植物非專一性脂質運輸蛋白質第一型已被證實其在活體外 (in vitro) 具有在膜間運輸脂質的功能。然而，關於其運送脂質的詳細機制卻尚未清楚。本實驗室先前已確認並分析綠豆種子中所純化出的非專一性脂質運輸蛋白質第一型，研究發現，在蛋白質中一些疏水性胺基酸可能參與和脂質的相互作用。因此，在這篇研究中，我們建構綠豆非專一性脂質運輸蛋白質的表現載體，並利用定點突變的技術來闡明不同的胺基酸對於蛋白質生物化學和生物物理方面的影響。我們選擇並突變圍繞在與脂質作用位點附近的疏水性胺基酸 (Leu10、Ile14、Val31、Ile34、Asp43、Arg44、Leu51、Leu69、Val75、Tyr79和Ile81) 和半胱胺酸 (Cys48、Cys48/Cys87、 Cys3/Cys50、Cys13/Cys27和Cys28/Cys73) 。我們對這十六個突變蛋白質進行二級結構、穩定性和脂質運輸能力的分析。結果證明這些疏水性胺基酸對於蛋白質的穩定性和其運輸脂質的能力是很重要的。同時我們也建構破壞雙硫鍵的突變蛋白質(C48S和C48A/C87A)來研究結構彈性對於綠豆非專一性脂質運輸蛋白質第一型的影響。結果顯示，這兩個突變的蛋白質具有較高的運輸脂質能力，我們推測，可能是由於這兩個失去雙硫鍵突變蛋白質的結構較鬆散，進而增加了與脂質作用的機會。藉由這一系列定點突變蛋白質的結果發現：疏水性作用力和結構的可塑性在非專一性脂質運輸蛋白質的生物功能方面可能扮演著重要的角色。

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.

Contents 1
Contents of Tables and Figures 2
Abstract 3
中文摘要 4
Abbreviations 5
Chapter 1. Introduction 6
1.1 Plant nonspecific lipid transfer proteins (nsLTPs) 6
1.2 Structural characteristics of nsLTPs 7
1.3 Multiple biological roles 8
1.4 Previous study in our laboratory 9
1.5 The theme of this thesis 10
Chapter 2. Materials and Methods 12
2.1 Materials 12
2.2 Cloning of MB nsLTP1 gene into recipient vector 12
2.3 Construction of MB nsLTP1 mutants 13
2.4 Expression of nsLTP1 and mutants 13
2.5 Purification of recombinant nsLTP1 and mutants 14
2.6 Mass Spectrometry 15
2.7 Circular Dichroism Experiments 15
2.8 Lipid Transfer Assay 16
2.9 S-Carboxymethylation of Cysteine 17
2.10 ANS Assay 17
Chapter 3. Results and discussion 18
3.1 Construction and Expression of recombinant MB nsLTP1 18
3.2 Protein purifications 19
3.3 Comparison between purified and recombinant MB nsLTP1 20
3.4 Characterization of mutants of the MB nsLTP1s 22
Mutants involved in ligand binding 22
Cysteine residue 48 mutation 26
Four disulfide-deficient mutants 27
Chapter 4. Conclusion 30
References 32

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