There is no evidence for the presence of a refolding intermediate
Curvature or ”roll-over” in the re-folding arm of a chevron is good evidence for the presence of a populated folding intermediate. However, it has been shown that curvature in the refolding arm can indicate the presence of transient aggregation at low concentrations of denaturant, as is observed in FADD DD. Any curvature present due to refolding from an intermediate will be masked by this aggregation. The non-concurrence of equilibrium and kinetic data is another indication of the presence of a refolding intermediate accumulating on the pathway.30 Where a Hammond fit is used, the agreement between kinetic and equilibrium free energies is good (Supplementary Data). Moreover, the average kinetic and equilibrium m-values, taken from WT and mutants, are the same within error (1.3 ± 0.1 kcal mol–1 M–1 and 1.4 ± 0.2 kcal mol–1 M–1, respectively). Furthermore, an amplitude analysis by CD showed no evidence for a dead-time change in amplitude, which would suggest that no helical structure is formed in the dead-time of the stopped-flow experiments (data not shown). Thus, our experiments suggest that, at least above 1.5 M urea where we analyse the kinetic data, the folding of FADD DD is essentially two-state.