臺灣博碩士論文加值系統

稻米非專一性脂質運輸蛋白在許多的植物中都被發現。脂質運輸蛋白可以和許多種的脂質結合且將這些脂質運輸來往於兩個膜之間。脂質運輸蛋白可分為兩類：一是LTP1，其分子量大約在9K左右；另一是LTP2，具有7K的分子量。LTP1的立體結構是由四條螺旋和一個C端的尾部所結合而成的。它們是具有一個疏水性的空洞可以結合脂質。另外LTP都具有4條雙硫鍵來維持其疏水性空洞。因為LTP具有疏水性空洞、四個雙硫鍵和四條的螺旋結構，LTP因此是很有趣的蛋白質來進行蛋白質折疊路徑的研究。
我們利用了oxidative folding, reductive unfolding 和scramble technique來做折疊路徑的研究。我們在不同的反應條件下進行摺疊和開散的實驗，之後利用HPLC分離摺疊及開散反應的中間物，並利用enzyme digestion及mass spectrometry來分析這些中間物的雙硫鍵配對。我們可以發現LTP1的折疊及開散過程較LTP2來得複雜許多；LTP2的折疊反應路徑是屬於簡單的two-state mechanism。同時我們除了利用了scramble technique來研究LTP1的折摺反應之外，我們也分離了四個變性的LTP結構。

Plant non-specific lipid transfer proteins (ns-LTPs) are isolated from various plants, including rice, barley and maize. Ns-LTPs can bind to variety of lipids and transfer phospholipids between membranes. Three dimensional structures of ns-LTP1s are composed of four helical segments and a C-terminal tail with four disulfide bonds connecting these secondary components together. The four helix segments forms a hydrophobic cavity inside the protein which is believed involved in the lipid binding activity. The property that ns-LTPs possess a hydrophobic cavity makes itself as an interesting model for protein folding study.
In this study, we investigated the folding pathway of rice ns-LTPs in terms of disulfide formation and breakage. We conducted oxidative folding, reductive unfolding and disulfide bond scrambling experiments to elucidate the folding pathway of rice ns-LTPs. We used HPLC to collect folding and unfolding intermediates and analyzed by the disulfide bond pattern determination and circular dichroism spectrometry. The disulfide bond patterns were determined by trypsin digestion, mass spectrometry, Edman sequencing, CN-induced cleavage and tandem mass. In our studies, folding pathway of rice LTP1 showed a higher complexity than rice ns-LTP2. We identified four denatured structures of scramble rice ns-LTP1 in scramble unfolding experiments and two reductive unfolding intermediates along the rice ns-LTP1 unfolding. Rice ns-LTP2 showed a simple two-state mechanism in reductive unfolding, scramble folding and unfolding.

Chapter One Introduction 2
1. Protein folding problem 4
2. Methods for protein folding 7
3. Rice non-specific lipid transfer protein 8
4. Theme of this study 12
Chapter Two Materials and Methods 13
2.1 Purification of ns-LTP1 13
2.2 Purification of rice ns-LTP2 13
2.3 Reduction of native ns-LTP1 14
2.4 Oxidative folding of ns-LTP1 14
2.5 Reductive unfolding of ns-LTPs. 14
2.6 Scramble unfolding of ns-LTPs 15
2.7 Scramble folding of ns-LTPs 15
2.8 Disulfide bond pattern determination 16
2.9 Circular Dichroism 18
Chapter Three Results 19
1. Oxidative folding of rice ns-LTP1 19
2. Reductive unfolding of ns-LTP1 23
3. Scramble unfolding of ns-LTP1 29
4. Scramble folding of ns-LTP1 43
5. Reductive unfolding, scramble unfolding and folding of ns-LTP2 48
Chapter Four Discussion 58
1. Oxidative folding of rice ns-LTP1 58
2. Reductive unfolding of rice ns-LTP1 59
3. Scramble unfolding of rice ns-LTP1 61
4. Scramble folding of rice ns-LTP1 64
5. Folding pathway studies of rice ns-LTP2 65
6. Summary 68
Reference 70

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