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

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