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研究生:黃宣意
論文名稱:Titin蛋白中含多脯胺酸之構形研究
指導教授:甘魯生
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
中文關鍵詞:含多脯胺酸之片段
外文關鍵詞:titinpolyproline
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人體肌纖維系統包含了thick和thin filaments及一巨大的蛋白質titin,其中titin分子量大約3000kDa,分為A-band與I-band。一般認為I-band與肌肉收縮機制有關,而 PEVK片段即在I-band中。本論文所選用之peptide乃由胚胎層肌肉細胞之PEVK peptide序列中篩選出重複率最高之28個胺基酸加以合成,稱之PEVK-28 peptide。
由以往的研究結果知含多個proline殘基會有PPI(cis:同邊)/ PPII(trans:對邊)構形轉換,為了瞭解PEVK-28 trans/cis構形間的轉換與肌肉彈性收縮-鬆弛過程的關聯性,將PEVK-28分為三小段(PR1:KPEVPPVKV,PR2:KKAPVAPPK,PR3:PEPPKEVVPE),其中PR1及PR2含有三個proline,PR3則含有四個;在NMR數據中發現,三段peptide中proline皆以trans構形存在之。而以圓二色差旋光分析儀探討PR1、PR2及PR3在水溶液中不同溫度下構形的改變,發現三個片段隨著溫度變化含有等色點(isodichroic point),表示有構形間的轉換;針對PPII在圓二色差旋光光譜特有的波長(223nm)作變溫實驗發現圖形並非一般典型熔點曲線,而是有一轉折點的曲折線,根據文獻(Alexander A. Makarov, v.12, p.85~91, 1993)判斷三片段結構應是屬於非協和性作用(non-cooperative transition);在NMR數據中,追蹤不同溫度NH的化學位移作圖也同樣可以看到此現象;而NMR NOE顯示此轉折並非表示proline的cis/trans構形轉換,而是與水合作用有關。
另外,以高壓液相層析儀研究peptide不同構形間的平衡,發現在低溫時,PR1、PR2、PR3皆出現兩個吸收峰 (major and minor peak);收集major和minor peak測質譜,發現分子量都相同,顯示低溫時PR1、PR2、PR3有構形異構物存在,在2D NMR數據中也可以發現低溫時有微弱的共振訊號出現。這些研究結果將有助於我們更深入瞭解PEVK-28在titin蛋白中的功能。

Titin is a giant elastic protein in striated muscle, which is composed of tandem, repeats of about 100 residue immunoglobin domains and the PEVK modules, which was believed to play a role of the elasticity of muscle. The structure of a 28-mer PEVK repeated module (HTR12) was proposed containing three short PPII helices region within -PXXP motif (X= a.a. residue), such as a semi-rigid, rod-shaped conformation like two solid bars with a flexible rope linked each other (Ma et al., Biochemistry, v.40, p.3427~3438, 2001).
In order to study the conformational properties of short PPII helix region in the 28-PEVK (HTR12) module, we designed and synthesized the three proline-rich subfragments; PR1: KPEVPPVKV, PR2: KKAPVAPPK, PR3: PEPPKEVVPE, which three sequences were derived from the 28-mer PEVK sequence. The CD and NMR were preformed the impact of the role of proline residue on the conformation of the PPII helix region in aqueous solution. These peptide fragments were displayed the characteristic PPII CD spectra (a weak positive shoulder band at 223 nm and a strong negative band near 200 nm). From temperature dependent experiment, the CD amplitude at 223 nm of three peptide showed a linear relationship at low and high temperature region, but a turning point is observed nearby 50℃. This turning point was not due to the cis/trans isomerization of proline residues, confirmed by the NMR NOE data. In addition, the chemical shift of amide proton with the temperature also have the similar result that in consistent with the temperature dependent CD experiment. According to our data, this phenomenon is due to the non-cooperative disordering of the conformation caused by destruction of the polypeptide hydration shell.
Our study will facilitate the understanding of function of PEVK in titin.

目錄
目次 頁次
圖目錄 -------------------------------------------------------------------------- I
表目錄 -------------------------------------------------------------------------- III
中文摘要 ----------------------------------------------------------------------- IV
英文摘要 ----------------------------------------------------------------------- VI
第一章緒論
1. 肌肉的簡介 ------------------------------------------------------------ 1
2. Titin的發現 ------------------------------------------------------------ 2
3. Titin與肌肉收縮 ------------------------------------------------------ 3
4. 脯胺酸之特性 ----------------------------------------------------------4
5. 研究主題 ----------------------------------------------------------------6
第二章原理簡介
1. Peptide合成 ----------------------------------------------------------- 7
2. 圓二色差旋光光譜術------------------------------------------------- 8
3. SELCON CD光譜分析軟體-----------------------------------------10
4. CD光譜之PPII二級結構比例計算方法 -------------------------13
5. 核磁共振光譜術 ------------------------------------------------------14
5.1核磁共振現象
5.2化學位移
5.3自旋-自旋耦合
5.4二維核磁共振
5.5多維核磁共振光譜中溶劑訊號的消除
5.6 2D NMR於peptide結構上之應用
6. HPLC ---------------------------------------------------------------------22
第三章儀器與藥品
1. 儀器 ----------------------------------------------------------------------25
2. 藥品 ----------------------------------------------------------------------26
第四章實驗步驟及方法
1. 實驗樣品備製 ----------------------------------------------------------27
2. 圓二色光譜實驗 -------------------------------------------------------27
3. 核磁共振光譜實驗 ----------------------------------------------------28
4. HPLC實驗 -------------------------------------------------------------28
第五章實驗結果
1. 圓二色差旋光光譜實驗 ----------------------------------------------30
2. 核磁共振光譜實驗 --------------------------------------------------- 33
2.1 1H NMR一維光譜實驗
2.2 1H NMR二維光譜實驗
3. HPLC實驗 ------------------------------------------------------------ 46
第六章 結論 ------------------------------------------------------------------ 48
參考文獻 ----------------------------------------------------------------------- 50

參考文獻資料:
1. 人體解剖生理概論 林貴福著
2. 國立中正大學化學所林庚龍碩士論文(1996)
3. 中國文化大學應用化學研究所鍾昆元碩士論文(1999)
4. 儀器分析 林敬二.林宗義譯
5. Amodeo, P., Morelli, M. A. C., and Motta, A., Biochemistry 33, 10754-10762 (1994)
6. Alexander A. Makarov, Ivan A. Adzhubei, Irina I. Protasevich, et al., Journal of Protein Chemistry 12,85-91(1993)
7. Chou, P. Y., and Fasman, G. D., Ann. Rev. Biochem. 47, 251-276 (1978)
8. Frommel, C., and Preissner, R., FEBS Lett. 277, 159-163 (1990)
9. Fox, R. O., Evans, P. A., and Dobson, C. M., Nature 320, 192-194 (1986)
10. Grathwohl, C., and Wuthrich, K., Biopolymers 15, 2043-2057 (1976)
11. Grathwohl, C., and Wuthrich, K., Biopolymers 20, 2623-2633 (1981)
12. Garel, J. R., Proc. Natl. Acad. Sci. USA 77, 795-798 (1980)
13. Higgins, K. A., Craik, D. J., Hall, J. G., and Andrews, P. R., Drug. Des. Delivery 3, 159-170 (1988)
14. Kan Ma, Lou-sing Kan, Kuan Wang, Biochemistry (2001)
15. Ladokhin A. S., Selsted ME, White SH, Biochemistry 38, 12313-12319 (1999)
16. Maruyama, K., Natori, R. & Nonomura, Y., New elastic protein from muscle. Nature 262, 58-60 (1976)
17. Maruyama, K., Biophys. Chem. 50, 73-85 (1994)
18. Schmid, F. X., Annu. Rev. Bio-phys. Biomed. 22, 123-142 (1993)
19. Soon-ho Park, William Shalongo, Earle Stellwagen, Protein Science 6, 1694-1700 (1997)
20. Sreerama, N., Woody, R. W., Anal. Biochem. 209, 32~44 (1993)
21. Trinick, J. Trends Biochem. Sci. 19, 405-409 (1994)
22. Tiffany, M. L., Krimm, S., Biopolymers 6, 1767-1770 (1968)
23. Rabanal, F., Ludevid, M. D., Pons, M., Giralt, E., Biopolymers 33, 1019-1028 (1993)
24. Ulf Reimer, Gerd Scherer, Mario Drewello, et al., J. Mol. Biol. 279, 449~460 (1998)
25. Wang, K., Advances in Biophysics. 33, 123-134 (1996)
26. Witt, C. C., et al., Journal of Structure Biology 122(1-2), 206-215 (1998)
27. Wuthrich, K. NMR of Protein and Nucleic Acids, John Wiley & Sons, New York, 1986
28. Wishart, D. S., Sykes, B. D., Richards, F. M., J. Mol. Biol. 222, 311~333 (1991)

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