All statistically significant mutations produce rational Φ-values. We calculated the Φ-values using the ∆∆G ‡-U values determined at both of the concentrations used for the kinetic experiments (Table This combined approach has been successfully applied to protein folding mechanisms, where specific structural features of the folding nucleus could be determined, including secondary structural elements , key contacts , the presence of a final-fold topology at the TSE , as well as transient interactions which stabilise the TS . More recently Φ-values integrated with simulations have also been used to capture the TS of binding reactions for intrinsically disordered proteins .