As a model β-strand-mediated PPI to explore the effect of N-methylation on peptide conformation and protein binding, we chose the interaction between human SUMO-118-97 (SUMO) and the SIM motif found in the M-IR2 region of RanBP2. Our chosen SIM sequence -Ac-Asp-Asn-Glu-Ile-Glu-Val-Ile-Ile-Val-Trp-Glu-Lys-Lys-NH2 (herein referred to as the 'parent SIM peptide')was taken from Namanja et al., who modified the wild-type RanBP2 M-IR2 SIM2705-2717 sequence to make it more amenable to biophysical study. This SIM peptide is intrinsically disordered in the absence of a binding partner, but binds to SUMO through β-augmentation with low micromolar affinity.(37) Key non-covalent interactions present in the bound complex (Fig. ) include hydrogen bonds from SUMO to the backbone of SIM residues Glu2709, Ile2711, and Val2713, and side chain H-bonds and π interactions to SIM Trp2714. Hydrophobic packing of isoleucine and valine side chains from SIM along the SUMO binding cleft also contributes to binding affinity; previously, we conducted an alanine scan on this sequence and identified a contiguous stretch of hot-spot residues from Val2710 to Trp2714 (VIIVW; ΔΔG ≥ 4.2 kJ/mol for each residue). This corresponds to the (V/I)X(V/I)(V/I) or (V/I)(V/I)X(V/I/L) consensus motif commonly found in SIMs. In addition, Glu2709 was just below the threshold for being classified as a hotspot residue.