To better understand the role of the modifications in increasing the affinity of the TACC3 peptidomimetics towards Aurora-A, we studied their unbound solution structure by NMR spectroscopy (Fig. ). At 5°C, we found that TACC3518-532 showed well-resolved 1 H resonances (Fig. , Table , Figs. S41-49), exhibiting amide-bond N-H vicinal coupling constants for each residue, JNH-αH, persistently in the range of 6-7 Hz, consistent with a general disordered/random coil secondary structure. Analysis of TACC3518-532 based on recently published random-coil chemical shifts for disordered proteins (ΔẟRC) supported this conclusion; both Cα (ΔẟCα) and Hα (ΔẟHα) secondary chemical shifts showed extended regions with small negative values (│ΔẟCα│≤ 1 ppm; │ΔẟHα│≤ 0.25 ppm), much lower and for ΔẟHα of opposing sign, to those of α-helical structures (ΔẟCα ≥ 2.0-3.5 ppm; Table , Figs. S50-52). We found no evidence of a folded (helical) structure in solution when the corresponding TOCSY/NOESY spectra were analyzed; only correlations between consecutive (i, i-1) amide bonds were observed. Modified peptide TACC3518-532-L/R-Bph-I/fP, exhibited broadened 1 H resonances of lower intensity, in particular for the constrained Bph group, residues Cys526 TACC3 , Glu523 TACC3 , and (4-I)Phe525 TACC3 (Fig. , Table and Fig. -60 for full spectral data at 5 °C). This indicates slower interconversion between conformers, in contrast to the unconstrained peptide. We have observed such behavior previously for constrained peptides that target helical interfaces. To further probe the effect of the constraint in TACC3518-532-L/R-Bph-I/fP, we carried out 1 H variable temperature (VT) NMR experiments from 5-50 °C (Figs. S61-62). Upon gradually increasing the temperature we measured a progressive shift to higher signal intensities (Fig. ), improved spatial resolution, and resonance narrowing, confirming that exchange between conformers occurs at low temperatures. TOCSY/NOESY spectra of TACC3518-532-L/R-Bph-I/fP revealed similar structural features to those found for the WT peptide, and were indicative of a disordered/random-coil structure based on 1 HNH and ẟRC chemical shifts (Table ; Figs. ); however, some key differences were observed. Firstly, we observed a clear conformational bias at the trans-(4-F)Pro528 TACC3 residue in the constrained peptide towards the desired exo-pucker/transamide population that persisted even at room temperature (Kexo/endo (5 °C)= 2.04; Kexo/endo (25 °C)= 1.22; insets Fig. ; Table ). Secondly, we observed more extensive nOe correlations for Phe525 TACC3 . Spectral correlations for the WT unconstrained peptide indicate Phe525 TACC3 contacts both the N-terminal residue (Ser524 TACC3 ) and the C-terminal residues (Asp527 TACC3 , Pro528 TACC3 ) (Figs. S38-41), whereas for TACC3518-532-L/R-Bph-I/fP the correlations were consistent with preferential orientation towards the N-terminus (Glu522 TACC3 and Ser524 TACC3 (Fig. , Figs. S58-60). These results indicate that whilst Phe525 TACC3 freely rotates about the Cα-Cβ dihedral in the WT sequence, in the constrained peptide its orientation is biased to that observed when bound to Aurora-A. We also examined the constrained variant TACC3518-532-L/R-Bph, where the (4-I)Phe525 and trans-(4-F)Pro528 are not present (Table -13, Figs. S63-71). We observed intermediate behavior for this peptide (Fig. ), with nOe correlations between residues on either side of Phe525, but with higher intensity for N-terminal residues. Finally, we analyzed our second constrained model peptide, TACC3518-532-S/E-Bph (Table Taken together, these NMR data indicate a combination of all three modifications restricts the conformational ensemble in the constrained peptidomimetics, presumably disfavouring some that are unproductive for binding and in effect predisposing the peptides towards higher affinity Aurora-A binding.