Structural and functional role of the disulfide bridges in the hydrophobin SC3.
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 the disulfide bridges on the self-assembly, the disulfides of the SC3 hydrophobin were reduced with 1,4-dithiothreitol. The free thiols were then blocked with either iodoacetic acid (IAA) or iodoacetamide (IAM), introducing eight or zero negative charges, respectively. Circular dichroism and infrared spectroscopy showed that after opening of the disulfide bridges SC3 is initially unfolded. IAA-SC3 did not self-assemble at the air-water interface upon shaking an aqueous solution. Remarkably, after drying down IAA-SC3 or after exposing it to Teflon, it refolded into a structure similar to that observed for native SC3 at these interfaces. Iodoacetamide-SC3 on the other hand, 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 bridges of SC3 are not directly involved in self-assembly but keep hydrophobin monomers soluble in the fungal cell or its aqueous environment, preventing premature self-assembly.
de Vocht
M L
ML
Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, NL-9747 AG Groningen, the Netherlands.
Reviakine
I
I
Wösten
H A
HA
Brisson
A
A
Wessels
J G
JG
Robillard
G T
GT
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D011138:Polytetrafluoroethylene / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary
D004220:Disulfides; D005656:Fungal Proteins; D011138:Polytetrafluoroethylene