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Sub-micromolar pulse dipolar EPR spectroscopy reveals increasing CuII-labelling of double-histidine motifs with lower temperature

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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 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.

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Original languageEnglish
Pages (from-to)11681-11685
Number of pages5
JournalAngewandte Chemie - International Edition
Volume58
Issue number34
Early online date18 Jul 2019
DOIs
Publication statusPublished - 19 Aug 2019

    Research areas

  • Dissociation constant, Double-histidine motif, EPR spectroscopy, Non-covalent interactions, RIDME

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