Proline 可以形成 exo 或是 endo 構型，且由於立體電子效應的關係當 4R- 位置有拉電子基取代時偏好 Cγ-exo 構形， 4S- 位置具有拉電子基取代時則偏好 Cγ-endo 構形。雖然 proline 不存在於 α-螺旋結構中，但他們常出現在 α-螺旋結構的氮端，所以我們以具有36個胺基酸的雞絨毛蛋白 ( HP36 ) 作為研究之蛋白模型。 HP36 為一個小的螺旋蛋白由三個 α-螺旋結構所組成且在其碳端的 α-螺旋上有 proline (Pro62) 位於此 α-螺旋結構的氮端。在 HP36 的原始結構中， Pro62 為 Cγ-exo 構形且會與 Trp64 有 aromatic-proline 作用力。本論文將 4 號位置具拉電子基團取代的 proline 衍生物包 (2S,4R)-4-hydroxyproline (Hyp), (2S,4R)-4-fluoroproline (Flp), (2S,4R)-4-methoxyproline (Mop), (2S,4S)-4-hydroxyproline (hyp), (2S,4S)-4-fluoroproline (flp), and (2S,4S)-4-methoxyproline (mop) 置換在雞絨毛蛋白中 Pro62 的位子，觀察 proline 的構型對 aromatic-proline 作用力和螺旋結構穩定性的影響。由 CD 和 NMR 實驗結果顯示被置換過的 HP36 其結構與 HP36 的原始結構 (wild type) 差不多，由 CD 變溫實驗與化學誘導變性實驗得知除了 flp 取代外，其他 proline 衍生物的取代均造成比原始結構不穩定。我們實驗結果顯示，以 proline 的衍生物置換 Pro62 不只有立體電子效應會影響 aromatic-proline 作用力， HP36 穩定性還會受到因 proline 衍生物嵌入所造成之立體障礙疏水性作用力的影響。

中文摘要 I
Abstract II
謝誌 IV
目錄 V
圖目錄 VII
表目錄 IX
一、 前言 1
1-1雞絨毛蛋白 (Villin) 簡介 1
1-2 Headpiece 介紹 5
1-3 立體電子效應 8
(1) 立體電子效應對proline 衍生物的影響 10
(2) 立體電子效應對α-螺旋蛋白穩定度的影響 12
1-4 固相胜肽合成法介紹19 14
1-4.1 酯化反應 (esterification) 16
1-4.2 去保護 (deprotection) 16
1-4.3 活化 (activation) 17
1-4.4 耦合 (coupling) 18
1-4.5 切除 (cleavage) 18
1-5 圓二色光譜儀 (Circular Dichroism)22,23 18
1-6 研究動機 23
二、 實驗步驟 25
2-1儀器型號 25
2-2使用藥品 26
2-3 Proline 衍生物的合成與鑑定 30
2-3.1 合成策略 30
2-3.2 化合物的合成 32
2-4 胜肽的合成、純化與鑑定 37
2-4.1 HP36 系列模擬胜肽 37
2-4.2 實驗步驟 38
2-5 光譜實驗 40
2-5.1 UV光譜判定濃度實驗 40
2-5.2 CD 光譜測量 41
2-5.3 NMR 光譜 43
2-5.4 螢光光譜 43
2-6 光譜數據分析 44
2-6.1 CD 變溫實驗的數據處理 44
2-6.2化學誘導變性 CD 實驗的數據處理 46
三、 結果與討論 49
3-1 HP36 系列胜肽 Far-UV CD 光譜探討 49
3-2 HP36 系列胜肽 NMR 實驗 52
3-2.1 1D 1H-NMR 光譜 52
3-2.2 2D 1H-NMR 光譜 54
3-3 HP36 系列胜肽之螢光光譜 60
3-4 變溫實驗與化學誘導變性 CD 實驗探討 62
3-4.1變溫實驗 62
3-4.2 化學誘導變性 CD 實驗 62
3-5 討論 67
第四章 結論 70
參考文獻 (Reference) 71
附錄 75

1. Friederich, E., Vancompernolle, K., Louvard, D., Vandekerckhove, J. (1999) Villin function in the organization of the actin cytoskeleton. J. Biol. Chem. 274, 26751-26760.
2. Bazari, L. W., Matsudaira, P., Wallek, M., Smeal, T., Jakes, R., and Ahmed, Y. (1988) Villin sequence and peptide map identify six homologous domains. Proc. Natl Acad. Sci. USA 81, 4986-4990.
3. Vardar, D., Chishti, A. H., Frank, B. S., Luna, E. J., Noegel, A. A., Oh, S. W., and McKnight, C. J. (2002) Villin-type headpiece domains show a wide range of F-actin-binding affinities. Cell Motil. Cytoskeleton 52, 9-21.
4. Meng, J., Vardar, D., Wang, Y., Guo, H. C., Head, J. F., and Mcknight, C. J., (2005) High-resolution crystal structures of villin headpiece and mutants with reduced F-actin binding activity. Biochemistry 44, 11963 -11973.
5. Panebra, A., Ma, S. X., Zhai, L. W., Wang, X. T., Rhee, S. G., Khurana, S. (2001) Regulation of phospholipase C-gamma(1) by the actin-regulatory protein villin. Am. J. Physiol., Cell Physiol. 281, 1046–58.
6. McKnight, C. J., Doering, D. S., Matsudaira, P. T., and Kim, P. S. (1996) A thermostable 35-residue subdomain within villin headpiece. J. Mol. Biol. 260, 126–134.
7. Xiao, S., Bi, Y., Shan, B., and Raleigh, D. P. (2009) Analysis of core packing in a cooperatively folded minature protein: the ultrafast folding villin headpiece helical subdomain. Biochemistry 48, 4607-4612.
8. Vermeulen, W., Vanhaesebrouck, P., Troys, M. V., Verschueren, M., Fant, F., Goethals, M., Ampe, C., Martins, J. C., and Borremans, F. A. M. (2004) Solution structures of the C-terminal headpiece subdomains of human villin and advillin, evaluation of headpiece F-actin-binding requirements. Protein Sci. 13, 1276-1287.
9. Vermeulen, W., Troys, M. V., Bourry, D., Rossenu, D. D. S., Goethals, M., Borremans, F. A. M., Vandekerckhove, J., Martins, J. C. and Ampe, C., (2006) Identification of the PXW sequence as a structural gatekeeper of the headpiece C-terminal subdomain fold. J. Mol. Biol. 359, 1277–1292.
10. Giacovazzo, C., Monaco, H. L., Artioli, G., Viterbo, D., Ferraris, G., Gilli, G. Zanotti, G., Monaco, G., Catti, M. (2002) Fundamentals of Crystallography 2nd ed., Oxford University Press, New York.
11. DeRider, M. L., Wilkens, S. J., Waddell, M. J., Bretscher, L. E., Weinhold, F., Raines, R. T., and Markley, J. L. (2002) Collagen stability: Insights from NMR spectroscopic and hybrid density functional computational investigations of the effect of electronegative substituents on prolyl ring conformations. J. Am. Chem. Soc. 124, 2497–2505.
12. Hinderaker, M. P. and Raines, R. T. (2002) An electronic effect on protein structure. Protein Sci. 12, 1188-1194.
13. Benzi, C., Improta, R., Scalmani, G., Barone, V. (2002) Quantum mechanical study of the conformational behavior of proline and 4R-hydroxyproline dipeptide analogues in vacuum and in aqueous solution. J. Comput. Chem. 23, 341-350.
14. Wolfe, S. (1972) Gauche effect. Stereochemical consequences of adjacent electron pairs and polar bonds. Acc. Chem. Res. 5, 102-111.
15. Improta, R., Benzi, C., Barone, V. (2001) Understanding the role of stereoelectronic effects in determining collagen stability. 1. A quantum mechanical study of proline, hydroxyproline, and fluoroproline dipeptide analogues in aqueous solution. J. Am. Chem. Soc. 123, 12568-12577.
16. Taylor, C. M., Hardre, R., Partrick, J. B. (2005) The impact of pyrrolidine hydroxylation on the conformation of proline-containing peptides. J. Org. Chem. 70, 1306-1315.
17. Taylor, C. M., Hardré, R., and Edwards, P. J. B., (2005). The impact of pyrrolidine hydroxylation on the conformation of proline-containing peptides. J. Org. Chem. 70, 1306-1315.
18. Naduthambi, D. and Zondlo, N. J. (2006) Stereoelectronic tuning of the structure and stability of the Trp cage miniprotein. J. Am. Chem. Soc. 128, 12430-12431.
19. Merrifield, R. B. (1986) Soild phase peptide synthesis. Science 232, 341-347.
20. 張湘戎，體抑素胜肽分子內雙硫鍵建構之研究，碩士學位論文，中原大學化學研究所，2003。
21. Chan, W. C., and White, P. D. (2004) Fmoc solid phase peptide synthesis. Oxford University press, New York.
22. Berova, N., Nakanishi, K., and Woody, R. (2000) Circular dichroism : Principles and applications. Wiley, Hoboken.
23. Skoog, D. A., Holler, F. J., and Nieman, T. A. (1998) Principles of instrumental analysis 5nd ed., Saunders College Press, Belmont.
24. Velluz, L., Legrand, M., and Grosjean, M. (1965). Optical circular dichroism. Academic Press, New York.
25. Fasman, G. D. (1996) Circular dichroism and the conformation analysis of biomolecules. Plenum Press, London.
26. Matsuo, K., Yonehara, R., and Gekko, K. (2005) Improved estimation of the secondary structures of proteins by vacuum-ultraviolet circular dichroism spectroscopy. J. Biochem. 138, 79-88.
27. Duan, Y., Wang, L., and Kollman, P. A. (1998). The early stage of folding of villin headpiece subdomain observed in a 200-nanosecond fully solvated molecular dynamics simulation. Proc. Natl. Acad. Sci. USA, 95, 9897–9902.
28. O'Neil, I. A., Thompson, S., Kalindjian, S. B., and Jenkins, T. C. (2003) The synthesis and biological activity of C2-fluorinated pyrrolo[2,1-c][1,4]benzodiazepines, Tetrahedron Letters 44, 7809-7812.
29. Kotch, F. W., Guzei, I. A., and Raines, R. T. (2008) Stabilization of the Collagen Triple Helix by O-Methylation of Hydroxyproline Residues. J. Am. Chem. Soc. 130, 2952-2953.
30. Wang, M., Tang, Y., Sato, S., Vugmeyster, L., McKnight, C. J., and Raleigh, D. P. (2003) Dynamic NMR line-shape analysis demonstrates that the villin headpiece subdomain folds on the microsecond time scale. J. Am. Chem. Soc. 125, 6032-6033.
31. 鄭統元，立體電子效應對雞絨毛蛋白 (HP36) 結構影響之探討，碩士學位論文，國立清華大學化學研究所，2009。
32. Julie, M. G., Yougjin, Z., Parmit, C. Jia, T., and Feng, G. (2008) Using an amino acid fluorescence resonance energy transfer pair to probe peotein unfolding： Application to the villin headpiece subdomain and the LysM domain. Biochemistry 47, 11070-11076.
33. Aune,K. C., Salahuddin, A., Zarlengo,M. H., and Tanford, C. (1967) Evidence for residual structure in acid- and heat-denatured proteins. J. Biol. Chem. 242, 4486–4489.
34. Deshpande, R. A., Khan, M. I., and Shankar, V. (2003) Equilibrium unfolding of RNase Rs from Rhizopus stolonifer: pH dependence of chemical and thermal denaturation. Biochim. Biophys. Acta 1648, 184-194
35. Farruggia, B., and Picó, G. A. (1999) Thermodynamic features of the chemical and thermal denaturations of human serum albumin. Int. J. Biol. Marcromol. 26, 317-323.
36. Tang, Y., Rigotti, D. J., Fairman, R., and Raleigh, D. P. (2004) Peptide models provide evidence for significant structure in the denatured state of a rapidly folding protein: the villin headpiece subdomain. Biochemistry 43, 3264–3272.
37. Luis, M., Nediljko, B. (2010) Synthetic Biology of protein folding. ChemPhysChem. 11, 1181-1187.
38. Thomas, S., Petra, H., Jaw, H. B., Birgit, W., Luis, M., Nediljko, B. Synthetic Biology of protein： Tuning GFPs folding and stability with Fluoroproline. PLos One. 3: e1680.