Structural and functional role of the disulfide bridges in the hydrophobinSC3

Hydrophobins function in fungal development by self-assembly at hydrophobic -hydrophilic interfaces such as the interface between the fungal cell wall and the air or a hydrophobic solid. These proteins contain eight conserved cysteine residues that form four disulfide bonds. To study the effect of th e disulfide bridges on the self-assembly, the disulfides of the SC3 hydroph obin were reduced with 1,4-dithiothreitol, The free thiols were then blocke d with either iodoacetic acid (IAA) or iodoacetamide (WM), introducing eigh t or zero negative charges, respectively. Circular dichroism and infrared s pectroscopy showed that after opening of the disulfide bridges SC3 is initi ally unfolded. IAA-SC3 did not self-assemble at the air-water interface upo n shaking an aqueous solution. Remarkably, after drying down IAA-SC3 or aft er exposing it to Teflon, it refolded into a structure similar to that obse rved for native SC3 at these interfaces. Iodoacetamide-SC3 on the other han d, which does not contain extra charges, spontaneously refolded in water in the amyloid-like beta-sheet conformation, characteristic for SC3 assembled at the water-air interface. From this we conclude that the disulfide bridg es of SC3 are not directly involved in self-assembly but keep hydrophobin m onomers soluble in the fungal cell or its aqueous environment, preventing p remature self-assembly.