臺灣博碩士論文加值系統

蛋白質的三級結構決定了其功能。因此，解析蛋白三級結構將有助於了解其功能。隨著快速成長的基因資料庫，結構基因體學將幫助我們了解生物體中，蛋白質的功能及其複雜的交互作用。Xanthomonas campestris屬革蘭氏陰性菌，是一株會感染十字花科並造成十字花科黑腐病的植物病原菌。研究該病原菌有其在學術上以及經濟價值。故我們致力於利用X-ray 結晶學的技術來解析Xanthomonas campestris中的蛋白質結構和確定其功能。
Xanthomonas campestris的基因體中被預測的基因約有7000個。本論文利用X-ray 結晶學的方法研究了10個目標蛋白質：XC449 (two-component system sensor protein)、XC743 (twitching motility protein)、XC708 (conserved hypothetical protein)、XC1008 (murein hydrolase D)、XC1313 (RNA-directed DNA polymerase)、XC1618 (type II secretion system protein like protein)、XC2895 (phage-related integrase)、XC3620 (Ham1 like protein)、XC4632 (conserved hypothetical protein)、XC7201 (3-hydroxyanthranilate 3,4-dioxygenase)。其中XC743、XC1008、XC1313、XC1618、XC3620、XC4632 經過測試了許多種誘發條件仍為不可溶或是沒有表現，將來可嘗試著使用其他的融合蛋白或是改變寄主(host)及表現方法來增加蛋白的可溶性。XC708為一被認為可能跟XCC的致病性調節有關，可以正向調控細胞外酵素、EPS、pathogenicity 等致病因子的合成。經由MBP fusion protein的載體大量表現，但是還無法完全被TEV酵素切除其融合的MBP蛋白而進行純化；並且在16℃水浴切除的過程中目標蛋白還是不斷的被沉澱出來。現正嘗試改變大量表現時的條件。XC2895其功能被預測是phage-related integrase，純化出來後在進行晶體篩選的步驟時發現蛋白質不斷的被降解，正嘗試篩選出好的buffer條件或進行其他的純化方法提高純度。XC449俱有351個胺基酸，其功能被預測為一two-component system sensor protein，此類型的蛋白常見於細胞膜上，兩個domain當中的一個domain到現在仍然沒有高相似度的三維結構被解析出來，經過E. coil BL21的大量表現純化後正在進行結晶條件的篩選。
XC7201由176個胺基酸所組成，比對已知物種資料庫後發現在原核真核菌中都有類似的序列，其功能被預測為3-hydroxyanthranilate 3,4-dioxygenase (3HAO)。3HAO是一種雙氧化酵素，在生物體進行把Tryptophan轉變成Quinolinic acid的生合成途徑中最後一個反應的催化。Quinolinic acid是一種內生性的神經毒素，活化N-methyl-D-aspartate (NMDA)接受器以及在生物合成NAD+的前驅物。在化學的意義上，3HAO扮演著把芳香環氧化開環的酵素。由於芳香環是一種非常穩定的化學結構，如果想要進行開環反應必要要經過極大的活化能，而了解生物體中是如何在常溫的環境下進行這樣的反應將會是非常有趣的題材。我們藉由研究此種蛋白的結構來了解其反應機制，並且希望能藉由這樣的啟發來幫助工業發展及學術上的應用。
我們利用Se-Met標定的XC7201蛋白已成功得到解析度達1.8 Å的晶體，並且使用在新竹同步輻射中心13B1的X-ray光源，利用多波長異常繞射的方法解決其相位問題，以得到其三維結構。解析其蛋白質結構後看到，XC7201在晶體結構中為二聚體聚合，而其聚合的力量主要為疏水性作用力。每一個蛋白質單體中有兩個金屬離子，經由X-ray吸收光譜得知為Ni2+金屬，其中一個金屬位於活性中心，由兩個His、一個Glu及兩個水分子組成八隅體配位鍵結。另一個金屬在蛋白質的C端，和4個Cys組成四面體配位鍵結，目前認為無生物活性，只具有穩定結構功能。經由結構上的比對發現，XC7201和另外兩物種的3HAO蛋白質結構有非常高度的相似性，較大的不同在於XC7201在C端有較長的序列，並且多了一個α螺旋，但是和活性區域距離較遠，可能不會影響到酵素活性，認為可能是生物特異性所造成的不同，還需要進一步的研究確認。

The functional of a protein is related to its three-dimensional structure. Due to the rapidly growing genomic database, structure genomics is paving a way to understand the function and intricate interaction among proteins in a whole organism. Xanthomonas campestris (Xcc) is a gram-negative bacterium that is phytopathogenic to cruciferous plants. Due to its academic and industrial values, we have endeavored to identify and characterize the structures and functions of proteins encoded in Xcc by using X-ray crystallography.
From the sequenced genome, approximately 7000 genes were predicted in Xcc. We have studied 10 of them: XC449 (two-component system sensor protein)、XC743 (twitching motility protein)、XC708 (consvered hypothetical protein)、XC1008 (murein hydrolase D)、XC1313 (RNA-directed DNA polymerase)、XC1618 (type II secretion system protein like protein)、XC2895 (phage-related integrase)、XC3620 (Ham1 like protein)、XC4632 (consvered hypothetical protein)、and XC7201 (3-hydroxyanthranilate 3,4-dioxygenase). Among those, XC743、XC1008、XC1313、XC1618、XC3620、XC4632 are insoluble or exhibit low expression after we have tried many different induction conditions. For other proteins, we have tried changing fusion partners or expressing in different hosts to further increase their solubility. XC708 are considered to be a regulator of pathogenicity pathway in Xcc. It can regulate the synthesis of extracellular enzyme, EPS, pathogenicity and other pathogenic factor. It has been fused to the MBP protein for overexpression, but cannot be completely cleaved by TEV; furthermore, it was found to precipitate during the cleavage reaction by TEV at 16℃. A better condition needs to be found for its overexpression. XC2896 was predicted as a phage-related integrase. Its overexpression is OK. However, we found that it degraded during crystallization screening. XC449 comprises 351 amino acid, and is predicted as a two-component system sensor protein. This type of protein usually locates on the cell membrane. It comprises two domain, one domain of that has no similar (identity more than 30%) sequence be found from Protein Data Bank structure. After expression in E. coil, a crystal screening is being performed to find crystallization condition.
XC7201 is an interesting protein comprising 176 amino acids. Many sequences in the prokaryote and eukaryote kingdom with high similarity score were detected by BLAST search. It was predicted to a 3-hydroxyanthranilate 3,4-dioxygenase (3HAO), which is an extradiol dioxygenase that catalyzes the final conversion step of tryptophan to quinolinic acid in the kynurenine pathway. Quinolinic acid is an endogenous neurotoxin, that activates the N-methyl-D-aspartate(NMDA) receptor and the biosynthesis of NAD+ precursor. Chemically speaking, 3HAO is an important enzyme that catalyzes the aromatic ring opening reaction. Since aromatic ring is a very stable compound, it would needs very high activation energy to begin the ring opening reaction. It is therefore interesting to investigate how the micro-organism can catalyze this reaction in room temperature at natural buffer condition. A detailed study of this enzyme structure can help understand the mechanism of this reaction, and help to development of its industrial application.
We have used the selenomethionine-labeled protein to get a good crystal that diffracted to at least 1.8 Å X-ray diffraction data at the remote and inflection wavelength have been collected. The phase problem has been solved using the MAD approach. A preliminary structure of XC7201 has been obtained. Further structural refinement is now undergoing! After analyzing the protein structure, we found that XC7201 exists as a dimer in the crystal, and stabilized mainly through hydrophobic interaction. Every monomer contains two metal ions, were found to be Ni2+ by X-ray absorption scpectrum. One of the metal ions locates in the active site, and is coordinated by two histidine and one glutamic acid. The other metal ion is located in the C-terminal region of the protein and coordinated by 4 cysteine. This metal center is possibly unrelated to the enzymatic activity, and mainly contributes to the enzyme stability. We found that the XC7201 structure has high similarity with 3HAO from other two species by structure alignment. The most difference from exists at C-terminal region XC7201 which has more amino acids, and forms a α helix. Because this helix is away from active site; it may not affect the enzyme activity ether. It’s importance needs further characterization.

中文摘要……………………………………………………………………………..i
Abstract………………………………………………………………………........iii
目錄…………………………………………………………………………………vi
附圖………………………………………………………………………………viii
附表…………………………………………………………………………….......viii
縮寫檢索表………………………………………………………………………...ix
實驗儀器設備……………………………………………………………………xi

第一章　前言…………………………………………………………………………1
一、Xanthomonas campestris pv.campestris之結構基因體計畫………...……....1
二、結構蛋質體學………………………………………………………………....2
第二章　材料與方法…………………………………………………………………3
一、目標基因選擇…….………………………………………………………...3
二、基因轉殖………………………………………………………………………3
(一) 引子的設計…………….……………..………………………………..4
(二) 聚合酶連鎖反應……………………..………………………………….4
(三) 表現載體……………….…………….………………………………… 5
(四) 質體DNA的抽取….……………...…………………..............................5
(五)載體的製備……….……………….…..………………………………....6
(六) E. coil勝任細胞之製備……………………..…………………………6
(七) Insert製備………………………………………………………………..7
(八) 接合.....................…………………………….………………………….7
(九) 轉殖作用……………………………………...…………………………7
(十) PCR Check 以及定序……….……………...……………………………8
三、蛋白質的大量表現與純化...………………………………………………….9
(一) SDS-PAGE的製備…………….……………………………………..…9
(二) 蛋白質之大量表現…………………………………………………….11
(三) 蛋白質的初步純化……………………………………………………12
(四) 以陰/陽離子交換樹脂純化蛋白………………………………………14
(五) 蛋白質濃度測定………………………………………………………15
(六) 蛋白質最適合溶液之篩選…………………………………………….15
(七) 分析級超高速離心機………………………………………………….16
四、蛋白質的結晶…………………………………………………………………16
(一) 蛋白質結晶實驗……………………………………………………16
(二) 結晶條件的篩選………………………………………………………17
(三) 手動微調和大量結晶…………………………………………………17
(四) 晶體的保護……………………………………………………………18
五、利用X-ray繞射技術解析蛋白質晶體結構………………………………….18
(一) X-ray繞射數據的收集………………………………………………...19
(二) 繞射數據的處理………………………………………………………19
(三) 結構因子………………………………………………………………19
(四) 相位問題………………………………………………………………20
(五) Patterson Method……………………………………………………….22
第三章　結果與討論………………………………………………………………24
一、XC7201的基因註解及功能分析…………………………………………...24
二、XC7201蛋白的製備、純化、結晶及繞射數據的收集……………………...26
三、晶體結構分析…………………………………………………………………27
四、討論……………………………………………………………………………30
第四章　結論………………………………………………………………………32
第五章　參考文獻…………………………………………………………………33
附表
表一、10個目標基因目前進度表………………………………………………..36
表二、XC7201基因基本資料……………………………………………………37
表三、XC7201的X-ray晶體繞射數據………………………………………….38
附圖
圖一、LIC (Ligation-Independent Cloning)示意圖………………………………39
圖二、氣相擴散法……..…………………..……………………………………..40
圖三、XC2895純化過程及發生降解情形………………………………………41
圖四、Kynureine pathway 路徑圖…………………………….…………………42
圖五、3HAO所進行的催化反應……………………………………….………..43
圖六、XC7201定序後序列與ATCC33913的XC1555做比較圖…………….44
圖七、XC7201的純化結果………………………………………………………45
圖八、使用分析級超高速離心機測量XC7201在溶液中的聚合情形………...46
圖九、XC7201的Se-Met標定蛋白結晶圖……………………………………47
圖十、XC7201二聚體 (dimer)的整體結構圖…………………………………48
圖十一、 XC7201的單一蛋白 (monomer)結構………….……………………..49
圖十二、位於dimer interface的疏水性胺基酸分佈……..…………………..50
圖十三、活性區域金屬配位之電子雲密度圖…………………………………51
圖十四、在C端的4個 Cystiene和Ni2+的配位鍵結電子雲密度圖………...52
圖十五、利用MUSTANG針對結構上做胺基酸序列的比對…………………53
圖十六、利用MISTANG針對結構上做重疊……..……………………………54
圖十七、XC7201與抑制劑ClHAA可能的錯合情況…..……………………..55
圖十八、3HAO的可能催化反應機制………………………………………….56

王旭川 (2002) 國立清華大學生命科學所碩士論文
胡玉真 (2004) 國立中央大學生命科學所碩士論文
黃照煒 (2006) 國立中興大學生化所碩士論文
Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P., & Ealick, S. E. (2002) Biochemistry 41 7659-7669.
Aslanidis, C. & de Jong, P. J. (1990) Nucleic Acids Res. 18, 6069–6074.
Brenner, Steven E. (2000) Nat. Struct. Biol. 7, 967-969.
Bugg, T. D., Sanvoisin, J., & Spence, E. L. (1997) Biochem. Soc. Trans. 25 81-85.
Cleasby, A., Wonacott, A., Skarzynaki, T., Hubbard, R. E., Davies, G. J., Proudfoot, A. E., Bernard, A. R. Payton, M. A. & Wells, T. N. (1996) Nat. Struct. Biol. 3 470-479.
Cole, James L., and Hansen Jeffrey C. (1999) J. Biomolecular Techniques 10, 163-176.
da Silca, A. C., Ferro, J. A. Reinach, F. C., Farah, C. S., Furlan, L. R., Quaggio, R. B., Monteiro-Vitorello, C. B., Van Sluys, M. A., Almeida, N. F., & Alves, L. M., et al. (2002) Nature 417, 459-463.
Delcher, A. L., Harmon, D., Kasif, S., White, O. & Salzberg, S. L. (1999) Nucleic Acids Res. 27 4636-4641.
Dodson, E. (2003) Acta. Cryst. D 59, 1958-1966.
Dow, J. M., Crossman, L., Findlay, K., He, Y. Q., Feng, J. X. & Tang, J. L. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10995-11000.
Frishman, D., Albermann, K., Hani, J., Heumann, K., Metanomski, A., Zollner, A. & Mewes, H. W. (2001) Bioinformatics 17, 44-57.
Frishman, D., Mironov, A., Mewes, H. W. & Gelfand, M. (1998) Nucleic Acids Res. 26 2941-2947.
Fritze, I. M., Linden, L., Frrigang, J., Auerbach, G., Huber, R., & Steinbacher, S. (2004) Plant Physiol. 134 1388-1400.
Gopal, P., Madan, L. L., Betz, S. F., & Khuri, S. (2004) Phytochemistry 65 7-17
Guidetti, P., Luthi-Carter, R. E., Augood, S. J. & Schwarcz, R. (2004) Neurobiol. Dis. 17 455-461.
Guerrero, S. A., Hecht, H. J., Hofmann, B., Biebl, H., Singh, M. (2001) Appl. Microbiol. Biotechnol. 56 718-723.
Hasegawa, Y., Muraki, T., Tokuyama, T., Iwaki, H., Tatsuno, M., & Lau, P. C. K. (2000) Microbiol. Lett. 190 185-190.
He, Y. W., Yu-Jin, A., Xu, M., Lin, K., Wang, L. H., Dong, Y. H. & Zhang, L. H. (2007) Mol. Microbiol. 64, 281-292.
Hendrickson, W., Ogata, C. (1997) Meth. Enzymol. 276 494-523.
Heyes, M. P., Saito, K., Jacobowitz, D., Markey, S. P., Takikawa, O., & Vickers, J. H. (1992) FASEB J. 6 2977-1989.
Holm, Liisa & Sander, Chris (1998) Nucleic Acids Research 26 316-319
Hwang, K. Y., Chung, J. H., Kim, S.-H., Ham, Y. S. & Cho, Y. (1999). Nature Struct. Biol. 6 691-696.
Jancarik, J., Pufan, R., Hong, C., Kim, Sung-Hou & Kim, R. (2004) Acta Cryst. D 60,1670-1673.
Kendrew, J. C., Bodo, G., Dintzis, H. M., Parrish, R. G., Wyckoff, H. & Phillips, D. C. (1958) Nature (London) 181 662-666.
Konagurthu, A. S., Whissstock, J. C., Stuckey P. J. & Lesk, A. M. (2006) Proteins 64 559-574.
Laskowski, R. A., Watson, J. D. & Thornton, J. M. (2003) J. Struct. Funct. Genomics 4, 167-177.
Li, X. W., Guo, K., Fan, J., Tang, W. Y., Wang, D. Q., Ge, H. G., Rong, H., Teng, M. K., Niu, L. W., Liu, Q. & Hao, Q. (2006) Protein Sci. 15 761-773.
Muraki, T., Taki, M., Hasegawa, Y., Iwaki., H., & Lau, P. C. K. (2003) Appl. Environ. Microbiol. 69 1564-1572.
Nandi, D., Lightcap, E. S., Koo, Y. K., Lu, X. L., Quancard, J. & Silverman, R. B. (2003) Int. J. Biochem. Cell Biol. 35 1085-1097.
Nogales, J., Canales, A., Jimenez-Barbero, J., Garcia, J. L., & Diaz, E. (2005) J. Biol. Chem. 280 35382-35390.
Patterson, A. L. (1935) Z. Krist. (A)90 517-542.
Pandey, J., Paul, D., & Jain, R. K. (2003) FEMS Microbiol. Lett. 229 231-236
Perutz, M. F., (1956) Acta. Cryst. 9 867-873
Rhodes, Gale (1999) Crystallography Made Crystal Clear: A Guide for Users of Macromolecular Models 2nd Edition. 92-132.
Rossmann, M. G., & Blow, D. M. (1962) Acta. Cryst. 15 24-31.
Salanoubat, M., Genin, S., Artiguenave, F. Gouzy, J., Mangenot, S., Arlat, M., Billault, A., Brottier, P., Camus, J. C., & Cattolico, L., et al. (2002) Nature 415, 497-502.
Schwarcz, R. & Pellicciari, R. (2002) J. Pharmacol Exp. Ther. 303 1-10.
Schwarcz, R., Okuno, E., White, R. J., Bird, E. D. & Whetsell Jr., W. O. (1988) Proc. Nat. Acad. Sci. 85 4079-4081.
Schwarcz, R., Whetsell, W.O. & Mangano, R.M. (1983) Science 219 316-318
Simpson, A. J., Reinach, F. C., Arruda, P., Abreu, F. A., Acencio, M., Alvarenga, R, Alves, L. M., J. E., Araya, J. E., Baia, G. S. & Baptista, C. S., et al. (2002) Nature 406, 151-157.
Solomon, E. I., Brunold, T. C., Davis, M. I., Kemsley, J. N., Lee, S. K., Lehnert, N., Neese, F., Skulan, A. J., Yang, Y. S., & Zhou, J. (2000) Chem. Rev. 100 235-349.
Spence, E. L., Kawamukai, M., Sanvoisin, J., Braven, H., & Bugg, T. D. H. (1996) J. Bacteriol. 178 5249-5256.
Stone, T. W. & Darlington, L. G. (2002) Nat. Rev. Drug Discov. 1 609-620.
Taylor, G.. (2003) Acta. Cryst. D 59 1881-1890.
Teplova, M., Tereshko, V., Sanishvili, R., Joachimiak, A., Bushueva, T., Anderson, W. F. & Egli, M. (2000). Protein Sci. 9 2557-2566.
Vetting, M. W., Wackett Jr., L. P., Que, L., Lipscomb, J. D., & Ohlendorf, D. H. (2004) J. Bacteriol. 186 1945-1958.
Woo, E. J., Dunwell, J. M., Goodenough, P. W., Marvier, A. C. & Pickersgill, R. W. (2000) Nat. Struct. Biol. 7 1036-1040.
Zhang, Y., Colabroy, K. L., Begley, T. P. & Ealick, S. E. (2005) Biochemistry 44 7632-7643.