The disulfide-coupled folding pathway of apamin as derived from diselenide-quenched analogs and intermediates

The sequence of apamin, an 18 residue bee venom toxin, encloses all the inf ormation required for the correct disulfide-coupled folding into the cystin e-stabilized alpha-helical motif. Three apamin analogs, each containing a p air of selenocysteine residues replacing the related cysteines, were synthe sized to mimic the three possible apamin isomers with two crossed, parallel , or consecutive disulfides, respectively. Refolding experiments clearly re vealed that. the redox potential of selenocysteine prevails over the sequen ce encoded structural information for proper folding of apamin. Thus, selen ocysteine can be used as a new device to generate productive and nonproduct ive folding intermediates of peptides and proteins. In fact, disulfides are selectively reduced in presence of the diselenide and the conformational f eatures derived from these intermediates as well as from the three-dimensio nal (3D) structures of the selenocysteine-containing analogs with their non natural networks of diselenide/disulfide bridges allowed to gain further in sight into the subtle driving forces for the correct folding of apamin that mainly derive from local conformational preferences.