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.

Pulse dipolar EPR spectroscopy reveals buffer modulated cooperativity of metal templated protein dimerization

“…First, measurements were performed as a nine-point pseudo-titration series (where each datapoint was a discrete sample) for Cu II , in the presence of phosphate buffer (150 mM NaCl, 42.4 mM Na2HPO4 and 7.6 mM KH2PO4, pH 7.4), having been used extensively for previous EPR methodological work involving GB1 and Cu II -NTA. 28,32,33 In Figure 2, the ∆ 𝑀−𝑅1 behaviour was consistent with a reduced apparent binding affinity of Cu II for the dHis motif in phosphate buffer, with <80% occupancy at a metal:protein ratio of 1:1. Indeed, ∆ 𝑀−𝑅1 was systematically lower for the phosphate buffer series than the Tris-HCl buffer series (ESI and vide infra for details).…”

The Advantages of EPR Spectroscopy in Exploring Diamagnetic Metal Ion Binding and Transfer Mechanisms in Biological Systems

“…dHis-Cu(II) spin-labeling has recently been shown to hold high affinity and stability against competitor metal ions (even in an acidic environment) to biomolecules. In this study, a nanomolar concentration of the protein was studied by EPR spectroscopy, which is also applicable for in-vivo research [80,108]. In addition, the combination of several techniques, such as microscopy [103,109], copper uptake experiments using 64 Cu [110], UV-Vis and NMR [13,111], can provide a better and more complete picture on Cu-related biological processes.…”

“…One such interaction is the dipolar interaction between two paramagnetic centers, which can report on nanometer distances (usually at a range of 2.0-10.0 nm) between two paramagnetic sites. The most common approaches to acquire nanoscale structural information are double electron electron resonance (DEER), also referred to as pulsed electron double resonance (PELDOR) [51,[71][72][73][74][75], Double Quantum Coherence (DQC) [76][77][78], and relaxation induced dipolar modulation enhancement (RIDME) [79,80]. In all experiments, the Fourier transform (FT) spectra yield characteristic lineshapes and splittings that may be analyzed for distances and distance distributions.…”

Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins: CuII–CuII and Nitroxide–Nitroxide Cases

“… 23 Thereby, demonstrating the high-affinity of genetically encoded double-histidine motifs to Cu II ions, 24 – 25 and their suitability as labelling sites for low concentration studies. 26 …”