在全球各地，敗血症是常見的致命性疾病之一且仍然具有很高的死亡率。而革蘭氏陰性細菌具有脂多醣的組成（LPS，也稱作內毒素）這是感染敗血症最常見的感染源。在治療上抗菌胜肽被稱為“天然抗生素”的新型廣譜抗菌藥物，不僅可以直接地殺滅細菌，也能藉由聚集LPS的形式去中和LPS的毒性。然而，並非所有的抗菌胜肽都具有聚集並中和LPS的能力。所以，我們想進一步探討的抗菌胜肽和中和LPS活性之間的關係。
我們設計的抗菌胜肽S1和S1Nal2是從矛頭蝮蛇毒液C-末端區域衍生的PEM-2而來。根據我們的實驗室的研究表明，含有非天然氨基酸β-萘基丙氨酸（NAL）的肽可以有效地提高抗菌活性和對鹽的耐受性。而這二個胜肽都有造成聚集LPS的能力，但只有S1Nal2具有中和LPS的效果。我們利用原二色光譜（CD）分析發現這二個胜肽分別在不存在或存在LPS的情況下顯示為隨機纏繞和α-螺旋構象。然後利用轉移NOE光譜（Tr-NOE）來提供胜肽結合上LPS的結構。且根據飽和轉移差譜（STD）顯示S1的色氨酸和S1Nal2的色氨酸和β-萘基丙氨酸都和LPS有著密切關係。最後，順磁弛豫增强（PRE）的結果，發現相比於S1，S1Nal2能更深地插入到LPS。綜上所述，我們發現S1Nal2的構象可以更有效地結合LPS。因此，我們希望能藉由S1和S1Nal2結構屬性的差異幫助我
們設計出更有效的LPS中和胜肽。

Sepsis is one of the most common fatal diseases in humans worldwide, with high rates of deaths. The most common source of infection resulting in sepsis is gram-negative bacteria that contain lipopolysaccharides (LPS, also known as endotoxins). Antimicrobial peptides, called “natural antibiotics,” are novel, broad-spectrum antimicrobial drugs that not only kill the bacteria directly but also neutralize LPS toxins by LPS aggregation. However, not all antimicrobial peptides have the ability to aggregate and neutralize LPS; therefore, we explored the relationship between antimicrobial peptide conformations and their LPS-neutralizing activity.
We designed two antimicrobial peptides, S1 and S1Nal2, derived from PEM-2, the C-terminal region of the venom from the snake Bothrops asper. Our previous laboratory studies have demonstrated that peptides containing the unnatural β-naphthylalanine (Nal) can effectively enhance the antibacterial activity and salt resistance. We also found that both S1and S1Nal2 have the ability to aggregate LPS, but only S1Nal2 has the ability to neutralize them. A CD spectroscopic analysis of each peptide in the absence or presence of LPS showed random coil and alpha-helical conformations, respectively. In addition, the LPS-bound structure of each peptide was determined by Tr-NOE spectroscopy. STD spectrum analysis revealed that the tryptophans of S1 and the tryptophans and Nal of S1Nal2 are closely associated with LPS. PRE results revealed that S1Nal2 has been inserted deeper into the LPS than S1. In summary, the conformation of S1Nal2 allows it to neutralize LPS more effectively than S1. The differences in the structural and functional properties of S1 and S1Nal2 enable us to design better LPS-neutralizing peptides.

中文摘要 2
Abstract 3
Acknowledgement 4
Contents 5
List of Figures 7
List of Tables 8
List of Abbreviations 9
Chapter 1 Introduction 10
1.1. Overview of Sepsis 10
1.2. Sepsis Treatment 11
1.3. The properties of AMPs 12
1.4. Hydrophobic characteristics of AMPs 13
1.5. Endotoxin-neutralizing activity of AMPs 14
1.6. Aim of the study 15
Chapter 2 Materials and Methods 17
2.1 Peptide and LPS preparation 17
2.2 Quantification of peptides 17
2.3 Circular dichroism (CD) spectroscopy 18
2.4 Transferred nuclear Overhauser enhancement (Tr-NOE) 20
2.5 Saturation transfer difference (STD) spectroscopy 22
2.6 Paramagnetic Relaxation Enhancement (PRE) 24
Chapter 3 Results 26
3.1 Binding structure of peptides by CD studies 26
3.2 Structure calculation and description of the peptides in LPS by Tr-NOE 27
3.3 Three-dimensional structure of the peptides in LPS by Tr-NOE 28
3.4 Structure characterization of the peptides in LPS by STD studies 28
3.5 Orientation of peptides in LPS by PRE studies 29
Chapter 4 Discussion 30
References 53

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