To determine more accurately the thermodynamic parameters for binding and investigate the binding kinetics of the parent SIM and its N-methylated variant peptides, NMR-relaxation based methods were used (Figs. and). Initially, a series of 1 H- 15 H NMR spectra were acquired using 15 N-enriched SUMO in the presence of natural abundance 14 N-SIM peptides. For the parent SIM peptide and for variants with similar values of ΔGbind (Me-Ile2708, Me-Ile2712, Me-Lys2717), peptide binding to SUMO was observed in the slow exchange regime on the chemical shift timescale, giving rise to two sets of 1 H- 15 N resonances for residues in the SIM-binding pocket (helix α1 and strand β2 of SUMO), corresponding to the bound and unbound species (Fig. ). For these tighter-binding variants (Me-Ile2708, Me-Ile2712, Me-Lys2717), bound-state chemical shift differences relative to SUMO/parent SIM (Δω) were only observed for residues expected to lie in proximity to the N-methylated SIM residue (Fig. and Fig. -S6, indicating that these peptides bind SUMO in the canonical binding conformation. Non-overlapping exchange cross-peaks were evident for a subset of residues when the 15 N magnetizations of the bound and unbound states were mixed following t1 ( 15 N) evolution (ZZ-exchange spectroscopy). Global fitting of the intensities of auto and cross peaks as a function of mixing time to McConnel equations (Fig. , Fig. ; SI for methods) gave apparent first-order association 𝑘 𝑜𝑛 𝑎𝑝𝑝 and first-order dissociation 𝑘 𝑜𝑓𝑓 rates for binding, which were converted to 𝑘 𝑜𝑛 and dissociation equilibrium constants (KD). For the parent, Me-Ile2708, Me-Ile2712 and Me-Lys2717 peptides, 15 N ZZ-exchange data at 5 °C fitted well to a 2-state bound-unbound model, yielding KD values in the low micromolar range (Table ). These confirm a significant increase in affinity for the N-methylated series (e.g. ΔΔGMe-Ile2712 vs parent ~ 2 kJ mol -1 ). For this set of four peptides, 𝑘 𝑜𝑓𝑓 values were similar (4-8 s -1 ) suggesting that 𝑘 𝑜𝑛 is the primary cause of changes in KD. Indeed, a positive correlation was observed between these two parameters (see Fig. ). The observed faster association rates, in the absence of any changes in the charge of the variant SIM peptides, could suggest that N-methylation at these locations conformationally predisposes these peptides for SUMO binding. For the weak peptide binders (Me-Val2710 and Me-Lys2716), only the unbound SUMO resonances were observed in 1 H- 15 N HSQC spectra, even at high SIM concentrations. This suggests that the population of the bound complex is small and/or binding does not take place in the slow chemical shift timescale. Thus, to investigate the binding kinetics of those variants, we used Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion that is sensitive to exchange between states with skewed populations on the millisecond timescale. Large CPMG profiles for SUMO residues in the SIM-binding site were observed at 5 °C upon addition of Me-Val2710 or Me-Lys2716 (Fig. and Fig. ). These profiles were absent for apo SUMO (Fig. ), suggesting that the observed millisecond dynamics are due to transient SIM binding. Global fitting of CPMG data at 600 and 750 MHz to a 2-state model yielded excellent fits (Fig. and Fig. ). CPMG-derived chemical shifts of the transiently populated, bound state of SUMO in the presence of Me-Val2710 or Me-Lys2716 corelated well with those of the stable, parent SIM-bound state, with the exception of some residues in the SIM binding pocket (see Fig. , S11b, S11b), suggesting that the bound state of SUMO in the presence of these SIM variants is not significantly different to that with parent SIM. However, 𝑘 𝑜𝑓𝑓 values for the weakly bound variants increased by more than 50fold (~200-550 s -1 ; Fig. )therefore, while the structure of SUMO in the bound state complex is unchanged by N-methylation, N-methylation at residues Val2710 and Lys2716 either prevents or hinders the ability of the SIM peptide to adopt its canonical binding conformation, leading to fast dissociation and a lower affinity interaction. It is surprising that Me-Val2710 does not show improved binding affinity as the NH is not involved in hydrogenbonding and methylation should restrict Ramachandran space to conformations that favour binding.( ) Therefore, it is likely that methylation adversely influences the accessible χ space of the isopropyl side chain of Val2710 (a hot-spot residue), making it incompatible with SUMO binding. In the case of Lys2716, we consider it more likely that methylation of this residue perturbs the allowable χ space for Trp2714. Table . Binding affinities and rates for the binding of N-methylated SIM peptides to SUMO. KD, kon, and koff values were determined for select SIM variants by NMR (using 15 All together, our results suggest that the restriction of the conformational space available to N-methylated peptides can increase association kinetics which correlates with enhanced binding. Conversely, in some cases, if the modified peptide cannot adopt a stable binding conformation, N-methylation can favour dissociation which correlates with diminished binding.