Abstract
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 Cu2+ 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 Cu2+ complexes of iminodiacetic acid or nitrilotriacetic acid. We discuss a computerized scan for native histidine pairs that might be prone to bind such Cu2+ complexes and spin-labelling site pair scans that can identify suitable double mutants for labelling. Predicted distance distributions between two Cu2+ labels are compared to experimental distance distributions. We also test the hypothesis that elastic network modelling of conformational transitions with Cu2+-dHis labels can provide more accurate structural models than with nitroxide labels.
Highlights
The combination of pulsed dipolar spectroscopy techniques, such as double electron electron resonance (DEER), known as PELDOR [1, 2], double-quantum coherence (DQC) [3, 4], relaxation-induced dipolar modulation enhancement (RIDME) [5, 6], or the single-frequency techniques for refocusing (SIFTER) dipolar couplings [7, 8], with site-directed spin labelling [9, 10] has developed into an important tool in structural biology [11,12,13,14], especially for proteins and protein complexes with
Most applications use nitroxide spin labels attached to a single cysteine residue, such as the methanethiosulfonate spin label (MTSSL), which has a moderately flexible linker between the backbone and the nitroxide moiety
In-silico spin labelling for labels that attach to two residues is feasible with a computational effort that allows for site scans of large proteins
Summary
The combination of pulsed dipolar spectroscopy techniques, such as double electron electron resonance (DEER), known as PELDOR [1, 2], double-quantum coherence (DQC) [3, 4], relaxation-induced dipolar modulation enhancement (RIDME) [5, 6], or the single-frequency techniques for refocusing (SIFTER) dipolar couplings [7, 8], with site-directed spin labelling [9, 10] has developed into an important tool in structural biology [11,12,13,14], especially for proteins and protein complexes with. There is a need to incorporate dHis-based distance restraints into the MMM software Such an implementation should allow to test whether native pairs of histidine residues may be prone to binding C u2+–IDA or C u2+–NTA and should be able to suggest site pairs for labelling in the spirit of the site scan feature [43] for nitroxide labels attached to a single cysteine residue. To this end, in this work we have generated histidine rotamer libraries and structural models for the Cu2+ complexes formed with a dHis motif and either of the two chelating ligands, IDA or NTA.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
