Cooperative stabilization of a molten globule apoflavodoxin fragment.

We have destabilized apoflavodoxin by site-specific excision of its C-terminal helix. The resulting flavodoxin fragment (Fld1-149) is compact and monomeric at pH 7.0, with spectroscopic properties of a molten globule and a low conformational stability. To study if Fld1-149 is cooperatively stabilized, we have measured the equilibrium urea unfolding by fluorescence, circular dichroism, and size-exclusion chromatography. The three techniques produced coincident unfolding curves. Furthermore, the thermal unfolding seems also to be cooperative as the same temperature of half-denaturation is obtained using fluorescence and circular dichroism. Fld1-149 displays cold denaturation. The equilibrium properties of Fld1-149 demonstrate that molten globules lacking well-defined tertiary interactions can still be cooperatively stabilized and that cooperatively may appear in protein conformations of very low stability. This suggests that protein folding intermediates, can, in principle, be cooperatively stabilized.