Abstract/Summary

The mechanism of action of the wheat defence proteins (puroindolines and purothionins) as antifungal agents was investigated by monitoring their interactions with the lipid systems mimicking the structures of the yeast cell membrane. The membrane models used were: monolayers at the air/liquid interface, flat bilayers at the solid/liquid interface and the folded bilayers in aqueous solutions (lipid vesicles). Each lipid model was interacted with defence proteins as single protein or mixed protein systems. The consequent changes in the structures of lipid membranes were then monitored by means of various biophysical techniques selected to probe different aspects of interfacial system. Surface pressure measurements on a Langmuir trough revealed the preference of wheat defence proteins towards membranes dominated by anionic, saturated phospholipids and, together with X-Ray Reflectometry (XRR) studies, revealed different interaction kinetics dependent on the phospholipid chain saturation. The monolayer studies also suggested that there existed antifungal synergy between the two groups of proteins: puroindolines and purothionins. Protein penetration to the flat lipid bilayers was analysed by Attenuated Total ReflectanceFourier-Transform Infrared Spectroscopy (ATR-FTIR) and Neutron Reflectometry (NR). The bilayer studies enabled to assess the lipid flipping between the leaflets and confirmed the protein’s strong adsorptive properties towards the anionic, saturated phospholipids. Protein interactions with the lipid vesicles were assessed by means of Differential Scanning Calorimetry (DSC) and revealed the dependence of the protein’s membrane-solubilising properties on the lipid net charge. The project also involved the development of the technique for the controlled flat lipid bilayer deposition on the ATR-FTIR solid support. For this purpose, a new Langmuir trough was designed and equipped with a dipping arm, allowing the covering of the support with the two-lipid-molecule-thick bilayers. The findings from the research provided an insight into the antifungal mode of action of wheat defence protein.