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研究生:郭乃瑋
研究生(外文):Nai-Wei Kuo
論文名稱:研究轉彎序列對於beta-摺板型胜肽結構、穩定度及摺疊動力學的影響
論文名稱(外文):Effects of turn mutation on the structure, stability and folding kinetics of a beta-sheet peptide
指導教授:陳長謙陳長謙引用關係
指導教授(外文):Sunney I. Chan Ph. D
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:82
中文關鍵詞:蛋白質摺疊貝他摺板型胜肽轉彎序列
外文關鍵詞:protein foldingbeta-sheetturn mutation
相關次數:
  • 被引用被引用:4
  • 點閱點閱:145
  • 評分評分:
  • 下載下載:8
  • 收藏至我的研究室書目清單書目收藏:0
中文摘要
氫鍵、疏水性作用、轉彎序列的影響是三個主要決定���{摺板穩定度的因素。一般來說,��-轉彎序列在影響���{摺板的穩定度上擔任一個重要角色。由我們實驗團隊之前以一個有三條反向平行長帶形成的��-摺板所做的結果顯示,將轉彎序列上的D-Pro以Asp代換後,可以降低原來��-摺板的穩定度。而且,在第一個轉彎序列上的這種突變,可以造成第一條與第二條長帶上相對應的胺基酸在骨架上往轉彎序列處移動一個胺基酸形成一個有TSDGK的轉彎序列,並且造成第一條胺基酸上的R基180度的翻轉至另一面。然而,這樣將第二個轉彎序列上的D-Pro以Asp代換的突變並沒有造成類似的胺基酸移動或是R基翻轉。於是,在這篇論文中我們想了解的是造成胺基酸往轉彎序列處移動的原因是因為受到新的轉彎序列影響還是新形成兩條長帶間疏水性作用的影響?我們將TSDGK的轉彎序列代換成在第二個轉彎序列突變後並沒有造成胺基酸移動的VDGO轉彎序列來探討這個作用力。
此外,我們也利用這個��-摺板來做早期蛋白質摺疊的動力學研究。我們的方法是利用一個對光不穩定的結合物來環化這個��-摺板使其結構改變,再以308-366nm波長的雷射光照射使對光不穩定結合物斷裂,讓��-摺板重新摺疊而恢復其原來的結構。在此過程中,我們以光聲波熱卡計來做動力學部分的研究。
Abstract
The hydrogen bonding, hydrophobic interactions, and the turn effect are three factors affecting the stability of ��-structures. Generally, the ��-turn is thought to play an important role in stabilizing the ��-structures. Previous studies in our group have disclosed that replacing DPro with Asp destabilizes the peptide whose DPG segment is in type II’ turn. More specifically, the DP to D mutation at DP-6 position caused an one amino-acid frameshift of the first ��-strand toward the turn region and resulted in side-chain inversion of the first ��-strand to give a five-residue TSDGK turn which is composed of a type I turn plus a �� bulge. However, a similar mutation at the DP-14 position did not change the hydrogen bond network and formed a VDGO turn instead of the VDPGO turn. The VDGO turn is in type II’ turn. The frameshift in the first hairpin raises the questions: whether the structural rearrangement is driven by the new turn sequence or by the hydrophobic interactions of new strand pairings. Here, we have replaced the TSDGK turn with the VDGO turn in the first hairpin to examine the driving force.
In this work, we used this small ��-sheet to study the early stage of protein folding. This method is based on photolysis of caged ��-sheet. The photolabile linker caged the ��-sheet to alter its structure. When peptide is exposed to the 308- and 366- nm light, the linker can be cleaved rapidly. The refolding begins as soon as the photolabile linker is cleaved. We used the technique of Photoacoustic Calorimetry to study the refolding kinetics of the ��-sheet.
Table of contents
Abstract I
Abstract Chinese II
Acknowledgements III
Abbreviation V
Table of Contents VII

Chapter 1: Introduction 1
1.1 The general concepts of protein folding and folding problem 1
1.2 The role of ��-turns on the stability and structure of ��-sheets 6
1.3 The classes of the turn type 9
1.4 Previous studies on 20-mer 11
1.5 The development of cyclized strategy 14
1.6 The aim of this project 18

Chapter 2: Materials and Methods 23
2.1 Materials 23
2.1.1 Water 23
2.1.2 Chemicals 23
2.1.3 Chromatography column, membranes, filters 25
2.1.4 pH meter 25
2.1.5 Peptide automated synthesizer 25
2.1.6 Centrifuge 25
2.1.7 Reverse-phase high performance liquid chrometagraphy 26
2.1.8 Lyophilizer 26
2.1.9 Mass spectroscopy 26
2.1.10 Circular dicroism spectroscopy 27
2.1.11 Ultraviolet spectroscopy 27
2.1.12 Phtotchemical reactor 27
2.1.13 Nuclear magnetic resonance spectroscopy 27
2.2 Methods 28
2.2.1 Solid phase peptide synthesis 28
2.2.2 Purified the linker 28
2.2.3 Head-to-side chain cyclization of peptide with linker 29
2.2.4 Cleavage peptide from resin 30
2.2.5 Peptide purification and identification 30
2.2.6 NMR spectra 31
2.2.7 NMR assignment 31
2.2.8 Hydrogen exchange rate 32
2.2.9 Structure calculation 32
2.2.10 Circular dichroism spectroscopy 33
2.2.11 Ultraviolet spectroscopy 33
2.2.12 Photochemical experiment 34
2.2.13 Photoacoustic Calorimetry 34

Chapter 3: Results and Discussion (I): structure calculation 38
3.1 Peptide synthesis, purification, and identification 38
3.2 Aggregation studies 38
3.3 Estimation of peptide 19-mer adopt a three-stranded antiparallel
��-sheet structure 40
3.4 19-mer adopted a left-handed twist 53
3.5 Structural stability of 19-mer by CD 54


Chapter 4: Results and Discussion (II): Cyclized peptide 59
4.1 Peptide synthesis 59
4.2 Purification of BrAc-CMB-OH 59
4.3 Synthesis of c-19merE11C (cyclized for 1 to 11) 60
4.4 Peptide purification, identification and CD data 61
4.5 The initial kinetic data of c-19merE11C 66
4.5.1 Introduction to Photoacousitc Calorimetry 66
4.5.2 The PAC signals of c-19merE11C 69
4.5.3 Introduction to Photothermal Beam Deflection 73
4.5.4 The PBD signals of c-19merE11C 74

Chapter 5: Conclusion and Future Plans 77

Reference 80
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