As anticipated, the backbone chemical shifts of the parent SIM were consistent with those for a fully unstructured (random coil) peptide (Fig. ). Comparison of chemical shifts within the N-methylated region of each variant, relative to parent SIM, is complicated by the fact that N-methylation will increase the electron density of the associated amide bond, due to the electron-donating character of the methyl group. In the absence of any structural changes, we expect the chemical shift of neighbouring HA atoms (i.e., HA of the Nmethylated residue and HA of the preceding residue) to be shifted downfield when compared to parent SIM chemical shifts. This is what is observed for Me-Val2710, Me-Ile2712, Me-Lys2716 and Me-Lys2717 (Fig. and Fig. ). Excluding atoms whose chemical environment is directly impacted by the introduction of the N-methyl group (i.e., atoms within six bonds of the methyl carbon), the measured backbone chemical shifts of all five Nmethylated variants differed very little from the shifts of the parent SIM, indicating that no long-range secondary structural elements (e.g., extended, β-rich structure) had been detected for these unbound peptides. In further support of this conclusion, we observed no NOE's indicative of helical or strand conformations. For Me-Ile2708, the HA chemical shift pattern surrounding the N-methylated peptide bond differs for both the trans and cis isomers; the residue before the N-methylated amide is significantly more upfield than in the other SIM variants, while the residue after the N-methylated amide is more downfield (Fig. ), suggesting the possibility of a turn-like collapse. An NOE between N2706-HA and E2709-HN was also observed for the trans isomer of Me-Ile2708 (Fig. ), indicating some localized structuring of residues at the N terminus of this variant. However, we note that the N terminus does not participate in SIM/SUMO recognition and thus it is difficult to predict how such ordering will affect binding.