關於抗菌胜肽結構與活性之間的關係已經被研究，有許多的結構參數表 明它們與活性之間具有關聯性。例如：極性角度與電荷對於胜肽的抗菌作用已 被證明是重要的。   
    MP-B 是本研究的模型胜肽，用來探討結構參數中包括：極性角度、電荷、 螺旋含量和疏水性與胜肽分子內的相互作用，我們提出新的結構參數為電荷密 度，討論陽離子氨基酸殘基的分佈對抗菌活性影響的效果，我們利用高解析的 二維核磁共振光譜觀察 MP-B 的構象變化與抗菌活性之間的關聯性，同時整理 先前的研究，並且分析胜肽結構參數對抗菌活性的影響。  
      胜肽的電荷集中 在 C 端時，電荷密度顯示出有一個較大的值，可能形成 較小的寡聚行為以及降低抗菌活性。同時我們在 RP-HPLC 滯留時間顯示，當 胜肽具有較小的極性角度時，疏水性與 RP-HPLC 的靜相相互作用力較強。而 另一方面，當胜肽具有較大的極性角度範圍時，若範圍內有更多電荷殘基與芳 香族殘基則顯示出與螺旋含量有著正相關。胜肽的屬性不是由特定的某個結構 參數所決定。這些胜肽的活性被認為是透過綜觀的結構參數所決定，並非是特 定的某個氨基酸殘基。

Relationship between structure and activity of the antimicrobial peptide has been reported in previous studies. Many structure parameters have been suggest to correlate with their activity. For example role of polar angle and charge for antibacterial have been demonstrated to be importance.    
    MP-B, as a model peptide in this study, is used to explore the correlation of the structural parameters, including polar angle, charge, helix content and hydrophobicity with their intramolecular and antimicrobial activity by using high-resolution two-dimensional NMR spectroscopy, we investigate to the conformational change of MP-B and an analog and analysis the impact of these parameters on the antimicrobial activity. We investigate in detail the influence of the angle subtended by the positively charged amino acids on amphipathic helical peptides.    
    The concentrated charge at C terminal, shows a larger value of the generalized order parameters relative to MP-B, and might form smaller oligomers and reduced antimicrobial activity. The reversed phase HPLC retention times showed that peptides with smaller polar angle have stronger interaction with the hydrophobic stationary phase. On the other hand, peptides with more charged residues and aromatic residues within a larger polar angle show a positive correlation with the content of helix content (%). Peptide property does not dominated by a specific structure parameter. The activity of these peptides is thought to be determined by global structural parameters rather than by a specific amino acid residue.

目錄……………………………………………………………………Ⅰ 
表目錄…………………………………………………………………Ⅲ 
圖目錄…………………………………………………………………Ⅴ 
縮寫表…………………………………………………………………XI 
第一章 緒論 ................................................................... 1 
1.1 胜肽結構參數(Peptides structural parameters) ............................................... 3 
1.1.1 極性角度(Polar angle) .......................................................................... 3 
1.1.2 疏水力矩(Hydrophobic moment) ......................................................... 5 1.1.3 疏水性(Hydrophobic) ........................................................................... 7 
1.1.4 電荷(Charge) ........................................................................................ 9 
1.1.5 螺旋(Helicity) ..................................................................................... 10 
1.2 模型胜肽 Mastoparans B 介紹 .................................................................... 11 
1.3 研究動機........................................................................................................ 13 
1.4 研究目的........................................................................................................ 13 
第二章 實驗方法 ........................................................ 15 
2.1 固相胜肽合成(Solid-Phase Peptide Synthesis, SPPS) ................................. 15 
2.1.1 固相胜肽合成方法............................................................................. 17 
2.1.2 胜肽純化............................................................................................ 20 
2.2 圓二色光譜儀（Circular Dichroism Spectrometer） .................................. 22 
2.2.1 圓二色光譜實驗方法......................................................................... 25 
2.3 核磁共振光譜簡介(Nuclear Magnetic Resonance Spectrum ) .................... 27 
2.4 化學位移(Chemical Shift) ............................................................................. 30 
2.5 化學位移指數(Chemical Shift Index) ........................................................... 31 
2.6 DOSY 光譜(Diffusion Ordered Spectroscopy) ............................................. 33 
2.7 黏度實驗........................................................................................................ 34 
2.8 NMR 光譜實驗方法 ..................................................................................... 35 
2.9 結構計算........................................................................................................ 40 
2.10 無模型法則 (Model-free approach) ........................................................... 42 
2.10.1 函數模型選擇 (Model selection) .................................................... 45 
2.10.2 無模型法則計算............................................................................... 47 
2.11 抗菌實驗 ...................................................................................................... 48 
2.11.1 細菌的培養 ....................................................................................... 48 
2.11.2 養菌 ................................................................................................... 49 
2.11.3 抗菌觀測 ........................................................................................... 50 
2.11.4 抑菌生長曲線 ................................................................................... 50 
2.11.5 劑量反應曲線(dose–response curves) ............................................. 50 
2.11.6 抗菌實驗步驟 ................................................................................... 53 
第三章 實驗結果 ........................................................ 55 
3-1 胜肽結構參數與抗大腸桿菌活性關係 ....................................................... 55 3-2 電荷密度對抗大腸桿菌活性關係 ............................................................... 62 
3-3 極性角度內的電荷分佈對抗大腸桿菌活性關係 ...................................... 65 
3-4 實驗胜肽實驗結果 ....................................................................................... 68 
3.5 胜肽純化與鑑定............................................................................................ 69 
3.6 胜肽二級結構分析圓二色光譜實驗............................................................ 71 
3.7NMR 光譜訊號辨認 ...................................................................................... 74 
3.8 二次化學位移................................................................................................ 87 
3.9 HSQC 實驗結果 ............................................................................................ 96 
3.10 DOSY 實驗結果 ........................................................................................ 101 
3.11 胜肽結構參數 ............................................................................................ 105 
3.12 抗菌活性實驗........................................................................................... 107 
第四章 實驗討論與結論 ...........................................112 
4.1 改變 MP-B 電荷位置對結構的影響 .......................................................... 112 
4.1.1 CD 光譜的觀察 ................................................................................ 112 
4.1.2 NMR 光譜的觀察 ............................................................................ 113 
4.2 胜肽結構參數統計...................................................................................... 116 
4.2.1Mastoparan B 及其衍生物的胜肽結構參數對抗大腸桿菌活性關係 .................................................................................................................... 119 
4.2.2 文獻中的胜肽與實驗胜肽的電荷密度對抗大腸桿菌活性之影響 .................................................................................................................... 121 
4.2.3 文獻中的胜肽與實驗胜肽的電荷分佈對抗大腸桿菌活性之影響 .................................................................................................................... 123 
4.2.4 文獻中的胜肽與實驗胜肽的疏水性對抗大腸桿菌活性之影響... 125 
4.3 胜肽結構參數的討論.................................................................................. 129 
第五章 結論 .............................................................. 130 
第六章 參考文獻 ...................................................... 132 

表目錄 
表 1.1 胺基酸側鏈的疏水值 ..................................................................... 8 
表 2.1 固相胜肽合成實驗藥品 .............................................................. 19 
表 2.2 固相胜肽合成實驗藥品 .............................................................. 20 
表 2.3 RP-HPLC 純化胜肽藥品 .............................................................. 21 
表 2.4 RP-HPLC 冲堤梯度 ...................................................................... 21 
表 2.5 圓二色光譜實驗藥品 ................................................................... 26 
表 2.6 常規核磁共振的原子核 .............................................................. 27 
表 2.7 無序殘繞 αH 及 NH 化學位移.................................................. 32 
表 2.8 核磁共振實驗藥品 ...................................................................... 35 
表 2.9 TOSCY 實驗參數 ......................................................................... 36 
表 2.10 NOESY 實驗參數 ....................................................................... 37 
表 2.11 DOSY 實驗參數 .......................................................................... 37 
表 2.12 HSQC-T1實驗參數 ..................................................................... 38 
表 2.13 HSQC-T2實驗參數 ..................................................................... 38 
表 2.14 HSQC-NOE 實驗參數 ................................................................ 39 
表 2.15 HSQC-T1與 HSQC-T2延遲時間 ............................................... 39 
表 2.16 Model-Free 函數模型 ................................................................. 45 
表 2.17 抗菌實驗藥品 ............................................................................ 54 
表 3.1 胜肽序列 ...................................................................................... 68 
表 3.2 MP-B 與 MP-BV11K13在變溫 CD 實驗所計算之螺旋含量 ........ 72 
表 3.3 MP-B 的 1H 化學位移表 .............................................................. 88 
表 3.4 MP-BV11K13的 1H 化學位移表 ....................................................... 89 
表 3.5 MP-B NOE 連線情形 .................................................................... 90 
表 3.6 MP-BV11K13 NOE 連線情形 ........................................................... 91 
表 3.7 MP-B 與 MP-BV11K13的 RMSD 值 ............................................... 95 
表 3.8 MP-B 在 310 K 的弛緩參數統計 ................................................ 99 
表 3.9 MP-BV11K13在 310 K 的弛緩參數統計 ..................................... 100 
表 3.10 MP-B 的 DOSY 實驗結果 ........................................................ 103 
表 3.11 MP-BV11K13的 DOSY 實驗結果 ............................................... 104 
表 3.12 胜肽結構參數 .......................................................................... 106 
表 3.13 胜肽抗菌活性半抑制濃度統計 ............................................... 109 
表 4.1 MP-B 衍生物序列 ....................................................................... 116 
表 4.2 MP-B 衍生物胜肽結構參數統計 .............................................. 117 
表 4.3 MP-B 衍生物胜肽結構參數統計 ............................................. 118     

圖目錄 
圖 1.1 A 類胜肽與 L 類胜肽微胞脂質雙層膜系統中，模擬胜肽在微胞
膜上的情形 ................................................................................................ 4 
圖 2.1(a) Fmoc 保護基去保護反應機制；(b) BOC 保護基去保護反應機
制 ............................................................................................................... 15 
圖 2.2 Ninhydrin Test ............................................................................... 16 
圖 2.3 CD 光譜所呈現的胜肽二級結構吸收訊號位置......................... 23 
圖 2.4 光通過樣品槽之前與之後的強度差異 ....................................... 24 
圖 2.5 氫原子在靜磁場中所產生的能階分裂示意 .............................. 28 
圖 2.6 原子核磁矩從 Z 軸偏移至 XY 軸產生進動 ............................... 29 
圖 2.7 不同時間尺度蛋白質動態運動 .................................................. 42 
圖 2.8 劑量反應曲線 .............................................................................. 51 
圖 3.1 疏水性對抗大腸桿菌情形 ........................................................... 55 
圖 3.2 疏水力矩對抗大腸桿菌情形 ....................................................... 55 
圖 3.3 極性角度對抗大腸桿菌情形 ....................................................... 56 
圖 3.4 電荷數對抗大腸桿菌情形 ........................................................... 56 
圖 3.5 螺旋含量對抗大腸桿菌情形 ....................................................... 56 
圖 3.6 極性角度為 80°疏水性與抗菌活性之間的關係 ...................... 57 
圖 3.7 極性角度為 100°疏水性與抗菌活性之間的關係 .................... 57 
圖 3.8 極性角度為 120°疏水性與抗菌活性之間的關係 .................... 58 
圖 3.9 極性角度為 140°疏水性與抗菌活性之間的關係 .................... 58 
圖 3.10 極性角度為 160°疏水性與抗菌活性之間的關係 .................. 59 
圖 3.11 極性角度為 180°疏水性與抗菌活性之間的關係 .................. 59 
圖 3.12 極性角度為 80°疏水力矩與抗菌活性之間的關係 ................ 60 
圖 3.13 極性角度為 100°疏水力矩與抗菌活性之間的關係 .............. 60 
圖 3.14 極性角度為 120°疏水力矩與抗菌活性之間的關係 .............. 60 
圖 3.15 極性角度為 140°疏水力矩與抗菌活性之間的關係 .............. 61 
圖 3.16 極性角度為 160°疏水力矩與抗菌活性之間的關係 .............. 61 
圖 3.17 極性角度為 180°疏水力矩與抗菌活性之間的關係 .............. 61 
圖 3.18 極性角度 80°胜肽的電荷密度與抗大腸桿菌情形 ................ 62 
圖 3.19 極性角度 100°胜肽的電荷密度與抗大腸桿菌情形 .............. 63 
圖 3.20 極性角度 120°胜肽的電荷密度與抗大腸桿菌情形 .............. 63 
圖 3.21 極性角度 140°胜肽的電荷密度與抗大腸桿菌情形 .............. 63 
圖 3.22 極性角度 160°胜肽的電荷密度與抗大腸桿菌情形 .............. 64 
圖 3.23 極性角度 180°胜肽的電荷密度與抗大腸桿菌情形 .............. 64 
圖 3.24 極性角度 80°胜肽的電荷分佈與抗大腸桿菌情形 ................ 65 
圖 3.25 極性角度 100°胜肽的電荷分佈與抗大腸桿菌情形 .............. 66 
圖 3.26 極性角度 120°胜肽的電荷分佈與抗大腸桿菌情形 .............. 66 
圖 3.27 極性角度 140°胜肽的電荷分佈與抗大腸桿菌情形 .............. 66 
圖 3.28 極性角度 160°胜肽的電荷分佈與抗大腸桿菌情形 .............. 67 
圖 3.29 極性角度 180°胜肽的電荷分佈與抗大腸桿菌情形 .............. 67 
圖 3.30 MP-BV11K13胜肽純化後的 RP-HPLC 圖譜 ............................... 69 
圖 3.31 MP-BV11K13胜肽純化後的 MALDI-TOF-MS 圖譜 ................... 70 
圖 3.32 MP-B 的 CD 光譜在不同溫度訊號吸收情形 ........................... 71 
圖 3.33 MP-BV11K13的 CD 光譜在不同溫度訊號吸收情形 .................. 72 
圖 3.34 MP-B TOCSY 光譜 ..................................................................... 74 
圖 3.35 MP-BV11K13 TOCSY 光譜 ............................................................ 75 
圖 3.36 MP-B 在 αH-NH 區的 NOE 連結 ........................................... 76 
圖 3.37 MP-BV11K13 在 αN 區訊號的 NOE 連結 ................................... 77 
圖 3.38 MP-B 在 βH-NH 區訊號的 NOE 連結 ...................................... 78 
圖 3.39 MP-BV11K13在 βH-NH 區的訊號 NOE 連結 .............................. 79 
圖 3.40 MP-B 在 NH-NH 區的訊號 NOE 連結 ..................................... 80 
圖 3.41 MP-BV11K13在 NH-NH 區訊號的 NOE 連結 ............................. 81 
圖 3.42 MP-B 在 αH-βH 區的訊號 NOE 連結 ................................... 82 
圖 3.43 MP-BV11K13在 αH-βH 區訊號的 NOE 連結 ........................... 83 
圖3.44 MP-B顯示Tyr 9芳香環側鏈及C端末端修飾的-NH2與其它殘
基間的 NOE ............................................................................................. 84 
圖3.45 MP-BV11K13顯示Tyr 9芳香環側鏈及C端末端修飾的-NH2與其
它殘基間的 NOE ..................................................................................... 85 
圖 3.46 MP-B 與 MP-BV11K13 的 αH 區域二次化學位移差值 .......... 92 
圖 3.47 MP-B 與 MP-BV11K13 的 NH 區域二次化學位移差值 ......... 93 
圖 3.48 MP-B 與 MP-BV11K13螺旋範圍分佈情形 .................................. 94 
圖 3.49 MP-B(●)與 MP-BV11K13 (○)T1弛緩速率 ................................. 96 
圖 3.50 MP-B(●)與 MP-BV11K13 (○)T2弛緩速率 ................................. 97 
圖 3.51 MP-B(●)與 MP-BV11K13 (○)NOE ............................................. 97 
圖 3.52 MP-B(●)與 MP-BV11K13 (○)S2值 .............................................. 98 
圖 3.53 NOE 數目統計 MP-B(■)與 MP-BV11K13(■)及 S2值統計
MP-B(▲)與 MP-BV11K13(▲) .................................................................... 98 
圖 3.54 MP-B(藍色)與 MP-BV11K13(紅色)在 310 K 的 DOSY 光譜疊圖 
................................................................................................................. 101 圖 3.55 MP-B(●)與 MP-BV11K13(△)在 30 % TFE-d3 / 70 % H2O，不同
溫度的擴散係數(D) ............................................................................... 102 
圖 3.56 MP-B(●)與 MP-BV11K13(▲)在 30 % TFE-d3 / 70 % H2O，不同
溫度的自結合狀態 ................................................................................ 102 
圖 3.57 實驗胜肽極性角度螺旋投影圖 ............................................... 105 
圖 3.58 MP-B 在 37 ℃下抗菌情形 .................................................. 107 
圖 3.59 MP-BV11K13在 37 ℃下抗菌情形 ........................................... 108  
圖 3.60 MP-B-COO-在 37 ℃下抗菌情形 ......................................... 108 
圖 3.61 MP-B(●)與 MP-BV11K13(▼)及 MP-B-COO-(○)，溫度 310 K 的
CD 光譜 .................................................................................................. 110 
圖 3.62 MP-B(綠色)與 MP-BV11K13(紅色)及 MP-B-COO-(藍色)在
RP-HPLC 滯留情形 ............................................................................... 111 
圖 4.1 Mastoparan B 及其衍生物胜肽的電荷密度與抗大腸桿菌情形 
................................................................................................................. 119 圖 4.2 Mastoparan B 及其衍生物胜肽的電荷分佈與抗大腸桿菌情形 
................................................................................................................. 119 圖 4.3 Mastoparan B 及其衍生物胜肽疏水性與抗大腸桿菌情形 ..... 120 
圖 4.4 Mastoparan B 及其衍生物胜肽疏水力矩與抗大腸桿菌情形 . 120 
圖 4.5 極性角度 80°胜肽的電荷密度與抗大腸桿菌情形 ................ 121 
圖 4.6 極性角 100°胜肽的電荷密度與抗大腸桿菌情形 .................. 121 
圖 4.7 極性角 120°胜肽的電荷密度與抗大腸桿菌情形 .................. 122 
圖 4.8 極性角 160°胜肽的電荷密度與抗大腸桿菌情形 .................. 122 
圖 4.9 極性角度 80°胜肽的電荷密度與抗大腸桿菌情形 ................ 123 
圖 4.10 極性角度 100°胜肽的電荷密度與抗大腸桿菌情形 ............ 123 
圖 4.11 極性角度 120°胜肽的電荷密度與抗大腸桿菌情形 ............ 124 
圖 4.12 極性角度 160°胜肽的電荷密度與抗大腸桿菌情形 ............ 124 
圖 4.13 極性角度 80°胜肽的疏水性與抗大腸桿菌情形 .................. 125 
圖 4.14 極性角度 80°胜肽的疏水力矩與抗大腸桿菌情形 .............. 126 
圖 4.15 極性角度 100°胜肽的疏水性與抗大腸桿菌情形 ................ 126 
圖 4.16 極性角度 100°胜肽的疏水力矩與抗大腸桿菌情形 ............ 126 
圖 4.17 極性角度 120°胜肽的疏水性與抗大腸桿菌情形 ................ 127 
圖 4.18 極性角度 120°胜肽的疏水力矩與抗大腸桿菌情形 ............ 127 
圖 4.19 極性角度 160°胜肽的疏水性與抗大腸桿菌情形 ................ 127 
圖 4.20 極性角度 160°胜肽的疏水力矩與抗大腸桿菌情形 ............ 128

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