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Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing Cu II ‐labelling of Double‐Histidine Motifs with Lower Temperature
Author(s) -
Wort Joshua L.,
Ackermann Katrin,
Giannoulis Angeliki,
Stewart Alan J.,
Norman David G.,
Bode Bela E.
Publication year - 2019
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201904848
Subject(s) - electron paramagnetic resonance , pulsed epr , chemistry , histidine , site directed spin labeling , nitroxide mediated radical polymerization , biomolecule , covalent bond , crystallography , spectroscopy , dipole , nuclear magnetic resonance , analytical chemistry (journal) , spin echo , physics , medicine , biochemistry , radical polymerization , organic chemistry , amino acid , quantum mechanics , chromatography , magnetic resonance imaging , copolymer , radiology , polymer
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 Cu II 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 Cu II coordination approach is vulnerable to low binding‐affinity. Herein, dissociation constants ( K D ) are investigated directly from the modulation depths of relaxation‐induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low‐ to sub‐μ m Cu II K D 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‐μ m protein concentrations in orthogonally labelled Cu II –nitroxide systems using a commercial Q‐band EPR instrument.