Self-assembly of protein monomeric units is of great interest in supramolecular complex design, but owing to the non-covalency of quaternary structural interactions, mimicking their functionality for the synthesis of new protein complexes is demanding. Metal coordination is used as a driving force for the assembly of protein monomers, leading to a degree of multimerization that depends on the metal ion coordination geometry. Different strategies employed the engineering of chelating motifs of natural and unnatural amino acid residues, the incorporation of non-natural ligands onto protein surfaces, and the construction of hybrid coordination motifs, resulting in metal-induced increased stability of the multimer due to higher metal-binding affinity. Proteinprotein interfaces nucleated by metal coordination have led to the formation of 2D and 3D crystalline protein lattices, and novel functional materials. Of particular interest is the formation of metal-bridged dimers because they are considered the precursor of more complex assemblies. Several techniques such as X-Ray crystallography, NMR, and sedimentation velocity have extensively characterized these binding processes. Dipolar electron paramagnetic resonance (EPR) spectroscopy has recently emerged as an excellent complementary tool for studying metal ion-binding equilibria with sub-micromolar sensitivity. The four-pulse DEER (double electron-electron resonance) and the five-pulse RIDME (relaxation-induced dipolar modulation enhancement) experiments (for pulse sequences see ESI section 1.3) allow detection of the weak dipolar interaction between paramagnetic centres, which is characterized by modulation with the dipolar frequency (ωAB) that encodes the inter-spin distance, rAB. The modulation depth (Δ) of these traces (i.e., the amplitude between the signal intensity at 𝑡 = 0 and the time when the signal is entirely damped in the limit of negligible intermolecular decay) informs the number of coupled spins. In previous studies, DEER and RIDME were employed individually for monitoring the metal-templated dimerization of a nitroxide labelled terpyridine-based ligand model system using different divalent metal ions as templates , and proved the feasibility of monitoring binding events at cryogenic temperatures, with the modulation depth informing on the degree of binding at a given metal-to-ligand ratio. Here, DEER and RIDME were employed complementarily for studying the metal-templated dimerization of a protein model system, the B1 immunoglobulin-binding domain of protein G of Streptococcus sp. Group G (GB1). Double-histidine (dHis) motifs are incorporated as artificial metal-binding sites, 26 making this system particularly suitable for this study. Previous works have used this system as a biological model for pulse dipolar electron paramagnetic resonance spectroscopy (PDS) studies, improving the precision and accuracy of distance measurements due to the increased rigidity of Cu II -chelate spin labels through bipedal attachment. The I6R1/K28H/Q32H and I6H/N8H/K28R1 constructs (Figure ) were selected for this work. Only the construct I6R1/K28H/Q32H gave appreciable DEER modulation depth when bound to Cu II and Zn II (see ESI section 2.1), and modulation depth was maximised for the Cu II series. This is not entirely surprising since it is consistent with the finding of metal-induced stabilization of the α-helix motif via histidine residues and its higher affinity towards Cu II than to other metal ions. 30 The DEER experiment measures the intermolecular nitroxide-nitroxide (R1-R1) distances within the metal-templated dimer and therefore provides direct information on the dimer formation. The modulation depth, ∆ 𝑅1-𝑅1 , depends on the dipolar interaction between nitroxide moieties of each GB1 monomer. The RIDME experiment measures the intramolecular metal-nitroxide distances (M-R1) and is a reporter of all metal-bound species. The modulation depth, ∆ 𝑀-𝑅1 provides information about the formation of dimers coordinated around the metal template and fractional saturation of the metal-binding site, the dHis motif. To characterize these binding equilibria, a cooperative binding model was used to fit simultaneously ∆ 𝑅1-𝑅1 and ∆ 𝑀-𝑅1 values. This allows simultaneous characterization of a dissociation constant (KD) for initial metal binding and a cooperativity factor (𝛼) for the metal-templated dimerization event.