Mutagenesis Study on the Structure Conformation and Binding Mechanism of Rice Nonspecific Lipid Transfer Protein 2

• Main Authors: Ming-Nan Chen, 陳明男
• Other Authors: Ping-Chiang Lyu
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/39365438099956700683

碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 93 === Plant nonspecific lipid transfer protein 2 (nsLTP2) is a soluble protein which is characterized by its ability to transfer phospholipids between membranes in vitro. Eight highly conserved cysteine residues can form four disulfide bonds to stabilize the folded conformation of nsLTP2. The three dimensional structure of nsLTP2 also reveals an inner hydrophobic cavity which may serves as a potential binding site for the hydrophobic or amphiphilic ligands such as fatty acids, phospholipids, and sterols. Although the structures of nsLTP2 have been reported before, the detailed properties/mechanisms of the ligand binding and lipid transfer activity are still unclear. In this study, a series of nsLTP2 mutants were made by site-directed mutagenesis to elucidate the importance of various hydrophobic residues on protein structure, stability, ligand binding and lipid transfer activity. Our results demonstrate that three individual mutations (L8A, F36A, and V49A) almost destroy the native tertiary structure of nsLTP2 and therefore lose its ability in ligand binding and lipid transfer. Besides, the aromatic side chains are usually critical in the hydrophobic packing. Two mutations in the tyrosine residues, Y45A and Y48A, were generated to exam their roles in nsLTP2. There is no obvious structural change in these two singly mutant proteins comparing with wild type protein. However, the ligand binding and lipid transfer activity are apparently decreased. This is suggested that these two residues play a significant role in biological activity. And especially point out that the F39A mutant not only increases the helical content, but also significantly enhances the phospholipid binding and lipid transfer activity even though the ergosterol binding ability was decreased. A change of Phe39 to Ala alters the conformation of nsLTP2, which may affect the binding and transfer mechanism. These mutants have provided significant information about the structure-to-function relationships of nsLTP2.