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研究生:楊凱傑
研究生(外文):Kai-Jay Yang
論文名稱:嗜熱菌Meiothermus taiwanensis醣甘油脂分子結構及模擬研究
論文名稱(外文):Study Molecular Structure and NMR-based Simulation of the Glycoglycerolipid from Meiothermus taiwanensis NTU-220
指導教授:吳世雄吳世雄引用關係
指導教授(外文):Shih-Hsiung Wu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:58
中文關鍵詞:醣脂質醣甘油脂分子模擬3J-氫碳偶合常數
外文關鍵詞:GlycolipidGlycoglycerolipidMolecular simualtion3J H-C coupling constant
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本論文的宗旨,在研究醣甘油脂藉由何種機制穩定細胞外膜,研究的素材為本實驗室所發現之嗜熱菌Meiothermus taiwanensis NTU-220外膜上的醣甘油脂,其主要的醣序列為α1,6-Galp-β1,6-Galp-β1,2-GalpNAc-α1,1-Glcp-glycerol,由文獻上的研究顯示,醣甘油脂的比例與環境溫度有密切的關係,隨著嗜熱菌生長環境溫度的增加,醣甘油脂的數量也隨之增加,然而,醣甘油脂藉由何種機制穩定細胞外膜,目前並無詳細報導,研究此醣甘油脂的三維結構將有助於了解其作用及功能。借由異核氫-碳J-HMBC脈衝序列取得橫跨醣苷鍵的3J-氫碳偶合常數,同時,搭配氫-碳 3J-Karplus方程式,計算出醣苷鍵二面角φ和ψ角度,搭配NOEs光譜所取的距離極限,計算模擬醣甘油脂於d6-二甲基亞砜溶液中的兩種可能構型。在另ㄧ方面,由氫核長距離同核位移相關譜(Long-Range COSY)及化學位移溫度變換係數等實驗所推測的氧-氫…氧氫鍵,均無法支持及佐證此兩種構型存在之可能性。

This thesis was aimed to study the solution structure of glycoglycerolipid from Meiothermus taiwanensis NTU-220. The primary sequence of this glycoglycerolipid was studied and reported in this group as following: α1,6-Galp-β1,6-Galp- β1,2-GalpNAc-α1,1-Glcp-glycerol. According to previously reported, the amount of glycolipid distributed on the cell membrane is increasing with the raising temperature. This result indicated that those glycolipids might play as chaperon toward the maintenance of cell membrane integrity, and this field is still fully understudied. Therefore, we considered that study the 3D structure of oligo-glycolipid could provide the initial understanding of possible biological property and function. Combined the distance restraints from NOESY spectra and the exocyclic φ andψ torsional restraints from heteronuclear 1H-13C 3J-HMBC experiment with NMR solvent DMSO-d6, the molecular simulation provided two possible structures. We also attempt to predict the possible O-H…O hydrogen bonds by temperature coefficient and long-range COSY experiments, however, those hydrogen bonds could not directly be explained and fitted into the simulated structures, reasonablely.

Contents

口試委員會審定書 ……………………………………………………II
致謝……………………………………………………………………III
中文摘要……………………………………………………………… VI
Abstract………………………………………………………………VII
Abbreviations………………………………………………………VIII
List of Figures and Tables………………………………………IX
1.Introduction
1.1 Thermophilic bacteria…………………………………………1
1.2 Glycolipids from thermophilic bacteria………………3
1.3 Glycolipids and its biological significant…………6
1.4 Immunogenic activities of glycolipids ………………8
1.5 Objectives …………………………………………………10
2. Materials and Methods
2.1 Materials
2.1.1 Bacterial strain………………………………………11
2.1.2 Medium for bacterial culture………………………11
2.1.3 Chemicals and reagents………………………………12
2.1.4 Instruments ……………………………………………12

2.2 Methods
2.2.1 Flow chart of experiments …………………………13
2.2.2 Bacterial growth condition…………………………14
2.2.3 Polar lipid extraction and GL1M isolation ……14
2.2.4 Monosaccharide composition analysis by
GC-MS ……………………………………………………15
2.2.5 NMR spectra analysis…………………………………17
2.2.6 Restrained simulated annealing calculation……18
3. Results and Discussion
3.1 Isolation of glycoglycerolipid GL1M and GC/MS
analysis …………………………………………………………19
3.2 Chemical shift assignment of glycoglycerolipid GL1M in
DMSO-d6……………………………………………………………20
3.3 NOEs and torsion angles of glycoglycerolipid GL1M in
DMSO-d6……………………………………………………………22
3.4 Molecular simulation of Glycoglycerolipid GL1M DMSO-d6
solution …………………………………………………………24
3.5 Chemical Shift Assignment and NOEs of Headgroup of GL1M
in SDS-d25 Micelles Solution ………………………………26
3.6 Evidence from NMR contradict the modeling
results……………………………………………………………27
4. Conclusion ………………………………………………………30
Figures and Tables …………………………………………………31
References ……………………………………………………………49
Appendix: Restraints for Modeling simulaiton


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