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Pulse Dipolar EPR Reveals Double-Histidine Motif Spin-labelling is Robust Against Competitor Ions

submitted on 19.01.2021, 00:57 and posted on 20.01.2021, 07:56 by Joshua Wort, Swati Arya, Katrin Ackermann, Alan J. Stewart, Bela Bode
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 CuII-chelates offers high precision distance determination in systems non-permissive to thiol-directed spin labelling. However, the non-covalency 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 labelling using CuII-nitrilotriacetic acid (CuII-NTA) is robust against the competitor ligand ZnII-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, particularly metalloproteins with divalent metal ion cofactors.



Leverhulme Trust RPG-2018-397

Wellcome Trust (099149/Z/12/Z)

BBSRC (BB/R013780/1)

ISSF support to the University of St Andrews from the Wellcome Trust


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University of St Andrews


United Kingdom

ORCID For Submitting Author


Declaration of Conflict of Interest

The authors declare no competing financial interests.