綠豆植物防禦素第一型 (VrD1)，是第一個被研究發現具有抗象蟲活性的植物防禦素，是一個由46個胺基酸組成 (富含半胱胺酸)，具有CSαβ motif及四對雙硫鍵來穩定結構的鹼性蛋白質。在我們實驗室之前的研究中，已利用核磁共振光譜儀得到綠豆植物防禦素第一型的水溶液結構；也利用定點突變方法來研究綠豆植物防禦素第一型對麵包蟲α-澱粉水解酶的影響。先前的實驗數據顯示綠豆植物防禦素第一型的第三環型結構以及310螺旋結構在其抑制麵包蟲α-澱粉水解酶的能力上扮演著重要的角色。在此論文中，使用了一項蛋白質工程技術 – 環狀序列重組。這個技術是將蛋白質原有的胺基端和羧基端接在一起，並在另一處產生新的開口，可用來研究蛋白質摺疊、結構穩定度、與蛋白質功能。我們得到兩個環狀序列重組蛋白質VrD1 CP36_G 和 VrD1 CP36_2G，他們都以甲硫胺酸36為新的胺基端，並各自以1個及2個甘胺酸連接舊有的胺基端和羧基端。分析結果發現VrD1 CP36_G 喪失了大部分的結構和抑制澱粉水解酶的功能，然而VrD1 CP36_2G保有了這兩項特性。已知在原有的胺基端和羧基端導入不同長度的鏈結可能會影響環型序列重組蛋白質的折疊，目前的結果顯示導入的鏈結長度必須大於原有胺基端和羧基端之間的距離。儘管VrD1 CP36_2G中新的胺基端和羧基端位於具有酵素抑制功能的第三環型結構上，VrD1 CP36_2G依然具有抑制麵包蟲α-澱粉水解酶的活性。野生型VrD1 及 VrD1 CP36_2G的IC50分別為1.93 μM 和 3.14 μM。因此推論，第三環型結構的主要用途是呈現其上胺基酸殘基所帶的正電荷，用以與麵包蟲α-澱粉水解酶作用進而達到抑制效果。

Mung bean (Vigna radiata) plant defensin 1 (VrD1) is the first reported plant defensin, which exhibits insecticidal activity against Callosobruchus chinensis (bruchid). VrD1 is a 46-residue basic peptide containing a cysteine-stabilized αβ (CSαβ) motif with four disulfide-bonds to stable its structure. Three dimensional structure determined by nuclear magnetic resonance (NMR) spectroscopy and alanine substitutions of noncysteine residues in VrD1 were done in our lab. Our previous studies showed that the loop L3 and the 310 helix played important roles in VrD1 insecticidal function. In this study, a protein engineering method – circular permutation (CP) – was employed on VrD1. The CP rearrangement in a protein is visualized as that the original termini of polypeptide are linked and new termini are created elsewhere. The CP rearranged proteins can be utilized to explore protein folding, stability of structure, and functions. Two CP-VrD1 proteins with residue M36 as the new N-terminus and linkers of one and two glycine (VrD1 CP36_G and VrD1 CP36_2G, respectively) were obtained and investigated. VrD1 CP36_G lost almost all structure and function, while VrD1 CP36_2G kept both characters. The length of linker for native termini may affect the protein folding, and our results revealed that the length of linker should be longer than the distance between the native N- and C-termini. VrD1 CP36_2G inhibited α-amylase although the new termini were created at the functional loop L3 and its IC50 was 1.93 μM while that of wild type VrD1 was 3.14 μM. So the important role of loop L3 may be to display the positively charged residues which participated in the electrostatic interaction between VrD1 and TMA.

中文摘要 1
Abstract 2
Chapter I Introduction 4
1.1 Plant defensins 4
1.2 Vigna radiata plant defensin 1 (VrD1) 5
1.3 Circular permutation (CP) 6
1.4 Aim of this study 7
Chapter II Materials and Methods 8
2.1 Construction of circular-permuted VrD1 (CP-VrD1) plasmids 8
2.2 Expression and purification of VrD1 proteins 9
2.3 Purification and activity assay of Tenebrio molitor α-amylase (TMA) 10
2.4 Protein identification by mass spectrometry 11
2.5 Determination of protein concentration 11
2.6 Assay for inhibitory function of VrD1 proteins against TMA 11
Chapter III Results & Discussion 14
3.1 Overexpression, purification, and identification of VrD1 proteins 14
3.2 Structure Analysis of wt-VrD1 and CP-VrD1 proteins 15
3.3 Inhibitory function of VrD1 proteins against TMA 16
Chapter IV Concusion 18
Reference 51

1. Bruix, M., Jimenez, M.A., Santoro, J., Gonzalez, C., Colilla, F.J., Mendez, E., and Rico, M. (1993). Solution structure of gamma 1-H and gamma 1-P thionins from barley and wheat endosperm determined by 1H-NMR: a structural motif common to toxic arthropod proteins. Biochemistry 32, 715-724.
2. Melo, F.R., Rigden, D.J., Franco, O.L., Mello, L.V., Ary, M.B., Grossi de Sa, M.F., and Bloch, C., Jr. (2002). Inhibition of trypsin by cowpea thionin: characterization, molecular modeling, and docking. Proteins 48, 311-319.
3. Osborn, R.W., De Samblanx, G.W., Thevissen, K., Goderis, I., Torrekens, S., Van Leuven, F., Attenborough, S., Rees, S.B., and Broekaert, W.F. (1995). Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae. FEBS letters 368, 257-262.
4. Terras, F.R., Eggermont, K., Kovaleva, V., Raikhel, N.V., Osborn, R.W., Kester, A., Rees, S.B., Torrekens, S., Van Leuven, F., Vanderleyden, J., et al. (1995). Small cysteine-rich antifungal proteins from radish: their role in host defense. The Plant cell 7, 573-588.
5. Stotz, H.U., Thomson, J., and Wang, Y. (2014). Plant defensins. Plant Signaling & Behavior 4, 1010-1012.
6. Bontems, F., Roumestand, C., Boyot, P., Gilquin, B., Doljansky, Y., Menez, A., and Toma, F. (1991). Three-dimensional structure of natural charybdotoxin in aqueous solution by 1H-NMR. Charybdotoxin possesses a structural motif found in other scorpion toxins. European journal of biochemistry / FEBS 196, 19-28.
7. Broekaert, W.F., Terras, F.R., Cammue, B.P., and Osborn, R.W. (1995). Plant defensins: novel antimicrobial peptides as components of the host defense system. Plant physiology 108, 1353-1358.
8. Cornet, B., Bonmatin, J.M., Hetru, C., Hoffmann, J.A., Ptak, M., and Vovelle, F. (1995). Refined three-dimensional solution structure of insect defensin A. Structure (London, England : 1993) 3, 435-448.
9. Maget-Dana, R., and Ptak, M. (1997). Penetration of the insect defensin A into phospholipid monolayers and formation of defensin A-lipid complexes. Biophysical journal 73, 2527-2533.
10. Zhang, H., and Kato, Y. (2003). Common structural properties specifically found in the CSαβ-type antimicrobial peptides in nematodes and mollusks: evidence for the same evolutionary origin? Developmental & Comparative Immunology 27, 499-503.
11. van der Weerden, N.L., and Anderson, M.A. (2013). Plant defensins: Common fold, multiple functions. Fungal Biology Reviews 26, 121-131.
12. Yang, Y.F. (2012). Development and Engineering of CSαβ Motif for Biomedical Application. In Development and Engineering of CSαβ Motif for Biomedical Application (Republic of China, InTech.
13. Chen, J.J., Chen, G.H., Hsu, H.C., Li, S.S., and Chen, C.S. (2004). Cloning and functional expression of a mungbean defensin VrD1 in Pichia pastoris. Journal of agricultural and food chemistry 52, 2256-2261.
14. Chen, K.C., Lin, C.Y., Kuan, C.C., Sung, H.Y., and Chen, C.S. (2002). A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid. Journal of agricultural and food chemistry 50, 7258-7263.
15. Liu, Y.J., Cheng, C.S., Lai, S.M., Hsu, M.P., Chen, C.S., and Lyu, P.C. (2006). Solution structure of the plant defensin VrD1 from mung bean and its possible role in insecticidal activity against bruchids. Proteins 63, 777-786.
16. Yang, Y.F., Cheng, K.C., Tsai, P.H., Liu, C.C., Lee, T.R., and Lyu, P.C. (2009). Alanine substitutions of noncysteine residues in the cysteine-stabilized alphabeta motif. Protein science : a publication of the Protein Society 18, 1498-1506.
17. Buonocore, V., and Poerio, E. (1976). Interaction of Tenebrio molitor L. alpha-amylase with a wheat flour protein inhibitor. FEBS letters 67, 202-206.
18. Machius, M., Vertesy, L., Huber, R., and Wiegand, G. (1996). Carbohydrate and protein-based inhibitors of porcine pancreatic alpha-amylase: structure analysis and comparison of their binding characteristics. Journal of molecular biology 260, 409-421.
19. Nahoum, V., Farisei, F., Le-Berre-Anton, V., Egloff, M.P., Rouge, P., Poerio, E., and Payan, F. (1999). A plant-seed inhibitor of two classes of alpha-amylases: X-ray analysis of Tenebrio molitor larvae alpha-amylase in complex with the bean Phaseolus vulgaris inhibitor. Acta crystallographica Section D, Biological crystallography 55, 360-362.
20. Pereira, P.J.B., Lozanov, V., Patthy, A., Huber, R., Bode, W., Pongor, S., and Strobl, S. (1999). Specific inhibition of insect α-amylases: yellow meal worm α-amylase in complex with the Amaranth α-amylase inhibitor at 2.0 Å resolution. Structure (London, England : 1993) 7, 1079-1088.
21. Strobl, S., Gomis-Rüth, F.-X., Maskos, K., Frank, G., Huber, R., and Glockshuber, R. (1997). The α-amylase from the yellow meal worm: complete primary structure, crystallization and preliminary X-ray analysis. FEBS letters 409, 109-114.
22. Yu, Y., and Lutz, S. (2011). Circular permutation: a different way to engineer enzyme structure and function. Trends in biotechnology 29, 18-25.
23. Cunningham, B.A., Hemperly, J.J., Hopp, T.P., and Edelman, G.M. (1979). Favin versus concanavalin A: Circularly permuted amino acid sequences. Proceedings of the National Academy of Sciences of the United States of America 76, 3218-3222.
24. Baird, G.S., Zacharias, D.A., and Tsien, R.Y. (1999). Circular permutation and receptor insertion within green fluorescent proteins. Proceedings of the National Academy of Sciences of the United States of America 96, 11241-11246.
25. Graf, R., and Schachman, H.K. (1996). Random circular permutation of genes and expressed polypeptide chains: application of the method to the catalytic chains of aspartate transcarbamoylase. Proceedings of the National Academy of Sciences of the United States of America 93, 11591-11596.
26. Hennecke, J., Sebbel, P., and Glockshuber, R. (1999). Random circular permutation of DsbA reveals segments that are essential for protein folding and stability. Journal of molecular biology 286, 1197-1215.
27. Qian, Z., Horton, J.R., Cheng, X., and Lutz, S. (2009). Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation. Journal of molecular biology 393, 191-201.
28. George, R.A., and Heringa, J. (2002). An analysis of protein domain linkers: their classification and role in protein folding. Protein engineering 15, 871-879.
29. Ivarsson, Y., Travaglini-Allocatelli, C., Brunori, M., and Gianni, S. (2009). Engineered symmetric connectivity of secondary structure elements highlights malleability of protein folding pathways. Journal of the American Chemical Society 131, 11727-11733.
30. Chalton, D.A., Musson, J.A., Flick-Smith, H., Walker, N., McGregor, A., Lamb, H.K., Williamson, E.D., Miller, J., Robinson, J.H., and Lakey, J.H. (2006). Immunogenicity of a Yersinia pestis vaccine antigen monomerized by circular permutation. Infection and immunity 74, 6624-6631.
31. Mitrea, D.M., Parsons, L.S., and Loh, S.N. (2010). Engineering an artificial zymogen by alternate frame protein folding. Proceedings of the National Academy of Sciences of the United States of America 107, 2824-2829.
32. Reitinger, S., Yu, Y., Wicki, J., Ludwiczek, M., D'Angelo, I., Baturin, S., Okon, M., Strynadka, N.C., Lutz, S., Withers, S.G., et al. (2010). Circular permutation of Bacillus circulans xylanase: a kinetic and structural study. Biochemistry 49, 2464-2474.
33. Turcotte, R.F., and Raines, R.T. (2008). Design and characterization of an HIV-specific ribonuclease zymogen. AIDS research and human retroviruses 24, 1357-1363.
34. Lo, W.C., Lee, C.C., Lee, C.Y., and Lyu, P.C. (2009). CPDB: a database of circular permutation in proteins. Nucleic acids research 37, D328-332.
35. Lo, W.C., and Lyu, P.C. (2008). CPSARST: an efficient circular permutation search tool applied to the detection of novel protein structural relationships. Genome biology 9, R11.
36. Whitmore, L., and Wallace, B.A. (2008). Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 89, 392-400.
37. Whitmore, L., and Wallace, B.A. (2004). DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic acids research 32, W668-673.
38. Vasas, A., Doka, E., Fabian, I., and Nagy, P. (2015). Kinetic and thermodynamic studies on the disulfide-bond reducing potential of hydrogen sulfide. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society 46, 93-101.
39. Lai, Y.T., Cheng, C.S., Liu, Y.N., Liu, Y.J., and Lyu, P.C. (2008). Effects of ligand binding on the dynamics of rice nonspecific lipid transfer protein 1: a model from molecular simulations. Proteins 72, 1189-1198.
40. Iwakura, M., and Nakamura, T. (1998). Effects of the length of a glycine linker connecting the N-and C-termini of a circularly permuted dihydrofolate reductase. Protein engineering 11, 707-713.
41. Lin, K.F., Lee, T.R., Tsai, P.H., Hsu, M.P., Chen, C.S., and Lyu, P.C. (2007). Structure-based protein engineering for alpha-amylase inhibitory activity of plant defensin. Proteins 68, 530-540.
42. Huang, S.Y., Chen, S.F., Chen, C.H., Huang, H.W., Wu, W.G., and Sung, W.C. (2014). Global disulfide bond profiling for crude snake venom using dimethyl labeling coupled with mass spectrometry and RADAR algorithm. Analytical chemistry 86, 8742-8750.