臺灣博碩士論文加值系統

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

1. Kader, J. C. (1996) Lipid transfer proteins in plant. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 627-654
2. Tai, S. P., and Kaplan, S. (1985) Phospholipid transfer proteins in microorganisms. Chem Phys Lipids 38, 41-50
3. Crain RC, Z. D. (1980) Two nonspecific phospholipids exchange proteins from beef liver. 1. Purification and characterization. Biochemistry 19, 1433-1439
4. Ostergaard, J., Vergnolle, C., Schoentgen, F. & Kader, J.C. (1993) Acyl-binding /lipid-transfer proteins from rape seedlings, a novel category of proteins interacting with lipids. Biochem. Biophys. Acta 1170, 109-117
5. Douady, D., Grosbois, M., Guerbette, F., and Kader, J.C. (1985) Purification of phospholipid transfer protein from maize seeds using a two-step chromatographic procedure. Physiol. Veg. 23, 373-380
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, 137-152
7. Douliez, J. P., Jegou, S., Pato, C., Molle, D., Tran, V., and Marion, D. (2001) Binding of two mono-acylated lipid monomers by the barley lipid transfer protein, LTP1, as viewed by fluorescence, isothermal titration calorimetry and molecular modelling. Eur J Biochem 268, 384-388
8. 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, 466-475
9. Lin, K. F., Liu, Y. N., Hsu, S. T., Samuel, D., Cheng, C. S., Bonvin, A. M., and Lyu, P. C. (2005) Characterization and structural analyses of nonspecific lipid transfer protein 1 from mung bean. Biochemistry 44, 5703-5712
10. Vignols, F., Lund, G., Pammi, S., Tremousaygue, D., Grellet, F., Kader, J. C., Puigdomenech, P., and Delseny, M. (1994) Characterization of a rice gene coding for a lipid transfer protein. Gene 142, 265-270
11. Gomar, J., Sodano, P., Ptak, M., and Vovelle, F. (1997) Homology modelling of an antimicrobial protein, Ace-AMP1, from lipid transfer protein structures. Fold Des 2, 183-192
12. Kader, J. C. (1985) Lipid binding proteins in plants. Chem Physics Lipids 38, 51-62
13. Kader, J. C. (1997) Lipid transfer proteins: a puzzling family of plant proteins. Trends Plant Sci. 2, 66-70
14. Rickers, J., Spener, F. & Kader, J.C. (1985) A phospholipid transfer protein that binds long-chain fatty acids. FEBS Lett. 180, 29-32
15. Gadella, T. W., Jr., and Wirtz, K. W. (1991) The low-affinity lipid binding site of the non-specific lipid transfer protein. Implications for its mode of action. Biochim Biophys Acta 1070, 237-245
16. Zachowski, A., Guerbette, F., Grosbois, M., Jolliot-Croquin, A., and Kader, J. C. (1998) Characterisation of acyl binding by a plant lipid-transfer protein. Eur J Biochem 257, 443-448
17. Guerbette, F., Grosbois, M., Jolliot-Croquin, A., Kader, J. C., and Zachowski, A. (1999) Comparison of lipid binding and transfer properties of two lipid transfer proteins from plants. Biochemistry 38, 14131-14137
18. Lullien-Pellerin, V., Devaux, C., Ihorai, T., Marion, D., Pahin, V., Joudrier, P., and Gautier, M. F. (1999) Production in Escherichia coli and site-directed mutagenesis of a 9-kDa nonspecific lipid transfer protein from wheat. Eur J Biochem 260, 861-868
19. Takishima, K., Watanabe, S., Yamada, M., Suga, T., and Mamiya, G. (1988) Amino acid sequences of two nonspecific lipid-transfer proteins from germinated castor bean. Eur J Biochem 177, 241-249
20. Gomar, J., Petit, M. C., Sodano, P., Sy, D., Marion, D., Kader, J. C., Vovelle, F., and Ptak, M. (1996) Solution structure and lipid binding of a nonspecific lipid transfer protein extracted from maize seeds. Protein Sci 5, 565-577
21. Tassin, S., Broekaert, W. F., Marion, D., Acland, D. P., Ptak, M., Vovelle, F., and Sodano, P. (1998) Solution structure of Ace-AMP1, a potent antimicrobial protein extracted from onion seeds. Structural analogies with plant nonspecific lipid transfer proteins. Biochemistry 37, 3623-3637
22. Samuel, D., Liu, Y. J., Cheng, C. S., and Lyu, P. C. (2002) Solution structure of plant nonspecific lipid transfer protein-2 from rice (Oryza sativa). J Biol Chem 277, 35267-35273
23. Pyee, J., Yu, H., and Kolattukudy, P. E. (1994) Identification of a lipid transfer protein as the major protein in the surface wax of broccoli (Brassica oleracea) leaves. Arch Biochem Biophys 311, 460-468
24. Park, S. Y., and Lord, E. M. (2003) Expression studies of SCA in lily and confirmation of its role in pollen tube adhesion. Plant Mol Biol 51, 183-189
25. Thoma, S., Hecht, U., Kippers, A., Botella, J., De Vries, S., and Somerville, C. (1994) Tissue-specific expression of a gene encoding a cell wall-localized lipid transfer protein from Arabidopsis. Plant Physiol 105, 35-45
26. Molina, A., and Garcia-Olmedo, F. (1997) Enhanced tolerance to bacterial pathogens caused by the transgenic expression of barley lipid transfer protein LTP2. Plant J 12, 669-675
27. Maldonado, A. M., Doerner, P., Dixon, R. A., Lamb, C. J., and Cameron, R. K. (2002) A putative lipid transfer protein involved in systemic resistance signalling in Arabidopsis. Nature 419, 399-403
28. Wang, S. Y., Wu, J. H., Ng, T. B., Ye, X. Y., and Rao, P. F. (2004) A non-specific lipid transfer protein with antifungal and antibacterial activities from the mung bean. Peptides 25, 1235-1242
29. Trevino, M. B., and MA, O. C. (1998) Three drought-responsive members of the nonspecific lipid-transfer protein gene family in Lycopersicon pennellii show different developmental patterns of expression. Plant Physiol 116, 1461-1468
30. Lindorff-Larsen, K., and Winther, J. R. (2001) Surprisingly high stability of barley lipid transfer protein, LTP1, towards denaturant, heat and proteases. FEBS Lett 488, 145-148
31. Sancho, A. I., Rigby, N. M., Zuidmeer, L., Asero, R., Mistrello, G., Amato, S., Gonzalez-Mancebo, E., Fernandez-Rivas, M., van Ree, R., and Mills, E. N. (2005) The effect of thermal processing on the IgE reactivity of the non-specific lipid transfer protein from apple, Mal d 3. Allergy 60, 1262-1268
32. Sorensen, S. B., Bech, L.M., Muldbjerg, M., Beenfeldt, T. & Breddam, K. (1993) Barley lipid transfer protein 1 is involved in beer foam formation. MBAA Technical Q. 30, 136-145
33. Hoffmann-Sommergruber, K. (2000) Plant allergens and pathogenesis-related proteins. What do they have in common? Int Arch Allergy Immunol 122, 155-166
34. Nieuwland, J., Feron, R., Huisman, B. A., Fasolino, A., Hilbers, C. W., Derksen, J., and Mariani, C. (2005) Lipid transfer proteins enhance cell wall extension in tobacco. Plant Cell 17, 2009-2019
35. Lerche, M. H., Kragelund, B. B., Bech, L. M., and Poulsen, F. M. (1997) Barley lipid-transfer protein complexed with palmitoyl CoA: the structure reveals a hydrophobic binding site that can expand to fit both large and small lipid-like ligands. Structure 5, 291-306
36. Han, G. W., Lee, J. Y., Song, H. K., Chang, C., Min, K., Moon, J., Shin, D. H., Kopka, M. L., Sawaya, M. R., Yuan, H. S., Kim, T. D., Choe, J., Lim, D., Moon, H. J., and Suh, S. W. (2001) Structural basis of non-specific lipid binding in maize lipid-transfer protein complexes revealed by high-resolution X-ray crystallography. J Mol Biol 308, 263-278
37. Xiong, A. S., Yao, Q. H., Peng, R. H., Li, X., Fan, H. Q., Cheng, Z. M., and Li, Y. (2004) A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res 32, e98
38. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150, 76-85
39. Gans, P. J., Lyu, P. C., Manning, M. C., Woody, R. W., and Kallenbach, N. R. (1991) The helix-coil transition in heterogeneous peptides with specific side-chain interactions: theory and comparison with CD spectral data. Biopolymers 31, 1605-1614
40. Kamal, J. K., and Behere, D. V. (2002) Thermal and conformational stability of seed coat soybean peroxidase. Biochemistry 41, 9034-9042
41. Douliez, J. P., Pato, C., Rabesona, H., Molle, D., and Marion, D. (2001) Disulfide bond assignment, lipid transfer activity and secondary structure of a 7-kDa plant lipid transfer protein, LTP2. Eur J Biochem 268, 1400-1403
42. Gadella, T. W., Jr., and Wirtz, K. W. (1994) Phospholipid binding and transfer by the nonspecific lipid-transfer protein (sterol carrier protein 2). A kinetic model. Eur J Biochem 220, 1019-1028
43. Somerharju, P., Brockerhoff, H., and Wirtz, K. W. (1981) A new fluorimetric method to measure protein-catalyzed phospholipid transfer using 1-acyl-2-parinaroylphosphatidylcholine. Biochim Biophys Acta 649, 521-528
44. Elmorjani, K., Lurquin, V., Lelion, A., Rogniaux, H., and Marion, D. (2004) A bacterial expression system revisited for the recombinant production of cystine-rich plant lipid transfer proteins. Biochem Biophys Res Commun 316, 1202-1209
45. Wirtz, P. S. a. K. W. A. (1982) Semisynthesis and properties of a fluorescent phosphatidyl-inositol analogue containing a cis-parinaroyl moiety. Chemistry and Physics of Lipids 30, 81-91
46. Douliez, J.-P. M., T.; Elmorjani, K.; Marion, D. (2000) Structure, Biological and Technological Functions of Lipid Transfer Proteins and Indolines, the Major Lipid Binding Proteins from Cereal Kernels. Journal of Cereal Science 32, 1-20
47. van Paridon, P. A., Gadella, T. W., Jr., Somerharju, P. J., and Wirtz, K. W. (1988) Properties of the binding sites for the sn-1 and sn-2 acyl chains on the phosphatidylinositol transfer protein from bovine brain. Biochemistry 27, 6208-6214
48. Cheng, H. C., Cheng, P. T., Peng, P., Lyu, P. C., and Sun, Y. J. (2004) Lipid binding in rice nonspecific lipid transfer protein-1 complexes from Oryza sativa. Protein Sci 13, 2304-2315
49. Subirade, M., Salesse, C., Marion, D., and Pezolet, M. (1995) Interaction of a nonspecific wheat lipid transfer protein with phospholipid monolayers imaged by fluorescence microscopy and studied by infrared spectroscopy. Biophys J 69, 974-988
50. Shoichet, B. K., Baase, W. A., Kuroki, R., and Matthews, B. W. (1995) A relationship between protein stability and protein function. Proc Natl Acad Sci U S A 92, 452-456
51. Meiering, E. M., Serrano, L., and Fersht, A. R. (1992) Effect of active site residues in barnase on activity and stability. J Mol Biol 225, 585-589
52. Sy, D., Le Gravier, Y., Goodfellow, J., and Vovelle, F. (2003) Protein stability and plasticity of the hydrophobic cavity in wheat ns-LTP. J Biomol Struct Dyn 21, 15-29
53. Mima, J., Jung, G., Onizuka, T., Ueno, H., and Hayashi, R. (2002) Amphipathic property of free thiol group contributes to an increase in the catalytic efficiency of carboxypeptidase Y. Eur J Biochem 269, 3220-3225