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The Engrailed Homeodomain folds on the microsecond time scale via an intermediate that is experimentally well characterised using structural Engrailed-Homeodomain mimics. Here, we analysed directly the changes in distance between key residues during the kinetics of unfolding and at equilibrium using fluorescence resonance energy transfer (FRET). Trp was the donor and 5-(((acetylamino)ethyl)amino) naphthalene-1-sulphate, the acceptor, substituted in positions that caused little change in stability. Distances calculated for the native state were in good agreement with those derived from the NMR structure. The distances between the N- and C-termini of Helix I and of Helix III increased, then decreased and finally increased again with increasing GdmCl concentration on equilibrium denaturation. This behaviour implied that there was a folding intermediate on the folding pathway and that this intermediate was populated at low concentrations of GdmCl concentration ( approximately 1 M). We analysed the changes in distance during temperature-jump relaxation kinetics, using a qualitative and very conservative procedure that drew conclusions only when changes in fluorescence of mutants containing either the donor or the acceptor alone would not obscure the change in the FRET signal when both donor and acceptor were present. The distance changes obtained under equilibrium and kinetic measurements were self-consistent and also consistent with the known high-resolution structures of the mimics of the folding intermediates. We showed that for analysing distances in disordered ensembles, it is important to use FRET probes with a critical distance close to the average separation in the ensemble. Otherwise, average distances could be over or underestimated.

Fluorescence resonance energy transfer analysis of the folding pathway of Engrailed Homeodomain