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

Automated summary

Pulse-dipolar EPR is an attractive method for structural characterization in solution, especially when paired with genetically encoded self-assembling spin labels and Cu-II-chelates. The combination of room-temperature isothermal titration calorimetry and cryogenic relaxation-induced dipolar modulation enhancement showed that the double-histidine motif spin labeling with Cu-II-nitrilotriacetic acid is robust against competition from Zn-II-NTA and maintains high nM binding affinity even under acidic and basic conditions. The strategy is well-suited for diverse biological applications, with the requirement of other metal ion cofactors or slightly acidic pH not necessarily being prohibitive.