Unfolding and inactivation of cutinases by AOT and guanidine hydrochloride

We present a comparative analysis of the unfolding and inactivation of three cutinases in the presence of guanidine hydrochloride (GdnHCl) and bis(2-ethylhexyl) sodium sulfosuccinate (AOT). Previous investigations have focused on the cutinase from Fusarium solani pisi (FsC). In addition to FsC, the present study includes the cutinase from Humicola insolens (HiC) and a mutant variant of HiC (μHiC) with increased activity and decreased surfactant sensitivity. Equilibrium and time-resolved denaturation by AOT were studied in aqueous solution and reverse micelles, and were compared with GdnHCl denaturation. The far-UV CD and fluorescence denaturation profiles obtained in the aqueous solutions of the two denaturants coincide for all three cutinases, indicating that unfolding is a co-operative two-state process under these conditions. In reverse micelles, the cutinases unfold with mono-exponential rates, again indicating a two-state process. The free energy of denaturation in water was calculated by linear extrapolation of equilibrium data, yielding very similar values for the three cutinases with averages of −11.6 kcal mol−1 and −2.6 kcal mol−1 for GdnHCl and AOT, respectively. Hence, the AOT denatured state (DAOT) is less destabilised than the GdnHCl denatured state (DGdnHCl), relative to the native state in water. Far-UV CD spectroscopy revealed that DAOT retains some secondary structure, while DGdnHCl is essentially unstructured. Similarly, fluorescence data suggest that DAOT is more compact than DGdnHCl. Activity measurements reveal that both DAOT and DGdnHCl are practically inactive (catalytic activity <1% of that of the native enzyme). The fluorescence spectrum of DAOT in reverse micelles did not differ significantly from that observed in aqueous AOT. NMR studies of DAOT in reverse micelles indicated that the structure is characteristic of a molten globule, consistent with the CD and fluorescence data.