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 Cla dosporiurn fulvum, is a 28-residue P-sheet peptide containing three disulfi de bridges. The folding of this peptide to its native conformation was exam ined in the presence of oxidized (GSSG) and reduced (GSH) glutathione at co ncentrations resembling those present in the endoplasmic reticulum. The con centrations 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 characteris tics, indicating that they have the same 3D structure and identical disulfi de bridges. Thus, AVR9 can be folded correctly in vitro. This folding can b e described by disulfide bridge formation leading to scrambled three disulf ide species, followed by disulfide reshuffling to acquire the native struct ure. The presence of urea significantly affected the folding of AVR9, indic ating that noncovalent interactions play a role in directing correct foldin g. Protein disulfide isomerase increased the folding rate at least 15-fold, but had no effect on the yield. The folding procedure has also been applie d successfully to two mutant AVR9 peptides, (K23A)AVR9 and biotinylated AVR 9. We conclude that the 28-residue sequence, without the preprosequence las present in vivo), contains sufficient information to direct correct foldin g and disulfide bridge formation in vitro.