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Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels
Published on: July 4, 2016
Sub-Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII -labelling of Double-Histidine Motifs with
Joshua L Wort1, Katrin Ackermann1, Angeliki Giannoulis1
1EaStCHEM School of Chemistry, Biomedical Sciences Research Complex, and Centre of Magnetic Resonance, University of St Andrews, North Haugh, St Andrews, KY16 9ST, UK.
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.
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