Abstract
Plant nonspecific lipid transfer proteins (nsLTPs) are well known for their ability to bind and transfer lipids in vitro. However, the detailed mechanism of the lipid transferring is still unclear. In our previous study, mung bean (MB) nsLTP1 purified from seed has been identified and analyzed. We found that certain hydrophobic residues are probably essential for lipid interaction. Therefore, in this study, we constructed the expression vector carrying MB nsLTP1 gene and employed site-directed mutagenesis approach to investigate the biochemical and biophysical properties of the protein. The residues involved in the lipid binding (Leu10, Ile14, Val31, Ile34, Asp43, Arg44, Leu51, Leu69, Val75, Tyr79 and Ile81) and cysteine residues (Cys48, Cys48/Cys87, Cys3/Cys50, Cys13/Cys27 and Cys28/Cys73) were chosen to be mutated. Biophysical properties including secondary structure, stability and lipid transfer activity of these sixteen mutants were analyzed. Our results demonstrated that hydrophobic residues near lipid binding site are critical for protein stability and lipid transfer activity. We also constructed disulfide-deficient mutants (C48S and C48A/C87A) to study the flexibility of MB nsLTP1. The results showed that these two mutants, C48S and C48A/C87A, have higher lipid transfer activity, implying these mutants probably loose the protein structure to enhance the lipid interactions. All together, these observations revealed that the hydrophobic interaction and structural plasticity may play important roles of biological function in nsLTP1.