Folding and conformational analysis of AVR9 peptide elicitors of the fungal tomato pathogen Cladosporium fulvum.
The race-specific elicitor AVR9, produced by the phytopathogenic fungus Cladosporium fulvum, is a 28-residue beta-sheet peptide containing three disulfide bridges. The folding of this peptide to its native conformation was examined in the presence of oxidized (GSSG) and reduced (GSH) glutathione at concentrations resembling those present in the endoplasmic reticulum. The concentrations of GSH and GSSG, and the applied temperature strongly affected the folding efficiency. The effect of temperature appeared reversible. The conditions for in vitro folding were optimized and a maximum yield of 60-70% of correctly folded peptide was obtained. In vitro folded AVR9 is equally as active as native fungal AVR9. They both display similar NMR characteristics, indicating that they have the same 3D structure and identical disulfide bridges. Thus, AVR9 can be folded correctly in vitro. This folding can be described by disulfide bridge formation leading to scrambled three-disulfide species, followed by disulfide reshuffling to acquire the native structure. The presence of urea significantly affected the folding of AVR9, indicating that noncovalent interactions play a role in directing correct folding. Protein disulfide isomerase increased the folding rate at least 15-fold, but had no effect on the yield. The folding procedure has also been applied successfully to two mutant AVR9 peptides, (K23A)AVR9 and biotinylated AVR9. We conclude that the 28-residue sequence, without the preprosequence (as present in vivo), contains sufficient information to direct correct folding and disulfide bridge formation in vitro.
van den Hooven
H W
HW
Department of Biomolecular Sciences, Laboratory of Biochemistry, Wageningen Agricultural University, The Netherlands. h.hooven@organon.oss.abzonobel.nl
Appelman
A W
AW
Zey
T
T
de Wit
P J
PJ
Vervoort
J
J
D000595:Amino Acid Sequence; D002384:Catalysis; D002963:Cladosporium / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry; D018551:Solanum lycopersicum / Q000382:microbiology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D013057:Spectrum Analysis; D013696:Temperature
D004220:Disulfides; D005656:Fungal Proteins; C073605:AVR9 protein, Cladosporium fulvum; D005978:Glutathione