(3.238.235.155) 您好!臺灣時間:2021/05/11 19:37
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:蔡昆縉
研究生(外文):Kuen-Jin Tsai
論文名稱:利用高效能液相層析分析與XpsE蛋白結合之核苷酸及其水解ATP之過程
論文名稱(外文):Analysis of bound nucleotides on XpsE and its ATP hydrolysis activity by HPLC
指導教授:胡念台
指導教授(外文):Nien-Tai Hu
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:2009
畢業學年度:97
語文別:中文
論文頁數:71
中文關鍵詞:高效能液相層析
外文關鍵詞:XpsEHPLC
相關次數:
  • 被引用被引用:0
  • 點閱點閱:101
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
摘 要

  十字花科黑腐病菌 (Xanthomonas campestris pv. campestris) 利用第二型蛋白分泌機制,分泌各種水解酵素來破壞植物細胞的表面構造,此裝置主要是由 12 個專一性蛋白所負責,這些蛋白可能會在細菌內外胞膜間形成複合體,將位於胞質週緣區 (periplasm) 的水解酵素運送至胞外。其中,XpsE 蛋白是唯一位於胞內的非膜蛋白,其胺基酸序列包含四個 nucleotide binding motifs,本身具有 ATPase 的活性;XpsE 會聚合形成 multimer,並與內膜蛋白 XpsL 朝向胞內的 N domain 之間有交互作用,推測 XpsE 可能在第二型分泌機制中扮演著能量提供者的角色。為更深入了解 XpsE 蛋白水解 ATP 機制,本研究利用高效能液相層析系統 (HPLC) 分析與 XpsE 蛋白結合之核苷酸。結果發現在儀器偵測範圍內,不論是單獨表現的 XpsE 蛋白或者是 XpsE/MBP-XpsLN 中的 XpsE,在經由親合性管柱純化,並煮沸釋放結合的核苷酸後,於 HPLC 均無偵測到明顯訊號。進一步將 XpsE/MBP-XpsLN 複合體外加 ATP 以及 Mg2+ 於冰上反應,並以G-25 Sepharose 移除 free-nucleotide 之後,同樣煮沸後偵測,仍未見與蛋白結合的核苷酸訊號。然而,於低溫 (4°C) 狀態下,外加的 ATP 似乎會被 XpsE/MBP-XpsLN水解成 ADP,當 XpsE 與 ATP 摩耳數比約為 1:13 時,於 15 分鐘的反應時間內,約有 40% 的 ATP 被水解成 ADP,且這樣的水解活性會受到 vanadate 的抑制。進一步檢查此狀態下的 ATP 水解活性隨反應時間呈現的變化,發現在開始反應不久,ATP 的水解即達到飽和狀態。綜合以上訊息,說明經由親合性管柱所純化到的 XpsE 蛋白或 XpsE/MBP-XpsLN 複合體中的 XpsE可能處於未結合核苷酸,或者僅少數蛋白與核苷酸結合的狀態;而外加的 ATP 雖能於低溫下被複合體中的 XpsE 水解產生 ADP,但卻無法於水解進行過程中偵測到結合於蛋白中的 ATP/ADP,暗示反應產生的 ADP 可能與 XpsE 未緊密結合而迅速自蛋白中釋放。
Abstract

  Xanthomonas campestris pv. campestris utilizes the type II secretion system for secreting extracellular hydrolytic enzymes to degrade the surface structure of plant cells. The secretion apparatus is composed of 12 gene products that are thought to form a multiprotein complex, which spans the periplasmic compartment. It is specifically involved in translocation of the secreted proteins across outer membrane. Of all protein components, XpsE is the only cytoplasmic protein that lacks any membrane spanning region. There are four essential nucleotide binding motifs located within the C domain of XpsE, which is made capable of ATP binding and hydrolyzing activities. As suggested from previous studies, XpsE oligomerizes into mutilmer before it interacts with the N domain of XpsL, an integral membrane protein. It has been proposed that XpsE may play an important role as energy supplier and/or signal transmitter. In order to gain better understanding about the molecular mechanism of how XpsE hydrolyzes ATP, I made use of HPLC for identifying protein-bound nucleotide. Following purification through affinity column, XpsE or XpsE/MBP-XpsLN complex was boiled to release the protein-bound nucleotide and separated through HPLC. As suggested from the HPLC profile, no significant signal that is above the machine detection limit was detected at the ADP/ATP retention time. To examine if bound nucleotide could be detected by incubating the XpsE/MBP-XpsLN with exogenously added ATP, I incubated the protein complex with ATP and Mg2+ on ice for 15 min followed by passing through the G25 Sepharose for the removal of free nucleotides and boiling for the release of protein-bound nucleotide before HPLC analysis. There was still no detectable protein-bound ATP/ADP. Unexpectedly, I found that the added ATP was hydrolyzed by the protein complex at 4 ºC. Within 15 minutes, about 40 % of the added ATP was converted into ADP. Moreover, such ATP hydrolysis activity was inhibited by vanadate. Further studies indicated that at the molecular ratio of XpsE to ATP of 1:13, ATP hydrolysis seems to reach its maximal level within a very short period of time. In summary, XpsE or XpsE/MBP-XpsLN complex, when purified from affinity column, may stay in a state with no bound or only trace amount of ATP/ADP. Although the added ATP could be hydrolyzed at 4 ºC, there was still no detectable protein-bound ATP/ADP implying the ADP generated from ATP hydrolysis by the XpsE/MBP-XpsLN was released from XpsE soon after the ATP was hydrolyzed.
目 錄

中文摘要 I
英文摘要 II
前言 3
一、 格蘭氏陰性菌的蛋白分泌系統 3
(一)、第一及第三類型分泌系統: 3
(二)、第二類型分泌系統、TPS 與 autotransporter: 4
(三)、第四類型分泌系統: 4
(四)、第六類型分泌系統: 4
二、 十字花科黑腐病菌中的第二型分泌系統 5
(一)、外胞膜 (outer membrane) 蛋白: 6
(二)、胞質週緣區 (periplasm) 蛋白: 7
(三)、內胞膜 (inner membrane) 蛋白 7
(四)、胞內 (cytosolic) 蛋白 8
1. XpsE與 XpsL 之間的交互作用關係 8
2. XpsE-XpsE之間的交互作用關係: 9
三、 XPSE 的 ATPASE 水解活性 10
材料與方法 12
一、 材料 12
二、 實驗方法 12
【1】蛋白的純化與分析 12
【1-1】 以 IPTG 誘導蛋白的大量表現 (overexpression) 12
【1-2】 以 Strep-Tactin 親合性管柱層析純化XpsE蛋白 12
【1-3】 以 Strep-Tactin 以及 amylose 親合性管柱進行 XpsE/MBP-XpsLN 複合體蛋白的接續純化 13
【1-4】 蛋白質電泳分析 (SDS-PAGE) 13
【1-5】 SDS-PAGE 銀染 (silver stain) 14
【1-6】 西方點墨法 (Western blot) 14
【1-7】 Bradford’s method 蛋白估量 15
【2】蛋白最適緩衝液之溶解度測試 15
【2-1】 Tris 緩衝液極限溶解度測試 15
【2-2】 Dynamic light scattering (DLS) 操作與分析 15
【3】ATPase 活性測定 16
【3-1】 利用〔γ-32P〕ATP 測試 ATP 水解活性 16
【3-2】 ATP 水解活性的計算方式 17
【4】FPLC 系統操作與分析 18
【4-1】 實驗前的準備與操作 18
【4-2】 Size exclusion chromatography 分析 19
【5】高效能液相層析儀 (HPLC) 操作與分析 19
【5-1】 實驗前的準備與操作 19
【5-2】 數據的分析及處理 20
【5-3】 雜訊及裂峰的處理 20
【5-4】 ATP水解過程的觀察及活性計算 20
結果 22
一、 HPLC 系統的建立 22
1. 分析管柱的選擇以及移動相的系統建立 22
2. 標準曲線的建立以及靈敏度測定 23
二、 蛋白純化系統的改善 — 最適緩衝溶液的搜尋 23
1. 以 DLS 搜尋使蛋白具有較高溶解度的緩衝液 24
2. 不同緩衝液對 XpsE ATPase 活性的影響 25
3. Tris 與 HEPES 緩衝液對蛋白產量及均質性的比較 26
三、 與 XpsE 結合的核苷酸測定 26
1. 單獨表現的 XpsE 中並未偵測到核苷酸 26
2. 與 MBP-XpsLN共表現而形成穩定複合體中的 XpsE 也未偵測到核苷酸 27
四、 藉由外加的 ATP 觀察 XPSE/MBP-XPSLN 水解 ATP 的情形 27
1. XpsE/MBP- XpsLN 複合體能將外加的 ATP 水解成 ADP 27
2. 微量的 ADP 及 ATP 可能結合於 XpsE/MBP-XpsLN 複合體當中 28
3. 加入 Vanadate 會阻斷外加 ATP 的水解過程 28
4. 加入 Vanadate 仍無法偵測到結合於 XpsE/MBP-XpsLN 複合體中的核苷酸 29
5. 低溫下 XpsE/MBP-XpsLN 複合體的 ATP 水解活性 29
參考文獻 35
圖……………………………………………………………………………………….43
表……………………………………………………………………………………….61
附錄…………………………………………………………………………………….63
參考文獻

Abendroth, J., Murphy, P., Sandkvist, M., Bagdasarian, M. and Hol, W.G. (2005) The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae. J Mol Biol, 348, 845-855.
Anderson, D.M. and Schneewind, O. (1999) Type III machines of Gram-negative pathogens: injecting virulence factors into host cells and more. Curr Opin Microbiol, 2, 18-24.
Auvray, F., Ozin, A.J., Claret, L. and Hughes, C. (2002) Intrinsic membrane targeting of the flagellar export ATPase FliI: interaction with acidic phospholipids and FliH. J Mol Biol, 318, 941-950.
Bingle, L.E., Bailey, C.M. and Pallen, M.J. (2008) Type VI secretion: a beginner''s guide. Curr Opin Microbiol, 11, 3-8.
Bitter, W. (2003) Secretins of Pseudomonas aeruginosa: large holes in the outer membrane. Arch Microbiol, 179, 307-314.
Bitter, W., Koster, M., Latijnhouwers, M., de Cock, H. and Tommassen, J. (1998) Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mol Microbiol, 27, 209-219.
Bochtler, M., Hartmann, C., Song, H.K., Bourenkov, G.P., Bartunik, H.D. and Huber, R. (2000) The structures of HsIU and the ATP-dependent protease HsIU-HsIV. Nature, 403, 800-805.
Braibant, M., Gilot, P. and Content, J. (2000) The ATP binding cassette (ABC) transport systems of Mycobacterium tuberculosis. FEMS Microbiol Rev, 24, 449-467.
Camberg, J.L., Johnson, T.L., Patrick, M., Abendroth, J., Hol, W.G. and Sandkvist, M. (2007) Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids. Embo J, 26, 19-27.
Camberg, J.L. and Sandkvist, M. (2005) Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J Bacteriol, 187, 249-256.
Chen, L.Y., Chen, D.Y., Miaw, J. and Hu, N.T. (1996) XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer. J Biol Chem, 271, 2703-2708.
Chen, Y., Shiue, S.J., Huang, C.W., Chang, J.L., Chien, Y.L., Hu, N.T. and Chan, N.L. (2005) Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system. J Biol Chem, 280, 42356-42363.
De Leon, M., Welcher, A.A., Nahin, R.H., Liu, Y., Ruda, M.A., Shooter, E.M. and Molina, C.A. (1996) Fatty acid binding protein is induced in neurons of the dorsal root ganglia after peripheral nerve injury. J Neurosci Res, 44, 283-292.
d''Enfert, C., Ryter, A. and Pugsley, A.P. (1987) Cloning and expression in Escherichia coli of the Klebsiella pneumoniae genes for production, surface localization and secretion of the lipoprotein pullulanase. Embo J, 6, 3531-3538.
Douville, K., Price, A., Eichler, J., Economou, A. and Wickner, W. (1995) SecYEG and SecA are the stoichiometric components of preprotein translocase. J Biol Chem, 270, 20106-20111.
Dums, F., Dow, J.M. and Daniels, M.J. (1991) Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria. Mol Gen Genet, 229, 357-364.
Filloux, A. (2004) The underlying mechanisms of type II protein secretion. Biochim Biophys Acta, 1694, 163-179.
Filloux, A., Bally, M., Ball, G., Akrim, M., Tommassen, J. and Lazdunski, A. (1990) Protein secretion in gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria. Embo J, 9, 4323-4329.
Filloux, A., Hachani, A. and Bleves, S. (2008) The bacterial type VI secretion machine: yet another player for protein transport across membranes. Microbiology, 154, 1570-1583.
Fullner, K.J., Lara, J.C. and Nester, E.W. (1996) Pilus assembly by Agrobacterium T-DNA transfer genes. Science, 273, 1107-1109.
Hanson, P.I. and Whiteheart, S.W. (2005) AAA+ proteins: have engine, will work. Nat Rev Mol Cell Biol, 6, 519-529.
Hu, N.T., Hung, M.N., Chen, D.C. and Tsai, R.T. (1998) Insertion mutagenesis of XpsD, an outer-membrane protein involved in extracellular protein secretion in Xanthomonas campestris pv. campestris. Microbiology, 144 ( Pt 6), 1479-1486.
Hu, N.T., Hung, M.N., Chiou, S.J., Tang, F., Chiang, D.C., Huang, H.Y. and Wu, C.Y. (1992) Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J Bacteriol, 174, 2679-2687.
Hu, N.T., Hung, M.N., Liao, C.T. and Lin, M.H. (1995) Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris. Microbiology, 141 ( Pt 6), 1395-1406.
Hu, N.T., Leu, W.M., Lee, M.S., Chen, A., Chen, S.C., Song, Y.L. and Chen, L.Y. (2002) XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes. Biochem J, 365, 205-211.
Kazmierczak, B.I., Mielke, D.L., Russel, M. and Model, P. (1994) pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer. J Mol Biol, 238, 187-198.
Lazarowski, E.R., Boucher, R.C. and Harden, T.K. (2000) Constitutive release of ATP and evidence for major contribution of ecto-nucleotide pyrophosphatase and nucleoside diphosphokinase to extracellular nucleotide concentrations. J Biol Chem, 275, 31061-31068.
Lee, H.M., Tyan, S.W., Leu, W.M., Chen, L.Y., Chen, D.C. and Hu, N.T. (2001) Involvement of the XpsN protein in formation of the XpsL-xpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus. J Bacteriol, 183, 528-535.
Lill, R., Dowhan, W. and Wickner, W. (1990) The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins. Cell, 60, 271-280.
Linderoth, N.A., Simon, M.N. and Russel, M. (1997) The filamentous phage pIV multimer visualized by scanning transmission electron microscopy. Science, 278, 1635-1638.
Mazar, J. and Cotter, P.A. (2006) Topology and maturation of filamentous haemagglutinin suggest a new model for two-partner secretion. Mol Microbiol, 62, 641-654.
Michel, G., Bleves, S., Ball, G., Lazdunski, A. and Filloux, A. (1998) Mutual stabilization of the XcpZ and XcpY components of the secretory apparatus in Pseudomonas aeruginosa. Microbiology, 144 ( Pt 12), 3379-3386.
Natale, P., Bruser, T. and Driessen, A.J. (2008) Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane--distinct translocases and mechanisms. Biochim Biophys Acta, 1778, 1735-1756.
Neuwald, A.F., Aravind, L., Spouge, J.L. and Koonin, E.V. (1999) AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res, 9, 27-43.
Nouwen, N., Ranson, N., Saibil, H., Wolpensinger, B., Engel, A., Ghazi, A. and Pugsley, A.P. (1999) Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation. Proc Natl Acad Sci U S A, 96, 8173-8177.
Ogura, T. and Wilkinson, A.J. (2001) AAA+ superfamily ATPases: common structure--diverse function. Genes Cells, 6, 575-597.
Planet, P.J., Kachlany, S.C., DeSalle, R. and Figurski, D.H. (2001) Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification. Proc Natl Acad Sci U S A, 98, 2503-2508.
Pukatzki, S., Ma, A.T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, W.C., Heidelberg, J.F. and Mekalanos, J.J. (2006) Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A, 103, 1528-1533.
Py, B., Loiseau, L. and Barras, F. (1999) Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL. J Mol Biol, 289, 659-670.
Ray, W.J., Jr. and Puvathingal, J.M. (1990) Characterization of a vanadate-based transition-state-analogue complex of phosphoglucomutase by kinetic and equilibrium binding studies. Mechanistic implications. Biochemistry, 29, 2790-2801.
Robien, M.A., Krumm, B.E., Sandkvist, M. and Hol, W.G. (2003) Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae. J Mol Biol, 333, 657-674.
Sandkvist, M. (2001) Type II secretion and pathogenesis. Infect Immun, 69, 3523-3535.
Sandkvist, M., Bagdasarian, M., Howard, S.P. and DiRita, V.J. (1995) Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae. Embo J, 14, 1664-1673.
Sandkvist, M., Keith, J.M., Bagdasarian, M. and Howard, S.P. (2000) Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae. J Bacteriol, 182, 742-748.
Satyshur, K.A., Worzalla, G.A., Meyer, L.S., Heiniger, E.K., Aukema, K.G., Misic, A.M. and Forest, K.T. (2007) Crystal structures of the pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility. Structure, 15, 363-376.
Savvides, S.N., Yeo, H.J., Beck, M.R., Blaesing, F., Lurz, R., Lanka, E., Buhrdorf, R., Fischer, W., Haas, R. and Waksman, G. (2003) VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion. Embo J, 22, 1969-1980.
Shiue, S.J., Kao, K.M., Leu, W.M., Chen, L.Y., Chan, N.L. and Hu, N.T. (2006) XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. Embo J, 25, 1426-1435.
Sianidis, G., Karamanou, S., Vrontou, E., Boulias, K., Repanas, K., Kyrpides, N., Politou, A.S. and Economou, A. (2001) Cross-talk between catalytic and regulatory elements in a DEAD motor domain is essential for SecA function. Embo J, 20, 961-970.
Smith, C.A. and Rayment, I. (1996) X-ray structure of the magnesium(II).ADP.vanadate complex of the Dictyostelium discoideum myosin motor domain to 1.9 A resolution. Biochemistry, 35, 5404-5417.
Strom, M.S., Bergman, P. and Lory, S. (1993) Identification of active-site cysteines in the conserved domain of PilD, the bifunctional type IV pilin leader peptidase/N-methyltransferase of Pseudomonas aeruginosa. J Biol Chem, 268, 15788-15794.
Thomas, J.D., Reeves, P.J. and Salmond, G.P. (1997) The general secretion pathway of Erwinia carotovora subsp. carotovora: analysis of the membrane topology of OutC and OutF. Microbiology, 143 ( Pt 3), 713-720.
Tsai, R.T., Leu, W.M., Chen, L.Y. and Hu, N.T. (2002) A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus. Biochem J, 367, 865-871.
Veenendaal, A.K., van der Does, C. and Driessen, A.J. (2004) The protein-conducting channel SecYEG. Biochim Biophys Acta, 1694, 81-95.
Voulhoux, R., Ball, G., Ize, B., Vasil, M.L., Lazdunski, A., Wu, L.F. and Filloux, A. (2001) Involvement of the twin-arginine translocation system in protein secretion via the type II pathway. Embo J, 20, 6735-6741.
Walker, J.E., Saraste, M., Runswick, M.J. and Gay, N.J. (1982) Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. Embo J, 1, 945-951.
Werber, M.M., Peyser, Y.M. and Muhlrad, A. (1992) Characterization of stable beryllium fluoride, aluminum fluoride, and vanadate containing myosin subfragment 1-nucleotide complexes. Biochemistry, 31, 7190-7197.
Yamagata, A. and Tainer, J.A. (2007) Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. Embo J, 26, 878-890.
Yeo, H.J., Savvides, S.N., Herr, A.B., Lanka, E. and Waksman, G. (2000) Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system. Mol Cell, 6, 1461-1472.
Zhang, Y. and Cremer, P.S. (2006) Interactions between macromolecules and ions: The Hofmeister series. Curr Opin Chem Biol, 10, 658-663.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔