綠豆中非專一性脂質運輸蛋白質及植物防禦素之結構,功能與蛋白質工程之研究

Structure, Function and Protein Engineering of Mung Bean Plant Nonspecific Lipid Transfer Protein and Plant Defensin

林谷峰(Ku-Feng Lin)

指導教授 ： 呂平江

國立清華大學/生命科學院/生命科學系/博士(2006年)

摘要

無資料

關鍵字

植物非專一性脂質運輸蛋白 ； 蛋白質穩定度 ； 疏水性中空腔 ； 同源結構模擬法 ； 蛋白質嵌合 ； 植物防禦素 ； 抗蟲功能 ； 核磁共振 ； alpha-澱粉酶抑制能力 ； 蛋白質結構

並列摘要

It is well recognized that environmental stresses, pathogenic infections, and insect damage significantly result in agricultural losses. Plants have developed defense mechanisms such as evolving stress-resistant system, developing barriers to entry and producing the plant poisons against these damage factors. Extensively studies have indicated that the modifications of the proteins related to the plant defense by protein engineering improve protein ability of defense functions. Thus, understanding the mechanisms as well as the biochemical properties of the proteins participate in regulating plant defense mechanisms is important.
Plant non-specific lipid transfer proteins (nsLTPs) are thermal stable proteins that are capable of transferring lipid molecules between membranes in vitro. This family of proteins is proposed to be involved in defense, pollination and germination; the in vivo biological function remains, however, elusive. Here we report the purification and sequencing of an nsLTP1 (Vr-nsLTP1) from sprouts of Vigna radiate (Vr). By determining and analyzing the solution structure of Vr-nsLTP1, some notable differences in the C-terminal tails and internal hydrophobic cavities were found between Vr-nsLTP1 and Os-nsLTP1 (purified from oryza sativa). Circular dichroism and fluorescence spectroscopy were used to compare the thermodynamics and lipid transfer properties of Vr-nsLTP1 with that of Os-nsLTP1. Docking of a lipid molecule into the solution structure of Vr-nsLTP1 reveals similar binding cavities and hydrophobic interactions as in Os-nsLTP1 consistent with their comparable lipid transfer properties measured experimentally.
Plant defensins are important constituents of the innate immune systems of plants. They are multi-function proteins reported to inhibit protease, protein synthesis in cell-free system or act against microbe and fungi. Here, the structure of a novel plant defensin isolated from the seeds of the Vigna radiata has been determined by 1H nuclear magnetic resonance spectroscopy. Interestingly, this protein exhibits neither insecticidal activity nor alpha-amylase inhibitory activity in spite of showing a similar global fold to that of VrD1, an insecticidal plant defensin that has been suggested to function by inhibiting insect alpha-amylase. Structural analyses and surface charge comparisons of VrD1 and VrD2 revealed that the charged residues of L3 correlate with the observed difference in inhibitory activities of these proteins. A VrD2 chimera produced by substitution of the proposed functional loop of VrD1 onto the structurally equivalent loop of VrD2 supported this hypothesis. These results clarify the mode of alpha-amylase inhibition of plant defensins and also represent a possible approach for engineering novel alpha-amylase inhibitors. The application of protein engineering to this protein family may provide an efficient method for protection against crop losses.

並列關鍵字

Plant non-specific lipid transfer proteins ； Protein stability ； Hydrophobic cavity ； Homology modelling ； Protein ducking ； Plant defensin ； Insecticidal activity ； Nuclear magnetic resonance ； Alpha-amylase inhibitory activity ； Protein structure

參考文獻

127.Chen, k. C. L., C. Y.; Chung, M. C.; Kuang, C. C.; Sung, H. Y.; Tsou, S. C.; Kuo, C. G.; Chen, C. S. (2002) Cloning and characterization of a cDNA encoding an antimicrobial protein from mung bean seeds. Bot. Bull. Acad. Sin. 43, 251-259.

1.Horvath, B. M., Bachem, C. W., Trindade, L. M., Oortwijn, M. E., and Visser, R. G. (2002) Expression analysis of a family of nsLTP genes tissue specifically expressed throughout the plant and during potato tuber life cycle Plant Physiol 129, 1494-506.

2.Douliez, J. P., Michon, T., Elmorjani, K., and Marion, D. (2000) Structure, biological and technological functions of lipid transfer proteins and indolines, the major lipid binding proteins from cereal kernels J. Cereal Sci. 32, 1-20.

4.Yu, Y. G., Chung, C. H., Fowler, A., and Suh, S. W. (1988) Amino acid sequence of a probable amylase/protease inhibitor from rice seeds Arch. Biochem. Biophys. 265, 466-75.

6.Desormeaux, A., Blochet, J. E., Pezolet, M., and Marion, D. (1992) Amino acid sequence of a non-specific wheat phospholipid transfer protein and its conformation as revealed by infrared and Raman spectroscopy. Role of disulfide bridges and phospholipids in the stabilization of the alpha-helix structure Biochim Biophys Acta 1121, 137-52.