In setting these results within the context of potential molecular mechanisms of recognition, we note the following additional considerations. First, there are likely subtle effects, such as a small increase in hydrogen-bond accepting ability of the carbonyl that might be expected upon N-methylation of the peptide bond. In turn, this would be anticipated to increase binding enthalpy. In addition, we cannot exclude the possibility that increased hydrophobicity or differential solvation of the methylated peptides influences affinity. Nonetheless, the entropy driven increased kon rates that we observe lead us to conclude that increased affinities are caused by restricting the accessible conformational space of the Nmethylated peptides. We simulated Ramachandran plots, which show that N-methylation significantly limits the phi(ϕ)/psi(ψ) angles accessible to residues on either side of the methylation site, such that residues are limited to extended or turn-like conformations (i.e., excluded from α-helical space). However, NMR analyses suggest the unbound peptides do not adopt a defined conformation in the absence of SUMO. We contend that the overall ensemble of SIM conformers has a higher ground state energy and that this lowers the entropy of activation for SUMO binding (see free energy diagrams in Fig. ). We note that the polyproline-II conformation is somewhat intermediate between the α-helix and β-strand conformations, and that N-methylation is known to restrict the conformational space of peptide backbones. Thus, whilst the methylated peptides cannot be considered as pre-organized for SUMO binding, the ensemble is expected to disfavour α-space and thus favour β-space localized around the N-methylated residue so is primed or predisposed towards SUMO recognition. Previously, pre-organization of a peptide that recognises its target through a bind-and-fold(20) mechanism (Fig. ) was shown not to enhance affinity for its target, because constraining limits "the number of ways to bind",(47) whilst for a peptide which recognises its target through conformational selection (Fig. ), constraining should increase affinity.( ) Given N-methylation does not seem to induce a specific extended conformer and instead favours an ensemble of conformers that are compatible with binding, the effect observed here may represent a useful strategy to drive affinity enhancement for ligands which bind their target through conformational selection or bind-and-fold mechanisms. Modulating peptide binding by tuning the entropy of activation/binding represents an untapped approach for design of peptidomimetic ligands; our future studies will apply this strategy to other β-strand mediated PPIs to explore the generality of these observations and for development of therapeutically relevant PPI inhibitors.