NLP proteins (Necrosis and ethylene inducing peptide 1 (Nep1)-like proteins) are important microbial virulence factors that enable plant pathogens to damage cells and invade tissues. They are secreted by numerous phytopathogenic bacteria, fungi, and oomycetes. Specific binding of NLP proteins to the sphingolipid glycosyl inositol phosphorylceramide (GIPC) in the plant membrane causes the formation of small unstable membrane pores due to protein aggregation, leading to cell death and tissue necrosis. In this master's thesis, we prepared and purified mutants of the NLP$_{Pya}$ protein from the oomycete Pythium aphanidermatum to investigate the impact of the C-terminal α-helix and a possible aromatic switch on the function of NLP proteins. Analysis of thermal stability by differential scanning fluorimetry and circular dichroism showed a slight change in the folding of the mutants compared to the wild type. The introduction of mutations does not affect the overall folding of the protein, as the mutants have a similar melting temperature to the wild type. Binding analysis with a sedimentation assay showed a lower binding capacity of the mutants to multilamellar vesicles composed of palmitoyl-oleoyl phosphatidylcholine, glycosyl inositol phosphorylceramide (GIPC), and plant sterols in a 1:6:3 molar ratio, which mimic plant cell membranes. The mutants also nonspecifically bound to control vesicles. An infiltration assay in tobacco leaves and measurement of ion leakage from cells using conductometry demonstrated lower cytotoxic activity of the mutants compared to the wild-type protein. Further investigation of the molecular mechanism of NLP protein function and their structure is important due to their potential role in the development of their inhibitors and herbicides.
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