Pulse Dipolar EPR Reveals Double-Histidine Motif Cu^II–NTA Spin-Labeling Robustness against Competitor Ions

Pulse-dipolar EPR is an appealing strategy for structural characterization of complex systems in solution that complements other biophysical techniques. Significantly, the emergence genetically encoded self-assembling spin labels exploiting exogenously introduced double-histidine motifs conjunction with CuII-chelates offers high precision distance determination nonpermissive to thiol-directed labeling. However, noncovalency this interaction exposes potential vulnerabilities competition from adventitious divalent metal ions, and pH sensitivity. Herein, a combination room-temperature isothermal titration calorimetry (ITC) cryogenic relaxation-induced dipolar modulation enhancement (RIDME) measurements are applied model protein Streptococcus sp. group G. G, B1 domain (GB1). Results demonstrate motif labeling using CuII-nitrilotriacetic acid (CuII–NTA) robust against competitor ligand ZnII–NTA at >1000-fold molar excess, nM binding affinity surprisingly retained under acidic basic conditions even though room temperature shows stronger dependence. This indicates well-suited diverse biological applications, requirement ion cofactors or slightly not necessarily being prohibitive.