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

ABSTRACT: 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.

SUBMITTER: Wort JL

PROVIDER: S-EPMC8006131 | biostudies-literature |

REPOSITORIES: biostudies-literature

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