Indeed, ∆ 𝑀-𝑅1 was systematically lower for the phosphate buffer series than the Tris-HCl buffer series (ESI and vide infra for details). Interestingly, the ∆ 𝑅1-𝑅1 behaviour suggested that in phosphate buffer, metal-templated dimer formation was optimised at a metal:protein ratio of 1:1 and persists even above stoichiometric Cu II concentration. Replicate measurements of the phosphate buffer series (see ESI) reproduced this observation, and the fitted parameters: 𝛼 = 2, and a 𝐾 𝐷 of 2.7 × 10 -5 further supported the positive cooperativity of dimerization. This affinity agrees with reported literature values for Cu II binding to histidine residues on a solvent-exposed α-helix. Being aware of the possible precipitation of Cu II in the presence of phosphate salts, buffers were screened that would retain free Cu II in solution under alkali conditions via CW-EPR measurements (see ESI section 2.4). For this purpose, Tris-HCl buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4) was most suitable, while for comparison the phosphate buffer retained ~15-60% of Cu II in solution when in presence of two equivalents of imidazole or half an equivalent of K28H/Q32H GB1, respectively (see ESI). Leaving a solution of CuCl2 in phosphate buffer to equilibrate led to negligible available Cu II (i.e., subsequent incubation with protein yielded very poor DEER modulation depths). From these observations we hypothesized that the changing availability of metal ions in solution would modify the binding equilibria reflected by the modulation depths of our measurements.