We next sought to rationalise the changes in potency observed as a consequence of Nmethylation. For some variants (e.g., Me-Ile2711 and Me-Val2713), disruption of binding could reasonably be attributed to loss of key backbone hydrogen bonds between SIM and SUMO, and/or the steric hindrance conferred by the methyl group disrupting adjacent interactions (Fig. ). For the remaining variants, however, the observed changes in IC50 could not be readily explained based on changes to hydrogen bonding interactions or possible steric clashes between the variant peptide and the protein. We therefore considered that changes in binding affinity may instead arise from changes in backbone conformation (which can in turn affect side-chain conformations) or changes in the electron density of amide bonds. With a selection of peptides from across the N-methylated SIM series (Fig. ), this was investigated using solution NMR methods and computational analyses for the unbound peptides.