Sub-micromolar pulse dipolar EPR spectroscopy reveals increasing CuII-labelling of double-histidine motifs with lower temperature
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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 motifs with CuII spin labels shows promise for further increasing the precision of distance measurements. It also appeals in proteins containing essential cysteines which can interfere with thiol‐specific labelling. However, the non‐covalent CuII coordination approach is vulnerable to low binding‐affinity. Here, dissociation constants (KD) are investigated directly from the cryoprotectant of relaxation induced dipolar modulation enhancement (RIDME) EPR distance experiments. The superb sensitivity of these experiments reveals low‐ to sub‐μM CuII KDs 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 Cu(II) ‐nitroxide systems using a commercial Q‐band EPR instrument.nd EPR instrument.
Wort , J , Ackermann , K , Giannoulis , A , Stewart , A J , Norman , D & Bode , B E 2019 , ' Sub-micromolar pulse dipolar EPR spectroscopy reveals increasing Cu II -labelling of double-histidine motifs with lower temperature ' Angewandte Chemie , vol. 131 , no. 34 , pp. 11807-11811 . https://doi.org/10.1002/ange.201904848
Copyright © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
DescriptionJLW is supported by the BBSRC DTP Eastbio. 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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