跳到主要內容

臺灣博碩士論文加值系統

(3.238.204.167) 您好!臺灣時間:2022/08/13 11:28
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:林祈宏
研究生(外文):Lin Chi-Hung
論文名稱:稻米脂質運輸蛋白質之摺疊路徑研究
論文名稱(外文):Folding Pathway Studies of Rice Non-specific Lipid Transfer Proteins Elucidated by Disulfide Bonds
指導教授:呂平江
指導教授(外文):Lyu Ping-Chiang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:71
中文關鍵詞:脂質運輸蛋白蛋白質摺疊雙硫鍵
外文關鍵詞:lipid transfer proteinprotein foldingdisulfide bond
相關次數:
  • 被引用被引用:0
  • 點閱點閱:115
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
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

1. Anfinsen CB: Principles that govern the folding of protein chains. Science 1973, 181:223-230.
2. Dill KA, Chan HS: From Levinthal to pathways to funnels. Nat Struct Biol 1997, 4:10-19.
3. Fersht A: Structure and Mechanism in Protein Science; 1998.
4. Dill KA: Theory for the folding and stability of globular proteins. Biochemistry 1985, 24:1501-1509.
5. Kim PS, Baldwin RL: Intermediates in the folding reactions of small proteins. Annu Rev Biochem 1990, 59:631-660.
6. Karplus M, Weaver DL: Protein folding dynamics: the diffusion-collision model and experimental data. Protein Sci 1994, 3:650-668.
7. Jackson SE, Fersht AR: Folding of chymotrypsin inhibitor 2. 1. Evidence for a two-state transition. Biochemistry 1991, 30:10428-10435.
8. Jackson SE, Fersht AR: Folding of chymotrypsin inhibitor 2. 2. Influence of proline isomerization on the folding kinetics and thermodynamic characterization of the transition state of folding. Biochemistry 1991, 30:10436-10443.
9. Dill KA, Bromberg S, Yue K, Fiebig KM, Yee DP, Thomas PD, Chan HS: Principles of protein folding--a perspective from simple exact models. Protein Sci 1995, 4:561-602.
10. Ptitsyn O: How molten is the molten globule? Nat Struct Biol 1996, 3:488-490.
11. Baldwin RL: Protein folding. Matching speed and stability. Nature 1994, 369:183-184.
12. Baldwin RL: The nature of protein folding pathways: the classical versus the new view. J Biomol NMR 1995, 5:103-109.
13. Balbach J, Forge V, van Nuland NA, Winder SL, Hore PJ, Dobson CM: Following protein folding in real time using NMR spectroscopy. Nat Struct Biol 1995, 2:865-870.
14. Radford SE, Dobson CM, Evans PA: The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature 1992, 358:302-307.
15. Miranker A, Robinson CV, Radford SE, Dobson CM: Investigation of protein folding by mass spectrometry. Faseb J 1996, 10:93-101.
16. Marti-Renom MA, Stuart AC, Fiser A, Sanchez R, Melo F, Sali A: Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct 2000, 29:291-325.
17. Sanchez R, Pieper U, Melo F, Eswar N, Marti-Renom MA, Madhusudhan MS, Mirkovic N, Sali A: Protein structure modeling for structural genomics. Nat Struct Biol 2000, 7 Suppl:986-990.
18. Creighton TE: Experimental studies of protein folding and unfolding. Prog Biophys Mol Biol 1978, 33:231-297.
19. Creighton TE: Intermediates in the refolding of reduced ribonuclease A. J Mol Biol 1979, 129:411-431.
20. Creighton TE: Disulfide bond formation in proteins. Methods Enzymol 1984, 107:305-329.
21. Weissman JS, Kim PS: Reexamination of the folding of BPTI: predominance of native intermediates. Science 1991, 253:1386-1393.
22. Chang JY: Controlling the speed of hirudin folding. Biochem J 1994, 300 ( Pt 3):643-650.
23. Chang JY: The disulfide folding pathway of tick anticoagulant peptide (TAP), a Kunitz-type inhibitor structurally homologous to BPTI. Biochemistry 1996, 35:11702-11709.
24. Chang JY, Canals F, Schindler P, Querol E, Aviles FX: The disulfide folding pathway of potato carboxypeptidase inhibitor. J Biol Chem 1994, 269:22087-22094.
25. Chang JY, Li L: The structure of denatured alpha-lactalbumin elucidated by the technique of disulfide scrambling: fractionation of conformational isomers of alpha-lactalbumin. J Biol Chem 2001, 276:9705-9712.
26. Chang JY: The folding pathway of alpha-lactalbumin elucidated by the technique of disulfide scrambling. Isolation of on-pathway and off-pathway intermediates. J Biol Chem 2002, 277:120-126.
27. Douliez JP, Pato C, Rabesona H, Molle D, Marion D: Disulfide bond assignment, lipid transfer activity and secondary structure of a 7-kDa plant lipid transfer protein, LTP2. Eur. J. Biochem. 2001, 268.
28. Jones BL, Marinac LA: Purification and partial characterization of a second cysteine proteinase inhibitor from ungerminated barley (Hordeum vulgare L.). J Agric Food Chem 2000, 48:257-264.
29. Douliez JP, Michon T, Elmorjani K, Marion D: Structure, biological and technological functions of lipid transfer proteins and indolines, the major lipid binding proteins from cereal kernels. J. Cereal Sci. 2000, 32:1-20.
30. Liu YJ, Samuel D, Lin CH, Lyu PC: Purification and Characterization of a novel 7-kDa non-specific Lipid Transfer Protein from Rice (Oryza sativa). Biochem.and Biophys.l Res. Commun 2002.
31. Samuel D, Liu Y-J, Cheng C-S, Lyu P-C: Solution Structure of Plant non-specific Lipid Transfer Protein-2 Purified from Rice (Oryza Satiya). J. Biol. Chem. 2002.
32. Baldwin RL, Rose GD: Is protein folding hierarchic? II. Folding intermediates and transition states. Trends Biochem Sci 1999, 24:77-83.
33. Baldwin RL, Rose GD: Is protein folding hierarchic? I. Local structure and peptide folding. Trends Biochem Sci 1999, 24:26-33.
34. Yu YG, Chung CH, Fowler A, Suh SW: Amino acid sequence of a probable amylase/protease inhibitor from rice seeds. Arch Biochem Biophys 1988, 265:466-475.
35. Wu J, Watson JT: A novel methodology for assignment of disulfide bond pairings in proteins. Protein Sci 1997, 6:391-398.
36. Qi J, Wu J, Somkuti GA, Watson JT: Determination of the disulfide structure of sillucin, a highly knotted, cysteine-rich peptide, by cyanylation/cleavage mass mapping. Biochemistry 2001, 40:4531-4538.
37. Yen TY, Joshi RK, Yan H, Seto NO, Palcic MM, Macher BA: Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry. J Mass Spectrom 2000, 35:990-1002.
38. Yen TY, Yan H, Macher BA: Characterizing closely spaced, complex disulfide bond patterns in peptides and proteins by liquid chromatography/electrospray ionization tandem mass spectrometry. J Mass Spectrom 2002, 37:15-30.
39. Ferran DS, Sobel M, Harris RB: Design and synthesis of a helix heparin-binding peptide. Biochemistry 1992, 31:5010-5016.
40. Chang JY, Li L, Bulychev A: The underlying mechanism for the diversity of disulfide folding pathways. J Biol Chem 2000, 275:8287-8289.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top