臺灣博碩士論文加值系統

δ-晶體蛋白為鳥類和爬蟲類的水晶體特有之晶體蛋白，扮演對光線折射的重要角色。在演化上此晶體蛋白乃由徵召精胺醯丁二酸裂解酶至眼睛內做為結構性蛋白質，除了與此酶具高序列類似性外並具酶活性。本實驗室之前的研究發現，在GdmHCl及pH值的改變下，晶體蛋白四聚體結構經由雙聚體解離而成各單體，而當置換雙單元間結合有關之胺酸成K315A、E267L及E327L，因為破壞雙單元和雙單元間氫鍵會使其不穩定，而有部份雙單元的形成。本論文進一步研究K315氫鍵及忌水性作用力對雙單元形成的重要性，及建構此三胺酸的位置之雙點及三點突變對-晶體蛋白結構安定性影響。用圓偏振二色旋光譜分析、丙烯醯胺螢光減弱法、ANS結合分析、溫度及變性劑存在下之變性實驗，超高速離心沉降速率分析等方法應用來研究其構形上的變化及穩定性。研究結果顯示K315扮演了很重要的角色於雙單元間提供氫鍵及忌水性結合之作用力，破壞氫鍵可以增加此蛋白質構形不穩定性，但是同時失去兩作用力反而增加彼此作用之氫鍵，而增加其穩定性。在尿素實驗結果上也發現在高濃度尿素下，K315A存在一較穩定的中間態。雙突變蛋白中E327L/E267L解離之雙單元體可達到58%。且Tm值降低17.50C。有趣的是三點突變E267L/K315L/E327L活性只剩下3 %。但解離之雙單元反而只有8%

目錄………………………………………………………………………………… II
圖目錄……………………………………………………………………………… IV
表目錄……………………………………………………………………………… VI
縮寫對照表…………………………………………………………………………VII
中文摘要………………………………………………………………………… VIII
英文摘要………………………………………………………………………………X
目 錄
序論……………………………………………………………………………………1
實驗材料……………………………………………………………………………… 5
壹、藥品試劑……………………………………………………………………5
貳、主要儀器及器材……………………………………………………………7
叁、試劑的配製…………………………………………………………………8
實驗方法…………………………………………………………………………… 11
壹、質體的製備……………………………………………………………… 11
貳、設計引子(Primer)……………………………………………………… 13
叁、聚合酶鏈連鎖反應(PCR)…………………………………………………13
肆、檢視PCR之成品……………………………………………………………15
伍、限制酶(restriction enzyme)切目標DNA和載體(vector)……………16
陸、DNA連結反應( Ligation)………………………………………………17
柒、轉殖作用(Transformation)…………………………………………… 18
捌、DNA序列的確認……………………………………………………………19
玖、勝任細胞(Competent cell；DH5α，BL21)的製備…………………… 19
拾、菌體培養………………………………………………………………… 21
拾壹、蛋白質純化…………………………………………………………… 22
拾貳、蛋白質純化(His Tagged)…………………………………………… 24
拾叁、蛋白質活性的測定…………………………………………………… 25
拾肆、圓偏振二色旋光譜分析……………………………………………… 26
拾伍、螢光光譜實驗………………………………………………………… 27
一、螢光減弱實驗…………………………………………………… 27
二、Gdn-HCl(urea)效應下之變性實驗………………………………28
三、ANS結合分析實驗…………………………………………………28
拾陸、分析級超高速離心實驗……………………………………………… 29
拾柒、蛋白質含量測定……………………………………………………… 30
拾捌、以SDS-PAGE鑑定蛋白質純度和分子量………………………………30
拾玖、突變鵝眼d晶體蛋白結構模擬……………………………………… 31
結果………………………………………………………………………………… 33
壹、野生型和突變型鵝眼δ-晶體蛋白之純化…………………………… 33
一、離子交換層析法………………………………………………………… 33
二、硫酸銨沉澱蛋白質……………………………………………………… 33
三、分子過篩層析法………………………………………………………… 33
貳、精胺醯丁二酸裂解酶活性分析………………………………………… 34
叁、圓偏振二色旋光譜分析………………………………………………… 34
肆、螢光減弱實驗…………………………………………………………… 35
伍、ANS結合分析………………………………………………………………35
陸、GdnHCl效應下之變性實驗……………………………………………… 36
柒、urea 效應下的變性實驗…………………………………………………37
捌、利用圓偏振二色旋光譜分析d-晶體蛋白突變位置對熱安性的影響…38
玖、分析型超高速離心分析………………………………………………… 38
討論………………………………………………………………………………… 40
壹、突變型鵝眼d-晶體蛋白的活性與構形關係分析………………………40
貳、胺酸K315支鏈對鵝眼d-晶體蛋白的結構安定性分析探討……………40
叁、雙點及三點突變對晶體蛋白的影響……………………………………42
參考文獻…………………………………………………………………………… 79

1.S. Meehan, Y. Berry, B. Luisi , C. M. Dobson, J. A. Carver, C. E. Macphee. (2004). Amyloid fibril formation by lens crystallin proteins and its implications for cataract formation . J. Biol. Chem. 279, 3413-3419.

2.L. M. Sampaleanu, F. Vall’ee, C.Slingsby, P. L. Howell. (2001). Structural studies of duck 1 and 2 crystallin suggest conformational changes occur during catalysis. Biochemistry. 40, 2732-2742.

3.J. Piatigorsky, W. E. O’Brien, B. L. Norman, K. Kalumuck, G. J. Wistow, T. Borras, J. M. Nickerson, E. F. Wawrousek. (1988). Gene sharing by -crystallin and argininosuccinate lyase. Proc. Natl. Acad. Sci. 85, 3479-3483.

4.L. M. Sampaleanu, A. R. Davidson, C. Graham, G. J. Wistow, P. L. howell. (1999). Domain exchange experiments in duck -crystallins: functional and evolutionary implications. Protein Sci. 8, 529-537.

5.H. J. Lee, Y. H. Lai, S.Y. Wu, Y. H. Chen. (2005). The effect of N-terminal truncation on double-dimer assembly of goose -crystallin. Biochem. J. 392, 545-554.

6.J. Horwitz. (1992). -crystallin can function as a molecular chaperone. Proc. Natl. Acad. Sci. 89, 10449-10453.

7.L. M. Sampaleanu, B. Yu, P. L. Howell. (2002). Mutational analysis of duck 2 crystallin and the structure of an inactive mutant with bound substrate provide insight into the enzymatic mechanism of argininosuccinate lyase. J. Biol. Chem. 277, 4166-4175.

8.M. Tsai, L. M. Sampaleanu, C. Greene, L. Creagh, C. Haynes, P. L. Howell. (2004). A duck 1 crystallin double loop mutant provides insight into residues important for argininosuccinate lyase activity. Biochemistry. 43, 11672-11682.

9.F. Vall’ee, M. A. Turner, P. L. Lindley, P. L. Howell. (1999). Crystal structure of an inactive duck  II crystalline mutant with bound argininosuccinate. Biochemistry. 38, 2425-2434.

10.M. Abu-Abed, M. A. Turner, F. Vall’ee, A. Simpson, C. Slingsby, P. L. Howell. (1997). Structure comparison of the enzymatically active and inactive form of crystalline and the role of histidine 91. Biochemistry. 36, 14012-14022.

11.L. M. Sampaleanu, F. Vall’ee, G. D. Thompson, P. L. Howell. (2001). Three-dimensional structure of the argininocuccinate lyase freguently complementing allele Q286R. Biochemistry. 40, 15570-15580.

12.A. R. Chakraborty, A. Davidson, P. L. Howell. (1999). Mutational analysis of amino acid residues involved in argininosuccinate lyase activity in duck  II crystalline. Biochemistry. 38, 2435-2443.

13.G. G. Chang, H. J. Lee, R. H. Chow. (1997). pH-incuced reversible dissociation of tetrameric duck lens delta-crystallin. Exp. Eye Res. 65, 653-659.

14.H. J. Lee, G. G. Chang. (2000). Guanidine hydrochloride induced reversible dissociation and denaturation of duck2-crystallin. Eur. J. Biochem. 267, 3979-3985.

15.H. J. Lee, S. W. Lu, G. G. Chang. (2003). Monomeric molten globule intermediate involved in the equilibrium unfolding of tetrameric duck delta2-crystallin. Eur. J. Biochem. 270, 3988-95.

16.B. Yu, G. D. Thompson, P. Yip, P. L. Howell, A. R. Davidson. (2001). Mechanisms for intragenic complementation at the human argininocuccinate lyase locus. Biochemistry. 40, 15581-15590.

17.B. Yu, P. Paroutis, A. R. Davidson, P. L. Howell. (2004). Disruption of a salt bridge dramatically accelerates subunit exchange in duck 2 crystallin. J. Biol. Chem. 279, 40972-40979.

18.黃志偉。 雙單元界面間胺酸基對鵝眼delta晶體蛋白安定性及單元合影響研究。國防醫學院生物化學研究所碩士論文。

19.A. J. Miles, L. Whitmore, B. A. Wallace. (2005). Spectral magnitude effects on the analyses of secondary structure from circular dichroism spectroscopic data. Protein Sci. 14, 368-374.

20.R. Boteva, A. Koek, N. V. Visser, A. J. W. G. Visser, E. Krieger, T. Zlateva, M. Veenhuis, I. V. D. Klei. (2003). Fluorescence analysis of the Hansenula polymorpha peroxisomal targeting signal-1 receptor, Pex5p. Eur. J. Biochem. 270, 4332-4338.

21.C. N. Pace. (1986). Determination and analysis of urea and guanidine hydrochloride denaturation curves. Meth enzymol. 131, 266-280.

22.Q. Zou, R. Habernann, K. P. Murphy. (1998). Urea effects on protein stability: hydrogen bonding and the hydrophobic effect. Proteins. Struct. Funct. & Genet. 31, 107-115.

23.C. A. Royer, C. J. Mann, C. R. Matthews. (1993). Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants. Protein. Sci. 2, 1844-1852.

24.S. Peter. (2000). Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Byophys. J. 78, 1606-1619.

25.M. M. Bradford. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

26.Burstein EA, Vedenkina NS, Ivkova MN. (1973). Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol. 18(4), 263–279.

27.Eftink MR, Ghiron CA. (1981). Fluorescence quenching studies with proteins. Anal Biochem. 114 (2), 199–227.

28.A. C. Wallace, R. A. Laskowski, J. M. Thornton. (1995). LIGPLOT: A program to generate schematic diagrams of protein-ligand interactions. Prot. Eng. 8, 127-134.