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研究生:林至凱
研究生(外文):Chih-Kai, Jacky Lin
論文名稱:對光不穩定連結物的合成及其對蛋白質摺疊
論文名稱(外文):The synthesis of photolabile linkers and their applications in studies of protein folding
指導教授:陳 長 謙
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:2002
畢業學年度:90
語文別:德文
論文頁數:5
中文關鍵詞:蛋白質摺疊對光不穩定連結物固相胜肽合成法圓二色光譜儀側鏈有保護基的胜肽環化蛋白質
外文關鍵詞:Protein foldingphotolabile linkersolid-phase peptide synthesiscircular dicroismsidechain caged peptidecyclized peptide
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利用傳統停止─流動裝置來觀測蛋白質摺疊動力學,一直受制於其過長的混合截止時間,並且變性劑的使用經常是不可避免的。事實上,變性劑對摺疊過程的影響一直未被討論。若變性劑解離及蛋白質摺疊是兩個速率相似的過程,則我們是無法分別這兩者的。此篇論文中,我們利用一種光不穩定化合物來偵測胜肽於奈秒至毫秒內的摺疊過程,此光不穩定化合物是一個有機的保護基或稱之為“籠”化合物。用於胜肽側鏈保護策略的稱為双甲氧─α─羥─α─苯基苯乙酮(Dimethoxy-benzoin: DMB),而用來環化蛋白質N端到胜肽側鏈的稱為溴乙醯基─羧基甲氧─α─羥─α─苯基苯乙酮(bromoacetyl-carboxymethoxybenzoin: BrAc-CMB),我們所用的胜肽都是由固相胜肽合成法所製造的。
我們試用不同條件與試劑合成DMB及Fmoc-Asp(DMB)-OH,並由紫外線光譜研究它們的光解特性。由圓二色光譜儀和二維核磁共振光譜儀所測得的資料,我們可以得知側鏈有保護基的胜肽之結構和其野生型之結構差異並不大,所以由DMB所保護的側鏈位置必須改變。環化蛋白質N端到胜肽側鏈策略有較大的潛能使蛋白質變性,我們合成了兩條環化的胜肽,並加以純化分離與鑑定。
The traditional stopped-flow methods for following the kinetic time course of protein folding reactions are limited by the dead-time of mixing. Moreover high concentrations of a denaturant are usually added to unfold proteins at the outset. In fact, how the denaturant affect folding pathways has been ignored. We cannot distinguish between the dissociation rate of denaturant and protein refolding rate. Using the photolabile linkers, a general method to monitor protein folding process shorter than 1 microsecond has been developed. The method is based on the photolysis of small organic protecting groups, or “cage” compounds. The compounds used in the studies are 3’,5’-dimethoxybenzoin (DMB) and bromoacetyl-carboxymethoxybenzoin (BrAc-CMB). These cage compounds are incorporated in to a three stranded β-sheet for the “sidechain” cage strategy and the N-terminal sequence of ubiquitin for “head-to-sidechain cyclization” strategy by Fmoc solid-phase peptide synthesis.
DMB and Fmoc-Asp(DMB)-OH were synthesized by different reactions and conditions. The properties of Fmoc-Asp(DMB)-OH and the “sidechain” caged peptide were further studied by UV spectroscopy. The “sidechain” caged peptide has no clear structural difference from its wildtype according to the CD spectroscopy and 2D-NMR data. So, the caged position had to be changed. The “cyclization” strategy has more potential to unfold the peptide. Two cyclized peptides were synthesized and purified.

Abbreviations i
Abstract iv
中文摘要 v
Chapter 1 Introduction 1
1. Protein folding problem 1
1.1 Significance of protein folding 1
1.2 Folding models 2
1.3 Kinetic studies of protein folding 6
1.3.1 Previous methodologies to study protein folding 6
(a) Stopped-flow, continuous-flow and quenched-flow 6
(b) Pressure jump 7
(c) NMR relaxation 8
1.4 Burst-phase in folding kinetics 8
1.4.1 Definition 8
1.4.2 Significance of early folding events 11
1.4.3 Methodologies for studying the early stage of protein folding 13
(a) Laser flash photolysis 14
(b) CO flash photolysis 14
(c) Photoreduction of cytochrome c heme (rapid electron transfer) 14
(d) Temperature-jump 15
(e) Ultrasonic absorption 16
1.4.4 Elementary steps in protein folding 17
1.5 Problems of using denaturant in folding studies 18
1.6 Our proposed new triggering method 20
1.7 Peptide models 25
1.8 Photoacoustic calorimetry 27
1.9 Aim of the project 29
Chapter 2 Materials and Methods 31
2.1 Materials 31
2.1.1 Water 31
2.1.2 Chemicals 31
2.1.3 Chromatography column, membranes and filters 34
2.1.4 Peptide automated synthesizer 35
2.1.5 Centrifugation 35
2.1.6 Peptide purification by HPLC 35
2.1.7 Lyophilizer 35
2.1.8 Mass spectrometry 36
2.1.9 Nuclear magnetic resonance spectroscopy 36
2.1.10 Ultraviolet and visible spectroscopy 37
2.1.11 Circular dicroism spectroscopy 37
2.2 Methods 37
2.2.1 Synthesis of 3’,5’-dimethoxybenzoin (DMB) 37
2.2.2 Synthesis of Fmoc-Asp(DMB)-OH 38
(a) Esterification (DCC/DMAP) 38
(b) Esterification (EDC/DMAP) 39
(c) Mitsunobu reaction 40
(d) Deprotection of allyl group 40
2.2.3 Purification of BrAc-CMB-OH 41
2.2.4 Solid phase peptide synthesis 41
2.2.6 Final cleavage 44
2.2.5 Head-to-side chain cyclization of peptide with BrAc-CMB-OH 45
2.2.7 Peptide purification and identification 46
Chapter 3 Results and Discussion (I): Side chain cage strategy 47
3.1 Synthesis of Fmoc-Asp(DMB)-OH 47
3.2 Characterization of Fmoc-Asp(DMB)-OH 55
3.3 Synthesis of 20-mer and 20-mer T9D 57
3.4 CD characteristics of 20-mer and 20-merT9D 58
3.5 Synthesis of 20-merT9D(DMB) 59
3.6 Characterization of 20-merT9D(DMB) 64
Chapter 4 Results and Discussion (II): Cyclization strategy 66
4.1 Purification of BrAc-CMB-OH 66
4.2 Synthesis of c-CMB-U(1-17)T9DE16KV17C (cyclized from 1 to17) 66
4.3 Synthesis of c-CMB-U(1-21)T9DE16KE18C (cyclized from 1 to 18) 70
Chapter 5 Conclusions and Future plans 73
References 74
Appendix 84
謝 誌 94

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