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研究生:黃世勳
研究生(外文):Huang, Shih-Hsun
論文名稱:鉤端螺旋體外膜脂蛋白LipL41 C端區域生物物理特性之研究
論文名稱(外文):Biophysical characteristics of C-terminal domain of LipL41 from pathogenic Leptospira
指導教授:孫玉珠
指導教授(外文):Sun, Yuh-Ju
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:51
中文關鍵詞:鉤端螺旋體外膜脂蛋白
外文關鍵詞:LeptospiraLipL41Tetratricopeptide repeatTPR
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鉤端螺旋體病是一種人畜共通的傳染病,好發於熱帶、亞熱帶地區。致病性鉤端
螺旋體感染宿主後,會引起腎小管間質炎(Tubulointerstital nephropathy, TIN),
造成腎臟慢性傷害,且潛伏於腎臟之病菌會藉由尿液傳播至環境當中。LipL41
是位於致病性鉤端螺旋體外膜的主要脂蛋白之一,也是重要的致病因子。過去的
研究中LipL41 可作為疫苗開發或感染疾病檢測之用。我們使用生物資訊分析軟
體分析序列後發現, LipL41 之C端區域(氨基酸位置256-355,簡稱為LipL41-C100)
為一段極親水之5 個連續的α-helix,此區域並包含有2 個Tetratricopeptide repeat
(TPR) motif (結構區域)。圓二色光譜儀(Circular Dichroism)之結果顯示,
LipL41-C100 之二級結構以α-螺旋為主(48%)。但NMR 之結果顯示 LipL41-C100
並未具有穩定的三級結構,這種具有二級結構但三級結構鬆散的蛋白,稱作熔球
態蛋白(molten globule state protein)。Thermal shift assay、膠體層析法(size
exclusion chromatography) 等實驗均證實LipL41-C100 符合熔球態蛋白之特性。
我們更進一步使用生物資訊軟體再次分析LipL41,並發現LipL41-C100 其所在
位置是無結構區域 (intrinsically unstructured domain),此類型domain 具有 molten
globule 之特性, 並與ligand 之結合密切相關。藉由添加三氟乙醇,
2,2,2-Trifluoroethanol (TFE) , 可以穩定LipL41-C100 之二級結構, 並模擬
LipL41-C100 與ligand 結合後之結構。圓二色光譜結果顯示,TFE 能協助
LipL41-C100 形成α-螺旋,LipL41-C100 在50% TFE 的環境下 ,α-螺旋之含量
上升至75.9%。核磁共振光譜顯示,添加50% TFE 後,LipL41-C100 形成較為穩
定且具有良好三級結構的螺旋構型,後續的NMR 相關實驗正在進行當中。
Leptospirosis, which is caused by pathogen Leptospira, is the most common
zoonotic disease emerged in the world. The organism enters the human body through
mucous membranes or broken skin contact with the urine of an infected animal.
Leptospira can cause damage of the kidney in the host and lead to tubulointerstitial
nephritis. LipL41 is one of the major lipoprotein and important virulence factor
located on the outer membrane of Leptosira. LipL41 was used to study
the vaccines against human leptospirosis and the diagnostic detection, but the
structural information is still unclear. Based on the sequence analysis of LipL41, we
found out that the C-terminal and hydrophilic region of LipL41 (residue 256-355,
denoted as LipL41-C100) contains five helices with two TPR motifs. Several studies
show that TPR motif is involved in protein-protein interaction and oligomerlization.
Analysis by circular dichroism (CD) showed that LipL41-C100 is a α-helix protein
(48%). The signals in the 1H-15N HSQC spectrum of LipL41-C100 were broad and
some of them were disappear and the observation represents that LipL41-C100 is a
molten globule protein. Thermal shift assay, size-exclusion chromatography (SEC)
and analytical ultracentrifugation (AUC) also revealed that LipL41-C100 has many
common characteristics of molten globule. We further analyzed the folding porosity
of LipL41 by Foldindex. The result predicted that LipL41 has an intrinsically
unfolded domain (residue 256-355) which is the same as LipL41-C100. The flexible
structure is related to the ligand binding ability. 2,2,2-Trifluoroethanol (TFE) have
been shown to stabilize the helical structure of the protein. The CD spectrum revealed
that the α-helix content of LipL41-C100 is increase from 48% to 75.9 % in the
presence of 50% TFE. The NMR spectra of LipL41-C100 in the presence of 50% TFE
also show that TFE stabilizes the regions in the molten globule state. The NMR
experiments of LipL41-C100 are underway.
Chapter 1 Introduction 1
Chapter 2 Materials and Methods
2.0 Bioinformatic analysis 5
2.1 Construction of Plasmid 5
2.2 Protein production and purification 6
2.3 Protein Crystallization 7
2.4 NMR sample preparation 7
2.5 NMR experiment 7
2.6 Circular dichroism 8
2.7 Size Exclusion Chromatography (SEC) 10
2.8 Mass Spectroscopy 10
2.9 Analytical Ultracentrifugation (AUC) 10
Chapter 3 Results and Discussion
3.1 Bioinformatic analysis of LipL41 full length 12
3.2 Expression, Purification and Characterization of LipL41-C100 13
3.3 The molecular weight of LipL41-C100 14
3.4 The major secondary structure of LipL41-C100 is α-helix 15
3.5 LipL41-C100 adopts a molten globule conformation 16
3.6 The tertiary structure of LipL41-C100 is relaxed 19
3.7 LipL41-C100 exposure more hydrophobic region 20
3.8 Thermal stability and reversibility of LipL41-C100 21
3.9 TFE assists the folding of LipL41-C100 22
3.10 The possible 3D model of LipL41-C100 23
Chapter 4 Conclusion 25
Chapter 5 Figures 26
Reference 48

1. Ko, A.I., C. Goarant, and M. Picardeau, Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol, 2009. 7(10): p. 736-47.
2. Trueba, G.A., C.A. Bolin, and R.L. Zuerner, Characterization of the periplasmic flagellum proteins of Leptospira interrogans. J Bacteriol, 1992. 174(14): p. 4761-8.
3. Evangelista, K.V. and J. Coburn, Leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune responses. Future Microbiology, 2010. 5(9): p. 1413-1425.
4. Goncalves-de-Albuquerque, C.F., et al., Leptospira and inflammation. Mediators Inflamm, 2012. 2012: p. 317950.
5. Johnson, R.C. and J.K. Walby, Cultivation of leptospires: fatty acid requirements. Appl Microbiol, 1972. 23(5): p. 1027-8.
6. Adler, B. and A. de la Pena Moctezuma, Leptospira and leptospirosis. Vet Microbiol, 2010. 140(3-4): p. 287-96.
7. Leptospirosis worldwide, 1999. Wkly Epidemiol Rec, 1999. 74(29): p. 237-42.
8. Yang, C.W., Leptospirosis renal disease: Understanding the initiation by Toll-like receptors. Kidney International, 2007. 72(8): p. 918-925.
9. Branger, C., et al., Identification of the hemolysis-associated protein 1 as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination. Infection and Immunity, 2001. 69(11): p. 6831-6838.
10. Haake, D.A., et al., Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immunoprotection. Infection and Immunity, 1999. 67(12): p. 6572-6582.
11. Hung, C.C., et al., Upregulation of chemokine CXCL1/KC by leptospiral membrane lipoprotein preparation in renal tubule epithelial cells. Kidney Int, 2006. 69(10): p. 1814-22.
12. Yang, C.W., et al., Leptospira outer membrane protein activates NF-kappaB and downstream genes expressed in medullary thick ascending limb cells. J Am Soc Nephrol, 2000. 11(11): p. 2017-26.
13. Alves, V.A.F., et al., Leptospiral Antigens (L-Interrogans Serogroup Ictero-Haemorrhagiae) in the Kidney of Experimentally Infected Guinea-Pigs and Their Relation to the Pathogenesis of the Renal Injury. Experimental Pathology, 1991. 42(2): p. 81-93.
14. Barnett, J.K., et al., Expression and distribution of leptospiral outer membrane components during renal infection of hamsters. Infection and Immunity, 1999. 67(2): p. 853-861.
15. Haake, D.A., et al., The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection. Infection and Immunity, 2000. 68(4): p. 2276-2285.
16. Tokuda, H. and S. Matsuyama, Sorting of lipoproteins to the outer membrane in E. coli. Biochim Biophys Acta, 2004. 1693(1): p. 5-13.
17. Hayashi, S. and H.C. Wu, Lipoproteins in bacteria. J Bioenerg Biomembr, 1990. 22(3): p. 451-71.
18. Inouye, S., et al., Amino acid sequence for the peptide extension on the prolipoprotein of the Escherichia coli outer membrane. Proc Natl Acad Sci U S A, 1977. 74(3): p. 1004-8.
19. Kamalakkannan, S., et al., Bacterial lipid modification of proteins for novel protein engineering applications. Protein Engineering Design & Selection, 2004. 17(10): p. 721-729.
20. Haake, D.A., et al., Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp. J Bacteriol, 1993. 175(13): p. 4225-34.
21. Shang, E.S., T.A. Summers, and D.A. Haake, Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species. Infect Immun, 1996. 64(6): p. 2322-30.
22. Cullen, P.A., et al., Surfaceome of Leptospira spp. Infect Immun, 2005. 73(8): p. 4853-63.
23. Barnett, J.K., et al., Expression and distribution of leptospiral outer membrane components during renal infection of hamsters. Infect Immun, 1999. 67(2): p. 853-61.
24. Monahan, A.M., J.J. Callanan, and J.E. Nally, Proteomic analysis of Leptospira interrogans shed in urine of chronically infected hosts. Infect Immun, 2008. 76(11): p. 4952-8.
25. Asuthkar, S., et al., Expression and characterization of an iron-regulated hemin-binding protein, HbpA, from Leptospira interrogans serovar Lai. Infect Immun, 2007. 75(9): p. 4582-91.
26. Nygaard, T.K., et al., Identification and characterization of the heme-binding proteins SeShp and SeHtsA of Streptococcus equi subspecies equi. BMC Microbiol, 2006. 6: p. 82.
27. Rouault, T.A., Microbiology. Pathogenic bacteria prefer heme. Science, 2004. 305(5690): p. 1577-8.
28. Schaible, U.E. and S.H. Kaufmann, Iron and microbial infection. Nat Rev Microbiol, 2004. 2(12): p. 946-53.
29. Lo, M., et al., Transcriptional response of Leptospira interrogans to iron limitation and characterization of a PerR homolog. Infect Immun, 2010. 78(11): p. 4850-9.
30. Velineni, S., S. Asuthkar, and M. Sritharan, Iron limitation and expression of immunoreactive outer membrane proteins in Leptospira interrogans serovar icterohaemorrhagiae strain lai. Indian J Med Microbiol, 2006. 24(4): p. 339-42.
31. Skaar, E.P., The battle for iron between bacterial pathogens and their vertebrate hosts. PLoS Pathog, 2010. 6(8): p. e1000949.
32. Sikorski, R.S., et al., A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell, 1990. 60(2): p. 307-17.
33. Prilusky, J., et al., FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics, 2005. 21(16): p. 3435-8.
34. Uversky, V.N., J.R. Gillespie, and A.L. Fink, Why are "natively unfolded" proteins unstructured under physiologic conditions? Proteins, 2000. 41(3): p. 415-27.
35. Rossmann, M.G., Preparation and Analysis of Protein Crystals - Mcpherson,A. Journal of the American Chemical Society, 1983. 105(12): p. 4120-4121.
36. Wishart, D.S., et al., H-1, C-13 and N-15 Chemical-Shift Referencing in Biomolecular Nmr. Journal of Biomolecular Nmr, 1995. 6(2): p. 135-140.
37. Markley, J.L., et al., Recommendations for the presentation of NMR structures of proteins and nucleic acids - (IUPAC Recommendations 1998). Pure and Applied Chemistry, 1998. 70(1): p. 117-142.
38. Bohm, G., R. Muhr, and R. Jaenicke, Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng, 1992. 5(3): p. 191-5.
39. Schuck, P., et al., Size-distribution analysis of proteins by analytical ultracentrifugation: Strategies and application to model systems. Biophysical Journal, 2002. 82(2): p. 1096-1111.
40. Altschul, S.F., et al., Basic local alignment search tool. J Mol Biol, 1990. 215(3): p. 403-10.
41. Karpenahalli, M.R., A.N. Lupas, and J. Soding, TPRpred: a tool for prediction of TPR-, PPR- and SEL1-like repeats from protein sequences. BMC Bioinformatics, 2007. 8: p. 2.
42. Jones, D.T., Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol, 1999. 292(2): p. 195-202.
43. Gautier, R., et al., HELIQUEST: a web server to screen sequences with specific alpha-helical properties. Bioinformatics, 2008. 24(18): p. 2101-2.
44. Lau, S.Y., A.K. Taneja, and R.S. Hodges, Synthesis of a model protein of defined secondary and quaternary structure. Effect of chain length on the stabilization and formation of two-stranded alpha-helical coiled-coils. J Biol Chem, 1984. 259(21): p. 13253-61.
45. Li, X., et al., Predicting Protein Disorder for N-, C-, and Internal Regions. Genome Inform Ser Workshop Genome Inform, 1999. 10: p. 30-40.
46. Romero, P., et al., Sequence complexity of disordered protein. Proteins, 2001. 42(1): p. 38-48.
47. Romero, Obradovic, and K. Dunker, Sequence Data Analysis for Long Disordered Regions Prediction in the Calcineurin Family. Genome Inform Ser Workshop Genome Inform, 1997. 8: p. 110-124.
48. Tompa, P., Intrinsically unstructured proteins. Trends in Biochemical Sciences, 2002. 27(10): p. 527-533.
49. Dyson, H.J. and P.E. Wright, Intrinsically unstructured proteins and their functions. Nature Reviews Molecular Cell Biology, 2005. 6(3): p. 197-208.
50. Dunker, A.K., et al., Function and structure of inherently disordered proteins. Current Opinion in Structural Biology, 2008. 18(6): p. 756-764.
51. Oldfield, C.J., et al., Comparing and combining predictors of mostly disordered proteins. Biochemistry, 2005. 44(6): p. 1989-2000.
52. Cliff, M.J., et al., Molecular recognition via coupled folding and binding in a TPR domain. Journal of Molecular Biology, 2005. 346(3): p. 717-732.
53. Uversky, V.N., What does it mean to be natively unfolded? European Journal of Biochemistry, 2002. 269(1): p. 2-12.
54. Erickson, H.P., Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy. Biological Procedures Online, 2009. 11(1): p. 32-51.
55. Lavinder, J.J., et al., High-Throughput Thermal Scanning: A General, Rapid Dye-Binding Thermal Shift Screen for Protein Engineering. Journal of the American Chemical Society, 2009. 131(11): p. 3794-+.
56. Kazakov, A.S., et al., Thermally induced structural changes of intrinsically disordered small heat shock protein Hsp22. Biophys Chem, 2009. 145(2-3): p. 79-85.
57. Reiersen, H. and A.R. Rees, Trifluoroethanol may form a solvent matrix for assisted hydrophobic interactions between peptide side chains. Protein Eng, 2000. 13(11): p. 739-43.
58. Diaz, M.D. and S. Berger, Preferential solvation of a tetrapeptide by trifluoroethanol as studied by intermolecular NOE. Magnetic Resonance in Chemistry, 2001. 39(7): p. 369-373.
59. Diaz, M.D., et al., Evidence of complete hydrophobic coating of bombesin by trifluoroethanol in aqueous solution: An NMR spectroscopic and molecular dynamics study. Chemistry-a European Journal, 2002. 8(7): p. 1663-1669.
60. Fioroni, M., et al., Solvation phenomena of a tetrapeptide in water/trifluoroethanol and water/ethanol mixtures: A diffusion NMR, intermolecular NOE, and molecular dynamics study. Journal of the American Chemical Society, 2002. 124(26): p. 7737-7744.
61. Levett, P.N., Leptospirosis. Clin Microbiol Rev, 2001. 14(2): p. 296-326.
62. Cecil, R.L., L. Goldman, and A.I. Schafer, Goldman's Cecil medicine. 24th ed. 2012, Philadelphia: Elsevier/Saunders/. xlii, 2569, 86 p.
63. Cuff, J.A. and G.J. Barton, Application of multiple sequence alignment profiles to improve protein secondary structure prediction. Proteins, 2000. 40(3): p. 502-11.
64. Ouali, M. and R.D. King, Cascaded multiple classifiers for secondary structure prediction. Protein Sci, 2000. 9(6): p. 1162-76.
65. Kyte, J. and R.F. Doolittle, A Simple Method for Displaying the Hydropathic Character of a Protein. Journal of Molecular Biology, 1982. 157(1): p. 105-132.
66. Prilusky, J., et al., FoldIndex((c)): a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics, 2005. 21(16): p. 3435-3438.
67. Uversky, V.N., Natively unfolded proteins: A point where biology waits for physics. Protein Science, 2002. 11(4): p. 739-756.
68. Nielsen, M., et al., CPHmodels-3.0-remote homology modeling using structure-guided sequence profiles. Nucleic Acids Research, 2010. 38: p. W576-W581.
69. Schrodinger, LLC, The PyMOL Molecular Graphics System, Version 1.3r1, 2010.


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