The building of activity in flavodoxin: interactions that drive the denatured state into the redox functional native conformation
Flavodoxin from Anabæna constitutes an interesting model to study protein folding and stability and to analyze the structural factors that govern the acquisition of its functional native conformation. The cofactor FMN can be easely separated from the holoprotein to yield the apo form. The apoflavodoxin formed in this way is less stable than the holo form, although it is well folded and shows a tridimensional structure very close to the holoflavodoxin, being the main difference the conformation of the two aromatic residues that sandwich FMN in the holo form. Unfolded apoflavodoxin can reach the native conformation in a few milliseconds. Its folding reaction has been studied by stopped-flow kinetics and site-directed mutagenesis. Although the urea unfolding equilibrium is two-state, a transient kinetic-trapped intermediate accumulates during the folding reaction. This well folded native apoflavodoxin binds the cofactor FMN to form the functional native conformation of holoflavodoxin. We have analyzed different side-chain and protein-ligand interactions that convert the unfolded polypeptide into the fully functional redox active native protein, by using site-directed mutagenesis, stopped flow techniques, equilibrium constant determinations and redox potential and redox kinetic measurements.