Rotamer Modelling of Cu(II) Spin Labels Based on the Double-Histidine Motif

Spin labels attached to two residues of a protein chain have less conformational flexibility than those attached to a single residue and thus lead to a narrower spatial distribution of the unpaired electron. The case of Cu 2+ labels based on the double-histidine (dHis) motif is of particular interest, as it combines the advantage of precise localization of the unpaired electron with a labelling scheme orthogonal to the more common cysteine-based labelling. Here, we introduce an approach for in silico spin labelling of a protein by dHis motifs and Cu 2+ complexes of iminodiacetic acid or nitrilotriacetic acid. We discuss a computerized scan for native histidine pairs that might be prone to bind such Cu 2+ complexes and spin-labelling site pair scans that can identify suitable double mutants for labelling. Predicted distance distributions between two Cu 2+ labels are compared to experimental distance distributions. We also test the hypothesis that elastic network modelling of conformational transitions with Cu 2+ -dHis labels can provide more accurate structural models than with nitroxide labels.