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 journalArticle

3 Citations (Scopus)
32 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 languageEnglish
Pages (from-to)11681-11685
Number of pages5
JournalAngewandte Chemie International Edition
Volume58
Issue number34
Early online date19 Jun 2019
DOIs
Publication statusPublished - 9 Aug 2019

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