透過您的圖書館登入
IP:3.238.87.31
  • 學位論文

用原子力顯微鏡觀察細菌蛋白MinE在雲母和雙層膜上的自組裝

Atomic Force Mcroscopy Charaterization of Fibrils Formed by Bacteria protein MinE on Mica and a Supported lipid bilayer

指導教授 : 黃英碩
若您是本文的作者,可授權文章由華藝線上圖書館中協助推廣。

摘要


生物系統中的蛋白、胜肽分子大部分都需要經過特殊的摺疊或自組裝之後才能發揮其應有的功能,然而這些生物分子的自組裝機制及過程到目前為止都還不是非常了解。在論文中,我們使用了最近被發現具有類β-蛋白澱粉的MinE蛋白質,用高靈敏度的頻率調制原子力顯微鏡觀察其全長以及其N端胺基酸序列1-31在雲母及單層雙層脂肪膜上的自組裝,MinE在大腸桿菌細胞分裂過程中扮演重要角色,而β-蛋白澱粉則在自然界中廣泛存在,大致可分為具功能性以及不具功能性β-蛋白澱粉,不具功能性的β-蛋白澱粉蛋白堆積在人體中會導致疾病,堆積在腦部會導致神經方面的疾病,例如愛滋海默症,而正常具功能性的β-蛋白澱粉則具高穩定性,通常扮演細胞骨架的功能。 從實驗中我們發現MinE其N端胺基酸序列1-31的合成胜肽在雲母上會自組裝成長的纖維狀巨分子,且因為頻率調制原子力顯微鏡的高靈敏度可以解出纖維狀巨分子是由一束四到六條較細的原纖維組成。纖維狀巨分子的型態會因為溶液中不同的鹽離子及蔗糖濃度而呈現直線、捲曲以及圓的型態,顯示溶液的環境對胜肽及蛋白質的自組裝有ㄧ定的影響,另外我們也觀察到MinE其N端在溶液中自組裝的動態行為,我們更進一步發現胜肽在雲母鋪附的雙層膜上也會自組裝成長的纖維狀巨分子,觀察結果顯示當雙層膜及雲母同時存在,胜肽自組裝的纖維結構只在雙層膜上出現,在雲母上則不會形成,顯示胜肽對雙層膜有高親合力。而在MinE全長的實驗中,MinE全長在雲母上的自組裝並沒有表現出特殊的可辨識的結構,當與在雲母上鋪附的雙層膜作用時則會在雙層膜的邊緣形成短的纖維化巨分子,與在雲母上的行為有所差異,也與MinE N端的表現不同。此篇論文對MinE這個具重要功能的蛋白用原子力顯微鏡做了詳盡的探討。

關鍵字

原子力顯微鏡

並列摘要


The placement of cell division site in model bacteria E.coli is tightly regulated by Min system. In E. Coli, Min system blocks the polar position for cell division, MinC, D and E are three members in Min system. Among the Min system, MinE can form a ring-like structure (the MinE-ring) in E. coli and oscillates with MinCD. MinE includes 88 amino acids and contains three domains: the N-terminal membrane targeting domain (MinE2-12), the anti-MinCD domain (MinE13-31) and the C-terminal dimerization domain (MinE32-88). In this study, we study the self-assembly behavior of MinE and MinE1-31 on mica and on supported lipid bilayer prepared from E-coli phospholipids extraction. To investigate the self-assembly behavior of MinE and MinE1-31, we utilize highly sensitive Frequency Modulation Atomic Force Microscopy (FM-AFM). FM-AFM applies a small force over the surface, thereby causes little deformation on soft and fragile biological samples. The results indicated that the features of both MinE and MinE1-31 filaments on supported lipid bilayer were very different from those assembling on mica. On mica, N-terminal MinE1-31 fragments self-assembled into filamentous bundles that contained 4~6 prorofilaments. However, the number of protofilament within filamentous bundles varied when appiled MinE1-31 on support lipid bilayer. For full length MinE, although the self-assembled structure shown on mica is amorphous, MinE was able to arrange into short protein filaments that decorated around edges of the membrane patches. We can conclude that MinEexhibits spatial preference to membrane edge but MinE1-31 does not. The results also hint that C-terminal MinE plays the main role in MinE membrane edge binding activity. Thesefindings may be cruicial to the MinE-ring formation in the midcell.

並列關鍵字

Atomic Force Mcroscopy

參考文獻


1. Lutkenhaus and J, Addinall S (1997) Bacterial cell division and the Z ring. Annual review of biochemistry 66: 93-116.
2. Adams DW, Wu LJ, Errington J (2014) Cell cycle regulation by the bacterial nucleoid. Current opinion in microbiology 22: 94-101.
4. Shih Y-L, Rothfield L (2006) The bacterial cytoskeleton. Microbiology and Molecular Biology Reviews 70: 729-754.
5. Raskin DM, de Boer PA (1997) The MinE ring: an FtsZ-independent cell structure required for selection of the correct division site in E. coli. Cell 91: 685-694.
8. BONNELL D (2001) Scanning probe microscopy and spectroscopy: theory, techniques, and applications: Wiley-vch.

延伸閱讀