蜘蛛毒蛋白對於生物膜結構變化影響之探討

Abstract

自然界中存在許多種由蜘蛛所分泌的多肽毒液，其中由智利蜘蛛毒液所分離出來的hanatoxin 1 (HaTx1)，為一種廣泛研究的雙親性多肽。HaTx1作為電壓感應開啟式鉀離子通道的抑制劑，可與離子通道進行結合。以往透過電生理與點突變的相關研究來闡述HaTx1與電壓感應開啟式鉀離子通道 Kv2.1的抑制機制；研究指出 HaTx1並非直接與 Kv2.1結合，而必須先與細胞膜進行作用。本論文利用生物物理方法來探討HaTx1與脂質膜之間的交互作用，提供一個良好的實驗平台，再結合高強度的同步輻射研究設施探索原子級的結構變化。以微觀的觀點，進一步探討 HaTx1與生物膜之間的作用關係。 本研究進行兩項實驗，分別瞭解 HaTx1作用於單層膜及雙層膜的方式。首先，以蘭牟爾膜 (Langmuir-Blodgett film) 來研究 HaTx1吸附及插入單層膜的方式以及膜組成成份是否造成影響；本文結果顯示HaTx1會吸附於單層膜，但是插入單層膜的情況會因膜組成成份不同 (POPC；POPC：DOPG = 3：1；POPC：DOPG = 2：1) 而有不同程度的膜面積擴張，意含著磷脂質的親水性頭部在HaTx1吸附及插入膜中扮演著重要的角色。其次，以多片層X光繞射的技術來測量雙層磷脂質膜的厚度；本文結果指出隨著HaTx1相對於脂質的濃度 (the molar ratio of peptide-to-lipid, P/L) 上升，HaTx1會造成不同組成成份 (POPC；POPC：POPG = 3：1；POPC：POPG = 2：1) 的雙層磷脂質膜變薄，也就是說無論組成份是純 POPC 或者是含有帶負電 POPG 所構成的雙層磷脂質膜，都會隨著 P/L 的上升，而使雙層磷脂質膜厚度有不同程度的變薄效應。結果顯示，HaTx1為一種雙親性胜肽且具有使雙層磷脂質膜厚度變薄的效應，如同其他已被廣泛研究的雙親性分子 (melittin, curcumin) ，皆會造成雙層磷脂質膜的形變。然而細胞膜的形變可能會進一步影響膜蛋白的功能，因此HaTx1可能會藉由改變雙層磷脂質的性質來影響電壓感應開啟式鉀離子通道。 倘若未來實驗技術突破，我們希望可以同時考慮HaTx1、脂質膜及電壓感應開啟式鉀離子通道，三者之間的交互作用，對於電壓感應開啟式鉀離子通道的詳細作用機制做更明確的推論及闡述。

A variety of polypeptide toxins are isolated from spider venom, and hanatoxin 1 (HaTx1) from a Chilean tarantula is one of the most extensively investigated peptides. HaTx1 might act as a voltage-gating modifiers to bind ion channels and has been used to characterize the blocking properties of the voltage-gated potassium channel Kv2.1 through electrophysiological and mutational studies. Recent studies have shown that HaTx1 may first interact with the cell membrane rather than directly bind onto voltage-sensing domain of Kv2.1. Thus, in this study the interactions between HaTx1 and bio-membranes were investigated with biophysical methods as an excellent experimental platform and high intensity synchrotron radiation source to explore the structural changes of bio-membranes at atomic level. Furthermore, we depicted the detailed mechanism between HaTx1 and bio-membranes. Two parts of experiments were designed to investigate the interactions between HaTx1 and monolayers at air/water interface and between HaTx1 and lipid bilayers. The Langmuir-Blodgett film was used to study the adsorption and the insertion into monolayers of HaTx1 and its effect on different compositions of lipids. The results indicated that HaTx1 first adsorbed to monolayers and caused the area expansion of membranes, which varied with different compositions of lipids (POPC；POPC：DOPG = 3：1；POPC：DOPG = 2：1), when inserting into monolayers. This implied that the headgroup of phospholipids plays a crucial role in the adsorption and insertion of HaTx1. In addition, Lamellar X-ray diffraction was used to measure the membrane thickness of phospholipid bilayers interacting with HaTx1. The results showed that the membrane thickness decreased as the molar ratio of peptide-to-lipid (P/L) increased in all three compositions of lipids (POPC；POPC：DOPG = 3：1；POPC：DOPG = 2：1); as the P/L increased, HaTx1 caused the thinning effect on the thickness of lipid bilayer in different extent. HaTx1 has been shown to be a amphipathic peptide and demonstrated the membrane-thinning effect. As other extensively investigated amphipathic molecules (e.g., melittin and curcumin), HaTx1 caused bilayer deformation. Such deformation of cell membrane might affect the function of membrane proteins, thus it is considerable that HaTx1 could affect the function of voltage-gated potassium channels by modifying the properties of phospholipid bilayers. In the future with the breakthrough of experimental techniques, we aim to reveal the interplay between three components, HaTx1, lipid membranes, and voltage-gated potassium channels, and to elucidate the detailed mechanism of voltage-gated potassium channels.