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. In Figure , 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.