幽門螺旋桿菌HP0242蛋白晶體結構與功能之研究

簡易檢索 / 詳目顯示

回結果列表

研究生：	陳柏蒼 Bo-Tsang Chen
論文名稱：	幽門螺旋桿菌HP0242蛋白晶體結構與功能之研究 Crystal Structure and Functional Studies of HP0242 from Helicobacter pylori
指導教授：	孫玉珠 Yuh-Ju Sun
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2005
畢業學年度：	93
語文別：	英文
論文頁數：	63
中文關鍵詞：	幽門螺旋桿菌、未知功能蛋白
外文關鍵詞：	HP0242, hypothetical protein, crystal structure, X-ray, Helicobacter pylori
相關次數：	點閱：3 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

HP0242是來自人類胃腸道病原菌 ─ 幽門螺旋桿菌（Helicobacter pylori）的一個未知功能蛋白，分子量為 11kDa。我們利用X光晶體繞射實驗來決定HP0242的立體結構，再透過多波長異常色散法 (Multiwavelength anomalous dispersion, MAD) 解決相位問題，最後得到解析度2.27Å晶體結構。 HP0242單元體 (monomer) 折疊成一個包含4個helices的三角形結構，兩個單元體則利用兩個Helix2之間強烈的疏水性作用力去形成雙元體 (dimer) 結構，其埋入的界面表面面積有3971 Å2，Helix2也利用疏水性及親水性作用力分別和Helix3及Helix4穩定雙元體結構。因此Helix2在HP0242雙元體的形成過程中扮演重要的角色。並且，根據膠體過濾層析法和分析型超高速離心機的結果顯示HP0242在水溶液中也是雙元體，我們推測雙元體結構應該是此蛋白執行生化功能的形式。在DALI 演算法上，根據三度空間結構所作的同源分析，顯示HP0242為一個新式摺疊 (novel fold) 的蛋白。 HP0242基因位在napA基因 (HP0243) 的下游，而napA基因上游直接有個鐵離子攝取調控蛋白 (ferric-uptake regulator) 的結合位。至今， HP0242與它的同源蛋白在細胞中所扮演的生物性功能仍未知。我們的實驗結果或許可以給予將來進行生物功能研究的一個方向。

HP0242 is a hypothetical protein from a human gastric pathogen, Helicobacter pylori. Here, we report the first crystal structure of HP0242 at 2.27 Å resolution were grown by the hanging drop vapor diffusion method and determined by multiwavelength anomalous dispersion (MAD) phasing. The overall structure of HP0242 folds like a musical instrument-triangle with four helices. Two monomers tightly interlock each other by Helix2 to form a dimer with extremely strong interactions, the total buried surface area of dimer is 3971 Å2. Helix2 also uses hydrophobic and hydrophilic residues to interact with Helix3 and Helix4 of another monomer, respectively. Helix2 is essential in the formation of HP0242 dimer, these interactions mentioned above stabilize the HP0242 structure. HP0242 is recognized as a dimer in solution according to the results of gel filtration and analytical ultracentrifugation. We suggest that dimer might represent the functional state for HP0242. A structure-based homology analysis with the DALI algorithm indicates that HP0242 has a novel fold. The HP0242 gene is next to the napA gene and there is a binding site for ferric-uptake regulator in upstream of napA gene. To date, none of the HP0242 and its homologues has been assigned a cellular function. Our results may shed a light on further functional studies based on the unique protein folding.

Abstract
Chinese    ................................................1
English    ................................................2
Chapter 1 Introduction ..................................3
Chapter 2 Materials and Methods .........................6
2.1 Cloning of HP0242 gene ..............................6
2.2 Protein expression and purification .................6
2.3. Protein characterization ...........................7
2.4 Crystallization .....................................8
2.5. X-ray data collection ..............................8
2.6. Self-rotation functions ............................9
2.7 Structure determination and refinement ..............9
Chapter 3 Results ......................................11
3.1 HP0242 characterization ............................11
3.2 Crystallization ....................................12
3.3 Space group determination ..........................12
3.4 Phase determination ................................13
3.5 Structure determination and refinement of HP0242 ...14
3.6 Overall structure ..................................15
3.7 Novel folding ......................................16
3.8 Oligomeric structure ...............................17
Chapter 4 Discussion ...................................19
Figures ................................................24
Tables .................................................45
Appendix ...............................................55
References .............................................56

                                

1. Ahn, H. J., S. J. Eom, H.-J. Yoon, B. I. Lee, H. Cho, and S. W. Suh. 2003. Crystal structure of class I acetohydroxy acid isomeroreductase from Pseudomonas aeruginosa. J. Mol. Biol. 328:505-515.
2. Alm, R. A., L.-S. L. Ling, D. T. Moir, B. L. King, E. D. Brown, P. C. Doig, D. R. Smith, B. Noonan, B. C. Guild, B. L. deJonge, G. Carmel, P. J. Tummino, A. Caruso, M. Uria-Nickelsen, D. M. Mills, C. Ives, R. Gibson, D. Merberg, S. D. Mills, Q. Jiang, D. E. Taylor, G. F. Vovis, and T. J. Trust. 1999. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176-180.
3. Baar, C., M. Eppinger, G. Raddatz, J. Simon, C. Lanz, O. Klimmek, R. Nandakumar, R. Gross, A. Rosinus, H. Keller, P. Jagtap, B. Linke, F. Meyer, H. Lederer, and S. C. Schuster. 2003. Complete genome sequence and analysis of Wolinella succinogenes. Proc. Natl. Acad. Sci. USA 100:11690-11695.
4. Bennett, M. J., and D. Eisenberg. 2004. The evolving role of 3D domain swapping in proteins. Structure 12:1339-1341.
5. Biou, V., R. Dumas, C. Cohen-Addad, R. Douce, D. Job, and E. Pebay-Peyroula. 1997. The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65 A resolution. EMBO J. 16:3405-3415.
6. Brunger, A. T., P. D. Adams, G. M. Clore, W. L. DeLano, P. Gros, R. W. Grosse-Kunstleve, J. S. Jiang, J. Kuszewski, N. Nilges, N. S. Pannu, R. J. Read, L. M. Rice, T. Simonson, and G. L. Warren. 1998. Crystallography & NMR System: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54:905-921.
7. Cendron, L., A. Seydel, A. Angelini, R. Battistutta, and G. Zanotti. 2004. Crystal structure of CagZ, a protein from the Helicobacter pylori pathogenicity island that encodes for a type IV secretion system. J. Mol. Biol. 340:881-889.
8. Coci, P., A. Ilari, E. Falvo, and E. Chiancone. 2003. The Dps protein of Agrobacterium tumefaciens does not bind to DNA but protects it toward oxidative cleavage. J. Biol. Chem. 278:20319-20326.
9. Elkins, P. A., J. M. Watts, M. Zalacain, A. van Thiel, P. R. Vitazka, M. Redlak, C. Andraos-Selim, F. Rastinejad, and W. M. Holmes. 2003. Insights into catalysis by a knotted TrmD tRNA methyltransferase. J. Mol. Biol. 333:931-949.
10. Evans, D. J. J., D. G. Evans, T. Takemura, H. Nakano, H. C. Lampert, D. Y. Graham, D. N. Granger, and P. R. Kvietys. 1995. Characterization of a Helicobacter pylori neutrophil-activating protein. Infect. Immun. 63:2213-2220.
11. Evansa, G., and R. F. Pettiferb. 2001. CHOOCH: a program for deriving anomalous-scattering factors from X-ray fluorescence spectra. J. Appl. Crystallogr. 34:82-86.
12. Holm, L., and C. Sander. 1993. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233:123-138.
13. Ilari, A., S. Stefanini, E. Chiancone, and D. Tsernoglou. 2000. The dodecameric ferritin from Listeria innocua contains a novel intersubunit iron-binding site. Nat. Struct. Mol. Biol. 7:38-43.
14. Jacobs, S. A., J. M. Harp, S. Devarakonda, Y. Kim, F. Rastinejad, and S. Khorasanizadeh. 2002. The active site of the SET domain is constructed on a knot. Nat. Struct. Mol. Biol. 9:833-838.
15. Jasin., M., L. Regan., and P. Schimmel. 1983. Modular arrangement of functional domains along the sequence of an aminoacyl tRNA synthetase. Nature 306:441-447.
16. Kraulis, P. J. 1991. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24:946-950.
17. Laskowski, R. A., M. W. Macarthur, D. S. Moss, and J. M. Thornton. 1993. Procheck - a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26:283-291.
18. Liang, J., H. Edelsbrunner, and C. Woodward. 1998. Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design. Protein Sci. 7:1884-1897.
19. Luo, Y., M. G. Bertero, E. A. Frey, R. A. Pfuetzner, M. R. Wenk, L. Creagh, S. L. Marcus, D. Lim, F. Sicheri, C. Kay, C. Haynes, B. B. Finlay, and N. C. J. Strynadka. 2001. Structural and biochemical characterization of the type III secretion chaperones CesT and SigE. Nat. Struct. Mol. Biol. 8:1031-1036.
20. Marchler-Bauer, A., and S. H. Bryant. 2004. CD-Search: protein domain annotations on the fly. Nucleic Acids Res. 32:W327-331.
21. McPherson, A. 1982. Preparation and Analysis of Protein Crystals. John Wiley, New York.
22. McRee, D. E. 1999. XtalView/Xfit - a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125:156-165.
23. Montecucco, C., and M. d. Bernard. 2003. Molecular and cellular mechanisms of action of the vacuolating cytotoxin (VacA) and neutrophil-activating protein (HP-NAP) virulence factors of Helicobacter pylori. Micro. Infect. 5:715-721.
24. N.I.H. consensus development panel on Helicobacter pylori in peptic ulcer and disease. 1994. Helicobacter pylori in peptic ulcer disease. J. Am. Med. Assoc. 272:65-69.
25. Nicholls, A., K. A. Sharp, and B. Honig. 1991. Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11:281-296.
26. Nureki, O., M. Shirouzu, K. Hashimoto, R. Ishitani, T. Terada, M. Tamakoshi, T. Oshima, M. Chijimatsu, K. Takio, D. G. Vassylyev, T. Shibata, Y. Inoue, S. Kuramitsub, and S. Yokoyamaa. 2002. An enzyme with a deep trefoil knot for the active-site architecture. Acta Crystallogr. D 58:1129-1137.
27. Nureki, O., K. Watanabe, S. Fukai, R. Ishii, Y. Endo, H. Hori, and S. Yokoyama. 2004. Deep knot structure for construction of active site and cofactor binding site of tRNA modification enzyme. Structure 12:593-602.
28. Orengo, C., A. Michie, S. Jones, D. Jones, M. Swindells, and J. Thornton. 1997. CATH - a hierarchic classification of protein domain structures. Structure 5:1093-1108.
29. Otwinowski, Z., and W. Minor. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276A:307-326.
30. Prasad Bahadur, R., P. Chakrabarti, F. Rodier, and J. Janin. 2004. A dissection of specific and non-specific protein-protein interfaces. J. Mol. Biol. 336:943-955.
31. Sara, B., and G. Partho. 2001. Structure of the Yersinia type III secretory system chaperone SycE. Nat. Struct. Mol. Biol. 8:974-978.
32. Schuck, P., M. A. Perugini, N. R. Gonzales, G. J. Howlett, and D. Schubert. 2002. Size-distribution analysis of proteins by analytical ultracentrifugation: Strategies and application to model systems. Biophysical Journal 82:1096-1111.
33. Shindyalov, I., and P. Bourne. 1998. Protein structure alignment by incremental combinatorial extension (CE) of the optimal path. Protein Eng. 11:739-747.
34. Stebbins, C. E., and J. E. Galan. 2001. Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type III secretion. Nature 414:77-81.
35. Suerbaum, S., C. Josenhans, T. Sterzenbach, B. Drescher, P. Brandt, M. Bell, M. Droge, B. Fartmann, H.-P. Fischer, Z. Ge, A. Horster, R. Holland, K. Klein, J. Konig, L. Macko, G. L. Mendz, G. Nyakatura, D. B. Schauer, Z. Shen, J. Weber, M. Frosch, and J. G. Fox. 2003. The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus. Proc. Natl. Acad. Sci. USA 100:7901-7906.
36. Swairjo, M. A., F. J. Otero, X.-L. Yang, M. A. Lovato, R. J. Skene, D. E. McRee, L. R. de Pouplana, and P. Schimmel. 2004. Alanyl-tRNA synthetase crystal structure and design for acceptor-stem recognition. Mol. Cell 13:829-841.
37. Taylor, W. R. 2000. A deeply knotted protein structure and how it might fold. Nature 406:916-919.
38. Terwilliger, T. C. 2000. Maximum-likelihood density modification. Acta Crystallogr. D 56:965-972.
39. Terwilliger, T. C., and J. Berendzen. 1999. Evaluation of macromolecular electron-density map quality using the correlation of local r.m.s. density. Acta Crystallogr. D 55:1872-1877.
40. Thompson, J., D. Higgins, and T. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.
41. Tomb, J.-F., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney, L. M. Fitzegerald, N. Lee, M. D. Adams, E. K. Hickey, D. E. Berg, J. D. Gocayne, T. R. Utterback, J. D. Peterson, J. M. Kelley, M. D. Cotton, J. M. Weidman, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky, P. D. Karp, H. O. Smith, C. M. Fraser, and J. C. Venter. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 389:539-547.
42. Tonello, F., W. G. Dundon, B. Satin, M. Molinari, G. Tognon, G. Grandi, G. Del Giudice, R. Rappuoli, and C. Montecucco. 1999. The Helicobacter pylori neutrophil-activating protein is an iron-binding protein with dodecameric structure. Mol. Microbiol. 34:238-246.
43. Tong, L., and M. Rossmann. 1990. The Locked Rotation Function. Acta Crystallogr A. 46:783-792.
44. Warren, J. R., and B. Marshall. 1983. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1:1273-1275.
45. Wen, Y., E. A. Marcus, U. Matrubutham, M. A. Gleeson, D. R. Scott, and G. Sachs. 2003. Acid-Adaptive Genes of Helicobacter pylori. Infect. Immun. 71:5921-5939.
46. Zanotti, G., E. Papinutto, W. G. Dundon, R. Battistutta, M. Seveso, G. D. Giudice, R. Rappuoli, and C. Montecucco. 2002. Structure of the neutrophil-activating protein from Helicobacter pylori. J. Mol. Biol. 323:125-130.

全文公開日期本全文未授權公開 (校內網路)
全文公開日期本全文未授權公開 (校外網路)

簡易檢索 / 詳目顯示

相關論文