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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳世雄(Shih-Hsiung Wu) | |
| dc.contributor.author | Yu-Ching Lin | en |
| dc.contributor.author | 林宇慶 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:11:28Z | - |
| dc.date.available | 2012-02-04 | |
| dc.date.copyright | 2010-02-04 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-01-27 | |
| dc.identifier.citation | 1. Buchanan, C.E., Hua, S.S., Avni, H. and Markovitz, A. (1973) Transcriptional control of the calactose operon by the capR (lon) and capT genes. J Bacteriol, 114, 891-893.
2. Chung, C.H. and Goldberg, A.L. (1981) The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La. Proc Natl Acad Sci U S A, 78, 4931-4935. 3. Chin, D.T., Goff, S.A., Webster, T., Smith, T. and Goldberg, A.L. (1988) Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La. J Biol Chem, 263, 11718-11728. 4. Amerik, A., Antonov, V.K., Ostroumova, N.I., Rotanova, T.V. and Chistiakova, L.G. (1990) [Cloning, structure and expression of the full-size lon gene in Escherichia coli coding for ATP-dependent La-proteinase]. Bioorg Khim, 16, 869-880. 5. Ito, K., Udaka, S. and Yamagata, H. (1992) Cloning, characterization, and inactivation of the Bacillus brevis lon gene. J Bacteriol, 174, 2281-2287. 6. Kutejova, E., Durcova, G., Surovkova, E. and Kuzela, S. (1993) Yeast mitochondrial ATP-dependent protease: purification and comparison with the homologous rat enzyme and the bacterial ATP-dependent protease La. FEBS Lett, 329, 47-50. 7. Wang, N., Gottesman, S., Willingham, M.C., Gottesman, M.M. and Maurizi, M.R. (1993) A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease. Proc Natl Acad Sci U S A, 90, 11247-11251. 8. Amerik, A., Petukhova, G.V., Grigorenko, V.G., Lykov, I.P., Yarovoi, S.V., Lipkin, V.M. and Gorbalenya, A.E. (1994) Cloning and sequence analysis of cDNA for a human homolog of eubacterial ATP-dependent Lon proteases. FEBS Lett, 340, 25-28. 9. Kuzela, S. and Goldberg, A.L. (1994) Mitochondrial ATP-dependent protease from rat liver and yeast. Methods Enzymol, 244, 376-383. 10. Roudiak, S.G., Seth, A., Knipfer, N. and Shrader, T.E. (1998) The lon protease from Mycobacterium smegmatis: molecular cloning, sequence analysis, functional expression, and enzymatic characterization. Biochemistry, 37, 377-386. 11. Ostersetzer, O., Kato, Y., Adam, Z. and Sakamoto, W. (2007) Multiple intracellular locations of Lon protease in Arabidopsis: evidence for the localization of AtLon4 to chloroplasts. Plant Cell Physiol, 48, 881-885. 12. Clemmer, K.M. and Rather, P.N. (2008) The Lon protease regulates swarming motility and virulence gene expression in Proteus mirabilis. J Med Microbiol, 57, 931-937. 13. Howard-Flanders, P., Simson, E. and Theriot, L. (1964) A Locus That Controls Filament Formation And Sensitivity To Radiation In Escherichia Coli K-12. Genetics, 49, 237-246. 14. Ogura, T. and Wilkinson, A.J. (2001) AAA+ superfamily ATPases: common structure--diverse function. Genes Cells, 6, 575-597. 15. Waxman, L. and Goldberg, A.L. (1982) Protease La from Escherichia coli hydrolyzes ATP and proteins in a linked fashion. Proc Natl Acad Sci U S A, 79, 4883-4887. 16. Rotanova, T.V., Melnikov, E.E., Khalatova, A.G., Makhovskaya, O.V., Botos, I., Wlodawer, A. and Gustchina, A. (2004) Classification of ATP-dependent proteases Lon and comparison of the active sites of their proteolytic domains. Eur J Biochem, 271, 4865-4871. 17. Tsilibaris, V., Maenhaut-Michel, G. and Van Melderen, L. (2006) Biological roles of the Lon ATP-dependent protease. Res Microbiol, 157, 701-713. 18. Rotanova, T.V., Botos, I., Melnikov, E.E., Rasulova, F., Gustchina, A., Maurizi, M.R. and Wlodawer, A. (2006) Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains. Protein Sci, 15, 1815-1828. 19. Lee, I. and Suzuki, C.K. (2008) Functional mechanics of the ATP-dependent Lon protease- lessons from endogenous protein and synthetic peptide substrates. Biochim Biophys Acta, 1784, 727-735. 20. Chung, C.H. and Goldberg, A.L. (1982) DNA stimulates ATP-dependent proteolysis and protein-dependent ATPase activity of protease La from Escherichia coli. Proc Natl Acad Sci U S A, 79, 795-799. 21. Sonezaki, S., Okita, K., Oba, T., Ishii, Y., Kondo, A. and Kato, Y. (1995) Protein substrates and heat shock reduce the DNA-binding ability of Escherichia coli Lon protease. Appl Microbiol Biotechnol, 44, 484-488. 22. Liu, T., Lu, B., Lee, I., Ondrovicova, G., Kutejova, E. and Suzuki, C.K. (2004) DNA and RNA binding by the mitochondrial lon protease is regulated by nucleotide and protein substrate. J Biol Chem, 279, 13902-13910. 23. 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. 24. Lu, B., Liu, T., Crosby, J.A., Thomas-Wohlever, J., Lee, I. and Suzuki, C.K. (2003) The ATP-dependent Lon protease of Mus musculus is a DNA-binding protein that is functionally conserved between yeast and mammals. Gene, 306, 45-55. 25. Maurizi, M.R., Trisler, P. and Gottesman, S. (1985) Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable. J Bacteriol, 164, 1124-1135. 26. Kuroda, A., Nomura, K., Ohtomo, R., Kato, J., Ikeda, T., Takiguchi, N., Ohtake, H. and Kornberg, A. (2001) Role of inorganic polyphosphate in promoting ribosomal protein degradation by the Lon protease in E. coli. Science, 293, 705-708. 27. Ngo, J.K. and Davies, K.J. (2007) Importance of the lon protease in mitochondrial maintenance and the significance of declining lon in aging. Ann N Y Acad Sci, 1119, 78-87. 28. Bota, D.A., Ngo, J.K. and Davies, K.J. (2005) Downregulation of the human Lon protease impairs mitochondrial structure and function and causes cell death. Free Radic Biol Med, 38, 665-677. 29. Higgins, J.J., Pucilowska, J., Lombardi, R.Q. and Rooney, J.P. (2004) A mutation in a novel ATP-dependent Lon protease gene in a kindred with mild mental retardation. Neurology, 63, 1927-1931. 30. Basel-Vanagaite, L. (2007) Genetics of autosomal recessive non-syndromic mental retardation: recent advances. Clin Genet, 72, 167-174. 31. Xin, W., Xiaohua, N., Peilin, C., Xin, C., Yaqiong, S. and Qihan, W. (2008) Primary function analysis of human mental retardation related gene CRBN. Mol Biol Rep, 35, 251-256. 32. Chen, S.H., Suzuki, C.K. and Wu, S.H. (2008) Thermodynamic characterization of specific interactions between the human Lon protease and G-quartet DNA. Nucleic Acids Res, 36, 1273-1287. 33. Charette, M.F., Henderson, G.W., Doane, L.L. and Markovitz, A. (1984) DNA-stimulated ATPase activity on the lon (CapR) protein. J Bacteriol, 158, 195-201. 34. Zehnbauer, B.A., Foley, E.C., Henderson, G.W. and Markovitz, A. (1981) Identification and purification of the Lon+ (capR+) gene product, a DNA-binding protein. Proc Natl Acad Sci U S A, 78, 2043-2047. 35. Simmons, L.A., Grossman, A.D. and Walker, G.C. (2008) Clp and Lon proteases occupy distinct subcellular positions in Bacillus subtilis. J Bacteriol, 190, 6758-6768. 36. Vasilyeva, O.V., Kolygo, K.B., Leonova, Y.F., Potapenko, N.A. and Ovchinnikova, T.V. (2002) Domain structure and ATP-induced conformational changes in Escherichia coli protease Lon revealed by limited proteolysis and autolysis. FEBS Lett, 526, 66-70. 37. Botos, I., Melnikov, E.E., Cherry, S., Khalatova, A.G., Rasulova, F.S., Tropea, J.E., Maurizi, M.R., Rotanova, T.V., Gustchina, A. and Wlodawer, A. (2004) Crystal structure of the AAA+ alpha domain of E. coli Lon protease at 1.9A resolution. J Struct Biol, 146, 113-122. 38. Patterson, J., Vineyard, D., Thomas-Wohlever, J., Behshad, R., Burke, M. and Lee, I. (2004) Correlation of an adenine-specific conformational change with the ATP-dependent peptidase activity of Escherichia coli Lon. Biochemistry, 43, 7432-7442. 39. Lee, A.Y., Hsu, C.H. and Wu, S.H. (2004) Functional domains of Brevibacillus thermoruber lon protease for oligomerization and DNA binding: role of N-terminal and sensor and substrate discrimination domains. J Biol Chem, 279, 34903-34912. 40. Wang, I., Lou, Y.C., Lin, Y.C., Lo, S.C., Lee, A.Y., Wu, S.H. and Chen, C. (2007) 1H, 13C and 15N resonance assignments of alpha-domain for Bacillus subtilis Lon protease. Biomol NMR Assign, 1, 201-203. 41. Ammelburg, M., Frickey, T. and Lupas, A.N. (2006) Classification of AAA+ proteins. J Struct Biol, 156, 2-11. 42. Smith, C.K., Baker, T.A. and Sauer, R.T. (1999) Lon and Clp family proteases and chaperones share homologous substrate-recognition domains. Proc Natl Acad Sci U S A, 96, 6678-6682. 43. Li, M., Rasulova, F., Melnikov, E.E., Rotanova, T.V., Gustchina, A., Maurizi, M.R. and Wlodawer, A. (2005) Crystal structure of the N-terminal domain of E. coli Lon protease. Protein Sci, 14, 2895-2900. 44. Woese, C.R. (1987) Bacterial evolution. Microbiol Rev, 51, 221-271. 45. Manachini, P.L., Fortina, M.G., Parini, C. and Craveri, R. (1985) Bacillus thevmovubev sp. nov. nom. rev. a Red-Pigmented Thermophilic Bacterium. International Journal of Systematic Bacteriology, 35, 493-496. 46. Shida, O., Takagi, H., Kadowaki, K. and Komagata, K. (1996) Proposal for two new genera, Brevibacillus gen. nov. and Aneurinibacillus gen. nov. Int J Syst Bacteriol, 46, 939-946. 47. Lee, A.Y., Tsay, S.S., Chen, M.Y. and Wu, S.H. (2004) Identification of a gene encoding Lon protease from Brevibacillus thermoruber WR-249 and biochemical characterization of its thermostable recombinant enzyme. Eur J Biochem, 271, 834-844. 48. Fu, G.K., Smith, M.J. and Markovitz, D.M. (1997) Bacterial protease Lon is a site-specific DNA-binding protein. J Biol Chem, 272, 534-538. 49. Fu, G.K. and Markovitz, D.M. (1998) The human LON protease binds to mitochondrial promoters in a single-stranded, site-specific, strand-specific manner. Biochemistry, 37, 1905-1909. 50. Lu, B., Yadav, S., Shah, P.G., Liu, T., Tian, B., Pukszta, S., Villaluna, N., Kutejova, E., Newlon, C.S., Santos, J.H. et al. (2007) Roles for the human ATP-dependent Lon protease in mitochondrial DNA maintenance. J Biol Chem, 282, 17363-17374. 51. Fried, M.G. (1989) Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis, 10, 366-376. 52. Revzin, A. (1989) Gel electrophoresis assays for DNA-protein interactions. Biotechniques, 7, 346-355. 53. Puapaiboon, U., Jai-nhuknan, J. and Cowan, J.A. (2000) Rapid and direct sequencing of double-stranded DNA using exonuclease III and MALDI-TOF MS. Anal Chem, 72, 3338-3341. 54. Sali, A. and Blundell, T.L. (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol, 234, 779-815. 55. Laskowski., R.A., Macarthur., M.W., Moss., D.S. and Thornton., J.M. (1993) PROCHECK: a program to check the stereochemicai quality of protein structures. J. Appl. Cryst., 26, 283-291. 56. Luthy, R., Bowie, J.U. and Eisenberg, D. (1992) Assessment of protein models with three-dimensional profiles. Nature, 356, 83-85. 57. Baker, N.A., Sept, D., Joseph, S., Holst, M.J. and McCammon, J.A. (2001) Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A, 98, 10037-10041. 58. http://www.pymol.org. 59. Cormack, B. (1997) Directed Mutagenesis Using the Polymerase Chain Reaction. Current Protocols in Molecular Biology, UNIT 8.5.1 John Wiley & Sons, Inc, New York. 60. Kelly, S.M. and Price, N.C. (1997) The application of circular dichroism to studies of protein folding and unfolding. Biochim Biophys Acta, 1338, 161-185. 61. Twining, S.S. (1984) Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes. Anal Biochem, 143, 30-34. 62. Goldberg, A.L., Moerschell, R.P., Chung, C.H. and Maurizi, M.R. (1994) ATP-dependent protease La (lon) from Escherichia coli. Methods Enzymol, 244, 350-375. 63. Lanzetta, P.A., Alvarez, L.J., Reinach, P.S. and Candia, O.A. (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem, 100, 95-97. 64. Dominguez, C., Boelens, R. and Bonvin, A.M. (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc, 125, 1731-1737. 65. Riethdorf, S., Volker, U., Gerth, U., Winkler, A., Engelmann, S. and Hecker, M. (1994) Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene. J Bacteriol, 176, 6518-6527. 66. Komazin-Meredith, G., Santos, W.L., Filman, D.J., Hogle, J.M., Verdine, G.L. and Coen, D.M. (2008) The positively charged surface of herpes simplex virus UL42 mediates DNA binding. J Biol Chem, 283, 6154-6161. 67. Sreerama, N. and Woody, R.W. (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem, 287, 252-260. 68. Hsu, C.H., Chen, C., Jou, M.L., Lee, A.Y., Lin, Y.C., Yu, Y.P., Huang, W.T. and Wu, S.H. (2005) Structural and DNA-binding studies on the bovine antimicrobial peptide, indolicidin: evidence for multiple conformations involved in binding to membranes and DNA. Nucleic Acids Res, 33, 4053-4064. 69. Kirchner, R., Vogtherr, M., Limmer, S. and Sprinzl, M. (1998) Secondary structure dimorphism and interconversion between hairpin and duplex form of oligoribonucleotides. Antisense Nucleic Acid Drug Dev, 8, 507-516. 70. Majka, J. and Speck, C. (2007) Analysis of protein-DNA interactions using surface plasmon resonance. Adv Biochem Eng Biotechnol, 104, 13-36. 71. Wickner, S., Maurizi, M.R. and Gottesman, S. (1999) Posttranslational quality control: folding, refolding, and degrading proteins. Science, 286, 1888-1893. 72. Nomura, K., Kato, J., Takiguchi, N., Ohtake, H. and Kuroda, A. (2004) Effects of inorganic polyphosphate on the proteolytic and DNA-binding activities of Lon in Escherichia coli. J Biol Chem, 279, 34406-34410. 73. McCauley, M.J., Shokri, L., Sefcikova, J., Venclovas, C., Beuning, P.J. and Williams, M.C. (2008) Distinct double- and single-stranded DNA binding of E. coli replicative DNA polymerase III alpha subunit. ACS Chem Biol, 3, 577-587. 74. Jones, S., Daley, D.T., Luscombe, N.M., Berman, H.M. and Thornton, J.M. (2001) Protein-RNA interactions: a structural analysis. Nucleic Acids Res, 29, 943-954. 75. Luscombe, N.M., Laskowski, R.A. and Thornton, J.M. (2001) Amino acid-base interactions: a three-dimensional analysis of protein-DNA interactions at an atomic level. Nucleic Acids Res, 29, 2860-2874. 76. Olson, W.K., Gorin, A.A., Lu, X.J., Hock, L.M. and Zhurkin, V.B. (1998) DNA sequence-dependent deformability deduced from protein-DNA crystal complexes. Proc Natl Acad Sci U S A, 95, 11163-11168. 77. Jones, S., van Heyningen, P., Berman, H.M. and Thornton, J.M. (1999) Protein-DNA interactions: A structural analysis. J Mol Biol, 287, 877-896. 78. Yang, L. and Schepartz, A. (2005) Relationship between folding and function in a sequence-specific miniature DNA-binding protein. Biochemistry, 44, 7469-7478. 79. Hashimoto, H., Horton, J.R., Zhang, X., Bostick, M., Jacobsen, S.E. and Cheng, X. (2008) The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix. Nature, 455, 826-829. 80. Garvie, C.W. and Wolberger, C. (2001) Recognition of specific DNA sequences. Mol Cell, 8, 937-946. 81. Snider, J. and Houry, W.A. (2008) AAA+ proteins: diversity in function, similarity in structure. Biochem Soc Trans, 36, 72-77. 82. Barnett, M.E., Zolkiewska, A. and Zolkiewski, M. (2000) Structure and activity of ClpB from Escherichia coli. Role of the amino-and -carboxyl-terminal domains. J Biol Chem, 275, 37565-37571. 83. Lupas, A.N. and Martin, J. (2002) AAA proteins. Curr Opin Struct Biol, 12, 746-753. 84. Guo, F., Maurizi, M.R., Esser, L. and Xia, D. (2002) Crystal structure of ClpA, an Hsp100 chaperone and regulator of ClpAP protease. J Biol Chem, 277, 46743-46752. 85. Botos, I., Melnikov, E.E., Cherry, S., Tropea, J.E., Khalatova, A.G., Rasulova, F., Dauter, Z., Maurizi, M.R., Rotanova, T.V., Wlodawer, A. et al. (2004) The catalytic domain of Escherichia coli Lon protease has a unique fold and a Ser-Lys dyad in the active site. J Biol Chem, 279, 8140-8148. 86. Zhang, X., Shaw, A., Bates, P.A., Newman, R.H., Gowen, B., Orlova, E., Gorman, M.A., Kondo, H., Dokurno, P., Lally, J. et al. (2000) Structure of the AAA ATPase p97. Mol Cell, 6, 1473-1484. 87. Niwa, H., Tsuchiya, D., Makyio, H., Yoshida, M. and Morikawa, K. (2002) Hexameric ring structure of the ATPase domain of the membrane-integrated metalloprotease FtsH from Thermus thermophilus HB8. Structure, 10, 1415-1423. 88. Melnikov, E.E., Andrianova, A.G., Morozkin, A.D., Stepnov, A.A., Makhovskaya, O.V., Botos, I., Gustchina, A., Wlodawer, A. and Rotanova, T.V. (2008) Limited proteolysis of E. coli ATP-dependent protease Lon - a unified view of the subunit architecture and characterization of isolated enzyme fragments. Acta Biochim Pol, 55, 281-296. 89. Thanbichler, M., Viollier, P.H. and Shapiro, L. (2005) The structure and function of the bacterial chromosome. Curr Opin Genet Dev, 15, 153-162. 90. Marsischky, G.T. and Kolodner, R.D. (1999) Biochemical characterization of the interaction between the Saccharomyces cerevisiae MSH2-MSH6 complex and mispaired bases in DNA. J Biol Chem, 274, 26668-26682. 91. Pierce, M.M., Raman, C.S. and Nall, B.T. (1999) Isothermal titration calorimetry of protein-protein interactions. Methods, 19, 213-221. 92. Peters, W.B., Edmondson, S.P. and Shriver, J.W. (2004) Thermodynamics of DNA binding and distortion by the hyperthermophile chromatin protein Sac7d. J Mol Biol, 343, 339-360. 93. Ceccarelli, D.F. and Frappier, L. (2000) Functional analyses of the EBNA1 origin DNA binding protein of Epstein-Barr virus. J Virol, 74, 4939-4948. 94. West, M. and Wilson, V.G. (2002) Hydrophobic residue contributions to sequence-specific DNA binding by the bovine papillomavirus helicase E1. Virology, 296, 52-61. 95. Ogura, T., Whiteheart, S.W. and Wilkinson, A.J. (2004) Conserved arginine residues implicated in ATP hydrolysis, nucleotide-sensing, and inter-subunit interactions in AAA and AAA+ ATPases. J Struct Biol, 146, 106-112. 96. Seeman, N.C., Rosenberg, J.M. and Rich, A. (1976) Sequence-specific recognition of double helical nucleic acids by proteins. Proc Natl Acad Sci U S A, 73, 804-808. 97. Gajiwala, K.S. and Burley, S.K. (2000) Winged helix proteins. Curr Opin Struct Biol, 10, 110-116. 98. Kodandapani, R., Pio, F., Ni, C.Z., Piccialli, G., Klemsz, M., McKercher, S., Maki, R.A. and Ely, K.R. (1996) A new pattern for helix-turn-helix recognition revealed by the PU.1 ETS-domain-DNA complex. Nature, 380, 456-460. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45265 | - |
| dc.description.abstract | Lon蛋白為一個多功能的單一聚合型酵素，且高度保留存在於各種生物體中。先前的研究指出Lon可以維持生物體中蛋白質完整的功能與結構，或是適時降解特定或非特定目標蛋白，進而參予調控體內多種新陳代謝活動。本論文以台灣本土所分離出的嗜熱短桿菌(Brevibacillus thermoruber WR-249)之Lon蛋白(Bt-Lon)為研究對象，探討其與DNA結合之間的關係，並同時與革蘭氏陽性標準菌株-枯草桿菌(Bacillus subtilis)及革蘭氏陰性標準菌株-大腸桿菌(Escherichia coli)之Lon蛋白進行比較，利用電泳凝膠遲滯法(GMSA)分析，得知原核細菌的Lon蛋白主要皆是利用其中的α-domain為DNA結合區域。利用DNase I配合基質輔助電射游離脫附飛行時間質譜(MALDI-TOF MS)分析，我們找到一段與Bt-Lon α-domain結合的DNA片段(5’-CTGTTAGCGGGC-3’)，我們將之命名為ms1。再利用表面電漿共振(SPR)及恆溫滴定熱分析(ITC)，確認ms1與Bt-Lon α-domain之間具有高專一性結合能力。參考Bt-Lon α domain結構的表面正電荷分佈，我們針對其中部份帶正電荷之胺基酸殘基進行定點突變，再進行與ms1 DNA之結合分析，結果顯示當Arginine 518殘基突變為丙胺酸後，結合力會明顯下降26倍，顯示在Bt-Lon α-domain與ms1 DNA之間的結合上，Arginine 518扮演重要的角色。 | zh_TW |
| dc.description.abstract | The multi-functional, homo-oligomeric, ATP-dependent Lon protease is highly conserved in prokaryotes and eukaryotic organelles. Previous studies have shown that Lon activity is essential for protein quality control and regulation of metabolic processes. Here we examined the DNA-binding activity of the Lon protease α-domains from Brevibacillus thermoruber, Bacillus subtilis, and Escherichia coli. Gel mobility shift assays indicated that the α-domain from Br. thermoruber has the highest DNA affinity. MALDI-TOF mass spectrometry showed that this α-domain binds to the nucleotide sequence 5’-CTGTTAGCGGGC-3’ (ms1). Surface plasmon resonance and isothermal titration calorimetry showed that a double-stranded DNA fragment of this sequence binds to the α-domain; double-stranded DNA fragments with 0 and 50% identity to the binding sequence had lower affinities for the α-domain. Five mutants of the α-domain from Br. thermoruber carrying single mutations (R537A, R546A, R553A, K580A and R584A) were constructed and showed only 1.2–2.0-fold lower DNA binding affinity; one mutant, R518A, displayed 26-fold lower affinity. The Bt-Lon R518A mutant also has lower affinity to DNA than wild type. These results revealed that Arg 518 of the Bt-Lon from Br. thermoruber plays a critical role in the DNA-binding activity. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T04:11:28Z (GMT). No. of bitstreams: 1 ntu-99-D91242005-1.pdf: 8363923 bytes, checksum: 0da2eda37bb893b82e4c2c699e55c73a (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
中文摘要 ii Abstract iii 1. Introduction - 1 - 2. Materials and Methods - 6 - 2-1. Subcloning of the α-domains - 6 - 2-2. Sequence alignment - 6 - 2-3. Expression and purification of Bt-Lon truncated proteins and α-domains - 6 - 2-4. Gel mobility shift assay - 7 - 2-5. Identification of the Bt-Lon α-domain DNA-binding sequence - 8 - 2-6. Homology modeling - 9 - 2-7. Site-directed mutagenesis - 9 - 2-8. Circular dichroism - 10 - 2-9. Surface hydrophobicity - 11 - 2-10. Native-PAGE of hairpin DNA - 11 - 2-11. Surface plasmon resonance (SPR) - 11 - 2-12. Isothermal titration calorimetry - 13 - 2-13. Protease assay - 13 - 2-14. Peptidase assay - 14 - 2-15. ATPase assay - 14 - 2-16. Docking model of protein and DNA complex - 15 - 3. Results - 17 - 3-1. Lon α-domains from Br. thermoruber, B. subtilis, and E. coli - 17 - 3-2. Structure of the α-domains - 18 - 3-3. Binding of the α-domain to DNA - 18 - 3-4. The sequence of the preferred DNA binding site of the Bt-Lon α-domain - 19 - 3-5. Kinetics of the interaction between the wild-type α-domain and DNA - 20 - 3-6. Thermodynamics of the interaction between the α-domain and DNA - 21 - 3-7. Interaction between DNA and Bt-Lon α-domain mutants - 21 - 3-8. DNA sequence specificity of Bt-Lon α-domain binding - 23 - 3-9. Interaction between DNA and Bt-Lon wild-type and R518A mutant - 23 - 3-10. Influence of plasmid or ms1 DNA on enzymatic activities of Bt-Lon - 24 - 3-11. The structure model of Bt-Lon α-domain/ds-ms1 complex - 25 - 4. Discussion - 26 - Figure - 36 - Figure 1. The amino acid sequence alignment of α-domains - 36 - Figure 2. SDS-PAGE of purified recombinant α-domains - 37 - Figure 3. Structure of α-domains - 38 - Figure 4. Far-UV CD spectra - 39 - Figure 5. Thermal denaturation - 40 - Figure 6. Near-UV spectra - 41 - Figure 7. Hydrophobicity measurement - 42 - Figure 8. Concentration dependent EMSA assay - 43 - Figure 9. GMSA assay of different α-domains - 44 - Figure 10. MALDI-TOF mass spectroscopy - 45 - Figure 11. Analysis of hairpin-duplex formation by Native PAGE - 46 - Figure 12. SPR sensorgrams of different α-domains - 47 - Figure 13. ITC result of the Bs-Lon α-domain - 48 - Figure 14. ITC result of the Bt-Lon α-domain - 49 - Figure 15. ITC result of the Ec-Lon α-domain - 50 - Figure 16. Site directed mutagenesis - 51 - Figure 17. SDS-PAGE of Bt-Lon α-domain mutants - 52 - Figure 18. Far-UV CD spectra of 6 different Bt-Lon α-domain mutants - 53 - Figure 19. SPR sensorgrams of Bt-Lon α-domain mutants - 54 - Figure 20. SPR sensorgrams of Bt-Lon α-domain to different hairpin DNA - 55 - Figure 21. GMSA assay of Bt-Lon wild type and mutant - 56 - Figure 22. SPR sensorgrams of the binding of Bt-Lon - 57 - Figure 23. Effect of plasmid DNA on enzymatic activities - 58 - Figure 24. Effect of ds-ms1 DNA on enzymatic activities - 59 - Figure 25. Structural model of Bt-Lon α-domain / ds-ms1 DNA - 60 - Table - 61 - Table 1. Oligonucleotides used in this study - 61 - Table 2. The ratios of the secondary structures in different α-domains - 62 - Table 3. Kinetic constants of the interaction - 63 - References - 64 - Appendix - 72 - Paper List - 72 - In Preparation List - 72 - Poster List - 73 - Paper - 74 - Poster - 107 - | |
| dc.language.iso | zh-TW | |
| dc.subject | 表面電漿共振 | zh_TW |
| dc.subject | 嗜熱短桿菌 | zh_TW |
| dc.subject | 恆溫滴定熱分析 | zh_TW |
| dc.subject | Surface Plasmon resonance | en |
| dc.subject | Isothermal titration calorimetry | en |
| dc.subject | α-domain | en |
| dc.subject | Lon | en |
| dc.subject | Brevibacillus thermoruber WR-249 | en |
| dc.title | 嗜熱短桿菌Lon蛋白酵素對DNA結合特性之研究 | zh_TW |
| dc.title | DNA-binding specificity of the Lon protease α-domain from Brevibacillus thermoruber WR-249 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 張文章(Wen-Chang Chang),梁博煌(Po-Huang Liang),羅禮強(Lee-Chiang Lo),余榮熾(Lung-Chih Yu),陳金榜(Chinpan Chen) | |
| dc.subject.keyword | 嗜熱短桿菌,表面電漿共振,恆溫滴定熱分析, | zh_TW |
| dc.subject.keyword | Brevibacillus thermoruber WR-249,Lon,α-domain,Surface Plasmon resonance,Isothermal titration calorimetry, | en |
| dc.relation.page | 109 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-01-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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