- 學位論文
利用場循環式核磁共振儀研究 Melittin 以及 Pardaxin 在磷脂膜上的效應
Effect of Melittin and Pardaxin on Phospholipids Membrane by Fast Field-Cycling NMR Relaxometry
摘要
抗菌胜肽( Antimocrobial peptides，AMPs ) 是由五到百個胺基酸所組成的低聚肽，為動物體對抗病原體的重要一環。到目前為止，多達5000 種 AMPs 已經被發現或合成出來。AMPs 可由二級結構區分為: β-sheet、α-helix、random coil 及 loop 四種型態，其中β-sheet 及α-helix 為AMPs最普遍的結構。AMPs 先吸附後進行插入，可使病原體表面積擴張及破壞外膜結構，導致病原體細胞膜表面穿孔進而死亡。其攻擊模式可分為以下三種: (1)環形穿孔模式 ( Toroidal mode ); (2)筒狀穿孔模式 ( Barrel-stave mode ); (3) 地毯模式 ( Carpet mode )。 本研究中兩種抗菌胜肽，根據文獻，Melittin 屬於環形穿孔模式；Pardaxin 屬於筒狀穿孔模式。兩者微小差異在於 Melittin 會促使脂雙層結構彎曲連接內外膜形成穿孔; Pardaxin 則直接穿過脂雙層形成穿孔，進而導致穿孔後膜內外滲透壓不平衡進而破裂。 兩種 AMPs 對電負性微脂粒磷脂分子的動態學，經由變場式核磁共振儀在不同溫度下紀錄自旋-晶格弛豫速率 ( R1 )，並藉由模型擬合實驗數據來得到欲觀測的動力學參數。並利用 500 MHz 的NMR磷譜，在POPG 及 AMPs 系統中加入錳離子，藉此觀察訊號的衰變來證明孔洞產生並且可了解兩模式差異。 模型擬合之R1 貢獻分為以下三部分貢獻: (1) 未鍵結區、(2) 鍵結區以及(3) 穿孔區。結果顯示，兩種 AMPs 一旦達P(peptide)/L(lipid) 臨界值 ( P/L* ) 皆會有上述三部分的出現。鍵結區的磷脂分子，會受到 AMPs 的影響導致彎曲彈性係數( κ )下降，代表有利於之後插入的行為; 而穿孔區的磷脂分子，其彎曲彈性係數( κ ) 上升，且不隨溫度變化，代表通道的穩定存在。值得注意的地方為，可以明顯看出 Pardaxin ( Barrel-stave ) 穿孔區的κ 明顯大於 Melittin ( Toroidal )穿孔區κ ，代表著 Pardaxin 通道形成會使得膜變得較硬。而由NMR 磷譜訊號的減弱，可以證明孔洞確實形成，且錳離子流入膜內。另一方面，由擬合部份以及磷譜實驗，可以看出Pardaxin 在極低 P/L 比例就已經有孔洞產生; Melittin 則是到達一定 P/L 比例才會有孔洞產生。平移擴散係數 ( D )的下降則是因為 AMPs 加入，吸附或插入的行為皆會對磷脂分子的擴散形成阻礙。
並列摘要
Antimicrobial peptides are small amphiphilic molecules with antibacterial activity, generally between 5 and over a hundred amino acids. Until now, over 5000 kinds of antimicrobial peptides are found. Antimicrobial peptides can be divided mainly into four groups according to its secondary structure: (1) β-sheet, (2)α-helix, (3)extended, and (4)loop. Among these mentioned above, the β-sheet and α-helix are the most common structure. The antimicrobial peptides perform the antibacterial activity by attacking the membrane, which can be divided into the following three attacking modes: (1) Toroidal mode, (2) Barrel-stave mode, and (3) Carpet mode. Two antimicrobial peptides, melittin and pardaxin, has been widely studied over the past two decades. The attacking mechanism are toroidal mode and barrel-stave mode for melittin and pardaxin, respectively. The difference between them is melittin leads to bending formation of the annular hole connecting the inner and outer lipid bilayer membrane while pardaxin directly penetrates the lipid bilayer to form a cylindrical hole. In addition, two attacking modes make membrane rupture due to the different osmotic pressure between inner and outer. The study of lipid bilayer dynamics is performed by fast field cycling nuclear magnetic resonance relaxometry. The spin-lattice relaxation rate of system consisting 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphoglycerol,sodium salt (POPG) and antimicrobial peptides are investigated by varing P/L ratio and temperatures. By using simulation, the detailed molecular dynamics is obtained. Furthermore, the 31P NMR spectroscopy is a powerful tool to inspect the pore-formation by observing the disappearance of the signal intensity.
參考文獻
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