with CuII spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol-specific labelling. However, the non-covalent CuII coordination approach is vulnerable to low binding-affinity. Herein, dissociation constants (KD ) are investigated directly from the modulation depths of relaxation-induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low- to sub-μm CuII KD s under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double-histidine motif for EPR applications even at sub-μm protein concentrations in orthogonally labelled CuII -nitroxide systems using a commercial Q-band EPR instrument.

AB - Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double-histidine motifs with CuII spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol-specific labelling. However, the non-covalent CuII coordination approach is vulnerable to low binding-affinity. Herein, dissociation constants (KD ) are investigated directly from the modulation depths of relaxation-induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low- to sub-μm CuII KD s under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double-histidine motif for EPR applications even at sub-μm protein concentrations in orthogonally labelled CuII -nitroxide systems using a commercial Q-band EPR instrument.

KW - dissociation constant

KW - double-histidine motif

KW - EPR spectroscopy

KW - non-covalent interactions

KW - RIDME

U2 - 10.1002/anie.201904848

DO - 10.1002/anie.201904848

M3 - Article

C2 - 31218813

SN - 1433-7851

VL - 58

SP - 11681

EP - 11685

JO - Angewandte Chemie International Edition

JF - Angewandte Chemie International Edition

IS - 34

ER -

Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII‐labelling of Double‐Histidine Motifs with Lower Temperature

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Keywords

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Data underpinning: Sub-micromolar pulse dipolar EPR spectroscopy reveals increasing Cu(II)-labelling of double-histidine motifs with lower temperature

Cite this