Measurements were then performed as an eight-point pseudo-titration series for Cu II in presence of Tris-HCl buffer. Figure shows the background corrected traces for RIDME and DEER measurements of the Tris-HCl buffer series. In Figure the ∆ 𝑀-𝑅1 behaviour indicated a high binding affinity of Cu II for the dHis motif, with >90% occupancy at a metal:protein ratio of 1:1. The ∆ 𝑅1-𝑅1 behaviour suggested that metal-templated dimer formation was optimised at a metal:protein ratio of 4:5, with marginally higher ∆ 𝑅1-𝑅1 than at a metal:protein ratio of 1:2 (see ESI), but was abolished entirely above stoichiometric ratios of Cu II , where either i) significant data cutting was required for processing or ii) the detected echo was free of dipolar modulation. Replicates of metal:protein ratios of 3:5 and 4:5 (see ESI) showed optimised dimer formation at a metal:protein ratio of 3:5, which is consistent with a negative cooperativity mode of templated dimerization (i.e., the initial dHis motif binding event outcompetes the formation of dimer construct), and the global fitting of the 𝐾 𝐷 and 𝛼 parameters support it further. Exploratory simulations validated the robustness of the cooperative binding model (see ESI section 2.5) and indicated a strongly negative cooperativity parameter (𝛼 = 0.15) and a 𝐾 𝐷 of 3.1 × 10 -6 .