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研究生： 劉芝伃
Yovela Z.-Y. Liu
論文名稱： 轉彎序列對b-摺板結構的影響
Effects of turn mutations on the structure of a three-stranded b-sheet peptide
指導教授： 俞聖法
Yu, Sheng-Fa
學位類別： 碩士
Master
系所名稱： 化學系
Department of Chemistry
論文出版年： 2006
畢業學年度： 94
語文別： 英文
論文頁數： 64
中文關鍵詞： b-摺板 、轉彎序列
英文關鍵詞： b-hairpin, turn sequence
論文種類： 學術論文
相關次數： 點閱：200  下載：0 
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    • 我們引用Gellman 在1998年設計出的一個可單獨存在且具有良好水溶性的b-摺板結構作為模板，將第六個位置的DP突變成D時，稱之為P6D，此時N端髮夾結構的轉彎序列由原本的DP-G ( Type II’ turn) 變成由五個氨基酸所組成的T-S-D-G-K ( Type I + b bulge )，造成N端髮夾結構氨基酸相對位置的改變；而繼續在第14個位置做相同突變時，稱之為P6DP14D，C端髮夾結構並無相對位置上的改變。本研究將Gellman胜肽N端髮夾的序列S-DP-G-K置換成P6DP14D在C端髮夾所形成的轉彎序列V-D-G-O，稱之為M-T20mer，探討其將會形成何種轉彎序列並探討其與P6D的差異何在，並將此胜肽C端髮夾位置做相同的置換，稱之M-TP14D，可完整探討轉彎序列和疏水性作用力對蛋白質摺疊的影響。我們主要利用核磁共振光譜、圓二色光譜，和結構的計算等方法進行研究，結果得到擁有V-D-G-O序列的胜肽在最終結構獲得較高的穩定性，但在摺疊的過程中因必須靠疏水性作用力 (此疏水性基團包含第三個b-長帶) 的幫助而較慢形成。

    We used the three-stranded b-sheet 20mer peptide, VFITSDPGKT
    YTEVDPGOKILQ, designed by Schenck and Gellman (1998), as the model peptide to exploit conformational stability of the structure between the effects of the turn sequence and the hydrophobic collapse using various mutation of the peptide. M-T20mer, one of the corresponding mutant peptides, wherein the TSDPGK turn sequence in the N-terminal hairpin was replaced by TVDGO, was synthesized. The turn structure was shown to altered with this change. We further mutated the DP to D in C-terminal hairpin of M-T20mer, denoted as M-TP14D, to compare the difference between our earlier studies on P6D and P6DP14D18. NMR spectra and simulated annealing methods were cored to solve the structure of these peptides. CD spectra were cored to show the different secondary structural features of these peptides. We concluded that the M-T20mer peptide has a 2:2 type I’ turn VDGO. The 20mer peptide with the V-D-G-O turn sequence in N-terminal hairpin was stabilized by hydrophobic collapse involving the third strand, whereas the 20mer peptide with T-S-D-G-K turn sequence (P6D mutation) was stabilized by the N-terminal turn.

    Table of contents Table of contents i 摘要 iv Abbreviations iv Chapter 1: Introduction 1 1.1 Protein Folding and Relevant Diseases 1 1.2 Folding Models and Mechanisms 2 1.3 Classes of the Beta Turn Types 8 1.4 Amino Acids and Common Characteristic of Secondary Structure 13 1.5 Our Previous Studies on the Refolding of a -hairpin on -sheet 15 1.5.1 The Head-to-Side Chain Strategy 15 1.5.2 Effects of Turn Sequence on 3-Strand  Sheets 17 1.6 De Novo Designed Peptide M-T20mer 20 Chapter2: Materials and Methods 21 2.1 Materials 21 2.1.1 General 21 2.1.2 Chromatography Columns, Membranes, and Filters 23 2.1.3 pH meter 23 2.1.4 Peptide automated synthesizer 24 2.1.5 Centrifuge 24 2.1.6 Reverse-phase High Performance Liquid Chromatography 24 2.1.7 Lyophilizer 24 2.1.8 Mass Spectroscopy 25 2.1.9 Circular Dichroism Spectroscopy 25 2.1.10 Ultraviolet Spectroscopy 25 2.1.11 Photochemical Reactor 25 2.1.12 Nuclear Magnetic Resonance Spectroscopy 26 2.2 Methods 26 2.2.1 Solid Phase Peptide Synthesis 26 2.2.2 Head-to-side chain Cyclization of Peptide with the Photolabile Linker 27 2.2.3 Cleavage of Peptide from Resin 27 2.2.4 Purification of “Caged” Peptides 30 2.2.5 Identification of the “Caged” Peptides by Mass Spectroscopy 30 2.2.6 Ultraviolet spectroscopy 31 2.2.7 CircularDichroism Spectroscopy 31 2.2.8 NMR Spectroscopy 31 2.2.9 NH exchange rate 32 2.2.10 Structure Analysis 32 Chapter3: Results 34 3.1 Synthesize and purify the M-T20mer 34 3.2 Chemical shifts and NOEs 35 3JNH-H coupling constants 43 3.4 NOE 45 3.5 Structure calculations and the constraints used 46 3.6 M-T20mer Adopt a Right-handed Twist 52 3.7 Synthesis and Purification of the other peptides and CD spectra 53 Chapter4: Discussion 56 4.1 Effects of mutations on the turn sequence 56 4.2 The result can correspond to the two mechanisms 61 4.3 The characteristic of c-M-TE12C 62 Reference 63

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