論文提要 當胞膜表面吸附抗菌胜肽時，若抗菌胜肽夠密集，就會使膜上產生穩定的孔洞。本文將從理論和實驗的層面來了解抗菌胜肽引發孔洞形成的機制。 當抗菌胜肽吸附於膜表面時，會展現雙親性(即親油/親水)的結構。並且會埋入膜的頭部區，使得膜的親油(碳鏈)區面積被延展，造成膜的厚度變薄。由於實驗證明膜厚度的變薄和胜肽濃度成正比，因此我們提出一個理論來說明胜肽濃度高時：1.膜厚度的變薄足以產生孔洞；2.部份胜肽會從膜表面轉移到孔洞的邊緣來穩定孔洞。這種胜肽轉移形成孔洞的過程類似一個相變。 實驗上，使用兩種被廣為研究的胜肽(alamethicin和melittin)與不同的脂質雙層膜作用來研究孔洞產生的過程。此兩種胜肽與膜作用分別會產生兩種不同型態的孔洞(barrel-stave孔洞和toroidal孔洞)。我們使用兩種實驗技術：1.指向性圓極化雙光譜技術，用來測定胜肽的方位--平行膜面，即吸附於膜表面；垂直膜面，即吸附於孔洞邊緣；2.多片層X光繞射技術，用來測量胜肽造成脂質雙層膜厚度的變化。實驗將測量一系列胜肽的方位和脂膜的厚度隨胜肽濃度的變化。 所有的實驗結果都非常符合我們理論的預測。結合理論與實驗結果可得到因?肽與膜作用而形成孔洞的相關參數值。我們討論這些參數的意義，而且比較不同胜肽，不同脂膜和不同型態的孔洞所具有的參數值。這些參數值在分子層次的分析上以及分子動力模擬的研究上非常有用。除此以外，本論文中的結果在基因與藥物輸送、藥物設計和抗感染的治療上也具有很高的應用價值。 Abstract When membrane surface adsorbs antimicrobial peptides, stable pores are found to emerge if the adsorbed peptides are sufficiently dense. This thesis presents both experimental and theoretical approaches to the understanding of such pore formation induced by antimicrobial peptides. When binding to membrane surface, antimicrobial peptides exhibit amphiphilic structure and embed themselves into the headgroup region of the membrane. As a result, the hydrophobic (carbon chain) region of the membrane is surface-stretched or equivalently is thinning. Based on the fact that the thinning effect is increased with peptide concentration, we proposed a theory to show: 1. The membrane thinning in high peptide concentration can be sufficiently strong to make pores on membrane; 2. The pores can be stabilized by transferring a fraction of peptides to the pore wall, resembling as a phase transition. Experimentally, we investigated the pore formation process in model bilayer membranes of various lipid compositions. Two of the best-study peptides, alamethicin and melittin, were used to represent peptides making two different types of pores, that is, barrel-stave pores and toroidal pores. Two methods were employed in experiments. They are: 1. Oriented circular dichroism (OCD) to monitor the peptide orientation, either parallel (surface adsorbed) or perpendicular (pore wall adsorbed), to the membrane surface; 2. Lamellar x-ray diffraction (LXD) to measure the membrane bilayer thickness. Experiments were conducted to measure the peptide orientation and the membrane thickness as a function of peptide concentration. All experimental results were in agreement with the prediction of the proposed theory. The parameters that characterize the peptide-membrane interaction related to the pore formation were extracted from the experimental results. We discussed the meaning of these parameters and compared their values for different lipids and for the two different types of pores. These parameters are useful for further molecular analysis and are excellent targets for molecular dynamic simulations studies. The results in this thesis are also potentially useful for gene and drug deliveries, drug design and anti-infective therapeutics.
