A general model to optimise CuII labelling efficiency of double-histidine motifs for pulse dipolar EPR applications
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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.
Wort , J , Ackermann , K , Norman , D & Bode , B E 2021 , ' A general model to optimise Cu II labelling efficiency of double-histidine motifs for pulse dipolar EPR applications ' , Physical Chemistry Chemical Physics , vol. 23 , no. 6 , pp. 3810-3819 . https://doi.org/10.1039/D0CP06196D
Physical Chemistry Chemical Physics
Copyright © 2021 The Author(s). Open Access article. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.
DescriptionJLW is supported by the BBSRC DTP Eastbio. We thank the Leverhulme Trust for support (RPG-2018-397). This work was supported by equipment funding through the Wellcome Trust (099149/Z/12/Z) and BBSRC (BB/R013780/1). We gratefully acknowledge ISSF support to the University of St Andrews from the Wellcome Trust.
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