Sub-Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII -labelling of Double-Histidine Motifs with Lower Temperature.

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-?m 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-?m protein concentrations in orthogonally labelled CuII -nitroxide systems using a commercial Q-band EPR instrument.

SUBMITTER: Wort JL 

PROVIDER: S-EPMC6771633 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature