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研究生:高光毅
論文名稱:由電子自旋共振順磁性物質標定技術探測色氨基酸包覆胜肽之自由能量位階圖
論文名稱(外文):Probing the Free Energy Landscape of Trp-cage Peptides by EPR Spin-Labeled Techniques
指導教授:陳長謙陳長謙引用關係
指導教授(外文):Sunney I. Chan
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:2007
畢業學年度:95
語文別:英文
論文頁數:56
中文關鍵詞:色氨基酸包覆胜肽順磁性物質
外文關鍵詞:Trp-cagespin-label
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色氨基酸包覆胜肽,NLYIQWLKDGGPSSGRPPPS,一個合成的20個殘基聚胜肽,是源自將一個39個殘基的胜肽,HGEGTFTSDLSKQMEEEAVRL
FIEWLKNGGPSSGAPPPS,名為exendin- 4(EX4)進行截取和突變。色氨基酸包覆胜肽未折疊至折疊不是ㄧ個簡單的兩態過程已被報告過。為了澄清這議題,我們合成了雙順磁性物質標定的色氨基酸包覆胜肽並且研究順磁性物質對於此胜肽中色氨基酸螢光的影響、圓二色以及電子自旋共振圖譜。順磁性物質之ㄧ是四甲吡氧胺酸(TOAC)被接在色氨基酸包覆胜肽的N端,而另ㄧ個是(1-氧-2,2,5,5-四甲基吡咯口林 -3-甲基)甲基硫代硫磺酸酯(MTSSL)被接在C端。我們利用穩定態圓二色光譜儀探測此胜肽的二級結構。我們發現在30度以上轉彎結構會一直存在但是螺旋結構會消失。在色氨基酸包覆胜肽的重新摺疊中,顯示轉彎是先形成的。這些測量也指出順磁性物質可以穩定α-螺旋結構。此外,由順磁性物質抑制色氨基酸的螢光,我們可以推斷出在C端的MTSSL比N端的TOAC更接近色氨基酸。最後,我們已經可以製備雙順磁性物質標定的色氨基酸包覆胜肽,並且也已使用電子自旋共振儀去探測兩個順磁性物質間的磁偶極-偶極影響。雙自由基色氨基酸包覆胜肽的電子自旋光譜訊號變寬,是兩個順磁性物質間的磁偶極-偶極影響所致,因此兩自由基的距離是在20Å範圍之內。
Trp-cage, NLYIQWLKDGGPSSGRPPPS, a synthetic 20 residue polypeptide, is obtained from the truncation and mutation of a 39 residue peptide, HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPS
SGAPPPS, called exendin-4 (EX4). It has been reported that folding/unfolding of the trp-cage is not a simple two-state process. Toward clarifying this issue, we have synthesized two spin-labeled trp-cage peptides and studied the effects of the spin-label on the tryptophan fluorescence in the peptides, circular dichroism, and the electron paramagnetic resonance spectrum. The one of the spin-labels is 2,2,6,6- tetramethylpiperidine-1-oxyl-4-amino-4- carboxylic acid (TOAC) coupled to the trp-cage at the N-terminal, and the other, (1-oxyl-2,2,5,5-tetramethyl- pyrroline-3-methyl)-methanethiosulfonate (MTSSL), at the C-terminal. We probe the secondary structure of the peptides using steady-state CD spectroscopy. We find that the turn structure always exists but the helix is lost at temperature above 30℃. In the refolding of the trp-cage, the turn appears to be formed first. These measurements indicate that the spin-labels stabilize the α-helix structure. Besides, from quenching of the tryptophan fluorescence by the spin-labels, we conclude that MTSSL at C-terminal is closer to the tryptophan than TOAC. Finally, we have prepared a doubly-labeled spin-label trp-cage and have used EPR to probe the magnetic dipole-dipole interaction between the two spin-labels. The EPR signal of the diradical trp-cage is broad, interacting onsite of magnetic dipole-dipole interaction between two spin-labels, and thus the distance is within 20Å.
Abstract I
中文摘要 III
謝誌 IV
Abbreviations VI
Table of Contents VII

Chapter 1 Introduction 1
1.1 Introduction to Protein Folding 1
1.2 Models and Theories of Protein Folding 3
1.3 Introduction to Fluorescence Spectroscopy 11
1.4 Background on the Trp-cage 13
1.5 The Aim of the Project 14

Chapter 2 Materials and Methods 16
2.1 Materials 16
2.1.1 Water 16
2.1.2 Chemicals 16
2.1.3 Chromatography Columns, Membranes, Filters 18
2.1.4 pH Meter 19
2.1.5 Peptide Automated Synthesizer 19
2.1.6 Centrifuge 19
2.1.7 High Performance Liquid Chromatography 19
2.1.8 Lyophilizer 20
2.1.9 Mass Spectroscopy 20
2.1.10 Circular Dichroism Spectroscopy 20
2.1.11 Ultraviolet Spectroscopy 21
2.1.12 Fluorescence Spectroscopy 21
2.1.13 Electron Paramagnetic Resonance Spectroscopy 21
2.2 Methods 21
2.2.1 Solid Phase Peptide Synthesis 21
2.2.2 Synthesis of Fmoc-TOAC-OH 22
2.2.3 Cleavage of Peptide from Resin 23
2.2.4 Peptide Purification and Identification 23
2.2.5 Synthesis of Trp-cage C-MTSSL 23
2.2.6 Electron Paramagnetic Resonance Spectroscopy 24
2.2.7 Circular Dichroism Spectroscopy 24
2.2.8 Ultraviolet Spectroscopy 25
2.2.9 Fluorescence Spectroscopy 25

Chapter 3 Results and Discussion 29
3.1 Peptide Synthesis, Purification, and Identification 29
3.2 The Structural Stability of Trp-cage by CD 33
3.3 Effect of Spin-Label Attachment on the Structural Stability of Trp-cage Peptide by CD 34
3.4 Thermal Denaturation of the Trp-cage Monitored by Fluorescence Quenching 35
3.5 Introduction to Electron Paramagnetic Resonance 40
3.6 The EPR Signals of Spin-Labeled Trp-cage Peptides 46
3.6.1 Peptide Synthesis, Purification, and Identification
46
3.6.2 The EPR of the Doubly-Spin-Labeled Trp-cage Peptide
49

Chapter 4 Conclusions 53

Reference 55
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