Clarification of the structure and molecular mechanism of action of Nep-1 like proteins (NLP) is imperative, due to their potential role of as herbicides and in the development of NLP protein inhibitors. In order to test the function of the C-terminal α helix in the binding capacity of model NLP protein from oomycete Pythium aphanidermatum (NLPPya) we prepared mutants; Phe211Ala, Phe212Ala, Phe211,212Ala, Asn194Ala and Asn196Ala. Analysis of thermal stability with differential scanning calorimetry and circular dichroism spectroscopy showed melting temperatures in mutants, which indicates their reduced thermal stability. In addition, the methods showed greater exposure of hydrophobic surfaces in the native state, differences in the native spectrum, as well as differences in the number and intensity of spectrum turns compared to NLPPya. The results therefore reveal changes in folding of mutants. Binding analysis by sedimentation tests showed that mutants have a higher binding ability to vesicles composed of palmitoyl-oleoyl-phosphatidylcholine, glycosyl inositol phosphoceramides (GIPC) and plant sterol in a 3:4:3 molar ratio). However, the mutants bound control vesicles without target lipids (GIPC), demonstrating that the binding was nonspecific. Although specific binding to GIPC by mutants and NLPPya was comparable, toxicity tests showed higher toxicity in NLPPya. Differences in aminoacid sequence and consequential differences in folding therefore lower membrane damage, caused by the mutants.
|
