Simulated annealing calculations of N-methylated peptides indicated that methylation of backbone amide bonds could restrict the accessible phi (ϕ) and psi (ψ) angles rendering the α-helical region of Ramachandran space inaccessible (Fig. ). As the parent SIM sequence has been shown to lack stable secondary structure in the absence of a binding partner, it is possible that some of the differences in binding affinity observed could be explained by changes to the secondary structure propensity of N-methylated SIM variants. NMR chemical shifts of backbone nuclei (Hα, Cα and Cβ) can be used as reporters of even small changes in secondary structure of disordered proteins/peptides,(40) and α-like or β-like chemical shifts are indicative of an increase in the population of those secondary structures. Therefore, the backbone and side chain chemical shifts of parent SIM and its N-methylated variants were assigned using 1 H-1 H TOCSY, 1 H-1 H NOESY spectra and natural abundance 1 H-13 C HSQC spectra.