By comparing the individual contributions of enthalpy and entropy for the interaction of constrained and unconstrained TACC3 variants to Aurora-A (Fig. ), the constrained variants (e.g. TACC3518-532_LR_Bph_IF/fP -TS = -22.7 kJ mol -1 ) were observed to exhibit a more favorable entropy of binding in comparison to the linear variants (TACC3522-536 -TS = -2.7 kJ mol -1 , TACC3518-532 TS = -15.0 kJ mol -1 ; Figs. ). This is compensated by a less favorable enthalpic contribution to binding (TACC3518-532 ΔH = 10.6 kJ mol -1 constrained variants ΔH ~ 7.2-8.5 kJ mol -1 ). To further assess the thermodynamic determinants of binding, we performed variabletemperature fluorescence anisotropy experiments (Figs. S97-98) and Van 't Hoff analyses under different conditions, specifically: buffer (Tris, HEPES), pH (pH = 6.0, 7.5 and 8.0) and salt content (NaCl = 0 mM, 150 mM). Although we observed subtle differences in absolute values of -TS and ΔH, the observed trends were consistent with the ITC experiments (i.e. more favorable -TS and less favorable ΔH for the constrained versus unconstrained peptide), indicating that the thermodynamic differences between unconstrained peptide and constrained peptidomimetic may be attributed to the constraint.