建立以單分子螢光顯微技術研究Shugoshin-1蛋白與潛力治療用胜肽間動態交互作用的平台

Abstract

蛋白質-蛋白質交互作用(Protein-Protein interactions, PPIs)促進大部分生物機能，蛋白質區域與區域之間的交互作用(Domain-Domain interactions, DDIs)引導功用性蛋白在特定區域作用，交互作用必須通過蛋白質的二級結構進行，二級結構中螺旋結構穩定且單純，探討螺旋結構之間的交互作用能夠得到更精確的結果。Shugoshin 1 (Sgo1)和蛋白磷酸酶2A (PP2A)之間的結合研究說明Sgo1募集PP2A在保護姊妹染色體中有重要的作用。為了發展一系列抗微生物胜肽，我們擷取Sgo1-PP2A交互作用的片段，將目標Sgo1胜肽和DNA交聯，並經由與生物素標記的互補DNA結合而固定在單分子檢驗平台上，並在緩衝溶液中添加螢光標記的游離潛力治療用胜肽進行交互作用，利用單分子螢光顯微鏡研究胜肽-胜肽交互作用動力學。和傳統的方法比較，需要的胜肽濃度相對較低(< 100 nM)，使我們可以對潛力治療用胜肽進行更廣泛的篩選。Protein-Protein Interactions (PPIs) facilitate many biological functions, and Domain-Domain Interactions (DDIs) induce functional protein activity in specific regions. The interactions between domains are usually dictated by their secondary structures . Helical structure is relatively simple and stable , hence , becomes a suitable candidate for studying domain-domain interactions . Previous study of Shugoshin 1(Sgo1)-Protein Phosphatase 2A(PP2A) binding demonstrated that recruitment of PP2A by Sgo1 plays an important role in the protection of sister chromatid , Hence , becomes a potent target for the development of antimicrobial peptides. We extract the fragments of Sgo1-PP2A interaction and establish a single-molecule-fluorescence-microscopy-based platform for studying the peptide-peptide interaction dynamics. Target peptide (Sgo1)-DNA hybrid was used in the immobilized single-molecule assays and interacted with the fluorescence-labeled free potential therapeutic peptides in buffer solution. The required concentration (< 100 nM) of peptides is relatively low compared to conventional methods, allowing us to perform a wider range of screening on the potential therapeutic peptides.