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 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 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 CuII–nitroxide systems using a commercial Q-band EPR instrument.
Wort , J L , Ackermann , K , Giannoulis , A , Stewart , A J , Norman , D G & Bode , B E 2019 , ' Sub-micromolar pulse dipolar EPR spectroscopy reveals increasing Cu II -labelling of double-histidine motifs with lower temperature ' Angewandte Chemie - International Edition , vol. 58 , no. 34 , pp. 11681-11685 . https://doi.org/10.1002/anie.201904848
Angewandte Chemie - International Edition
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