Eyring plots were obtained for the temperature-dependence of the kon and koff rates (Fig. and), from which the association/dissociation activation enthalpies (𝛥𝐻 𝑎𝑠𝑠 ‡ /𝛥𝐻 𝑑𝑖𝑠𝑠 ‡ ), activation/dissociation entropies (𝑇𝛥𝑆 𝑎𝑠𝑠 ‡ /𝑇𝛥𝑆 𝑑𝑖𝑠𝑠 ‡ ), and thus activation/dissociation free energies (𝛥𝐺 𝑎𝑠𝑠 ‡ /𝛥𝐺 𝑑𝑖𝑠𝑠 ‡ ), could be determined. The association of SIM and its variants with SUMO has an enthalpic activation barrier in all cases (Table ), while peptide dissociation has an enthalpic barrier for parent SIM and Me-Ile2712, and a smaller but entropic barrier for Me-Lys2716 (Fig. , Considering that the dissociation free energy barrier, 𝛥𝐺 𝑑𝑖𝑠𝑠 ‡ , is practically identical for the parent and Me-Ile2712 peptides (≈64 kJ/mol), the slightly more favourable 𝑇𝛥𝑆 𝑎𝑠𝑠 ‡ gives rise to the 3 kJ/mol decrease in ΔGbind for Me-Ile2712 (Fig. ). Taken together, these data suggest that entropy-driven lowering of the association activation barrier arising from an increase in the free energy of the N-methylated peptide (relative to the parent SIM) represents a plausible explanation for the increased affinity for Me-Ile2712.