A General Model to Optimise Copper(II) Labelling Efficiency of Double-Histidine Motifs for Pulse Dipolar EPR Applications
<div> <p>Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to studies of biomolecules underpinning health and disease by providing highly accurate and precise geometric constraints. Combining double-histidine (dH) motifs with Cu<sup>II</sup> spin labels shows promise for further increasing the precision of distance measurements, and for investigating subtle conformational changes. However, non-covalent coordination-based spin labelling is vulnerable to low binding affinity. Dissociation constants of dH motifs for Cu<sup>II</sup>-nitrilotriacetic acid were previously investigated <i>via </i>relaxation induced dipolar modulation enhancement (RIDME), and demonstrated the feasibility of exploiting the double histidine motif for EPR applications at sub-μM protein concentrations. Herein, the feasibility of using modulation depth quantitation in Cu<sup>II</sup>-Cu<sup>II </sup>RIDME to simultaneously estimate a pair of non-identical independent <i>K<sub>D</sub></i> values in such a tetra-histidine model protein is addressed. Furthermore, we develop a general speciation model to optimise Cu<sup>II </sup>labelling efficiency, in dependence of pairs of identical or disparate <i>K<sub>D</sub></i> values and total Cu<sup>II</sup> label concentration. We find the dissociation constant estimates are in excellent agreement with previously determined values, and empirical modulation depths support the proposed model. </p> </div> <br>