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 compac t and monomeric at pH 7.0, with spectroscopic properties of a molten g lobule and a low conformational stability. To study if Fld1-149 is coo peratively stabilized, we have measured the equilibrium urea unfolding by fluorescence, circular dichroism, and size-exclusion chromatograph y. The three techniques produced coincident unfolding curves. Furtherm ore, the thermal unfolding seems also to be cooperative as the same te mperature of half-denaturation is obtained using fluorescence and circ ular dichroism. Fld1-149 displays cold denaturation. The equilibrium p roperties of Fld1-149 demonstrate that molten globules lacking well-de fined tertiary interactions can still be cooperatively stabilized and that cooperativity may appear in protein conformations of very low sta bility. This suggests that protein folding intermediates can, in princ iple, be cooperatively stabilized.