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標題: Design the protein switches based on the kinetics of protein folding
作者: Shen-Yi Zheng
關鍵字: 蛋白質摺疊動力學
Rapidly unfolding/folding (RUF)
金黃色葡萄球菌protein A
IgG binding domains
螢光共振能量轉移效率(FRET efficiency)
Kinetics of protein folding
Rapidly unfolding/folding (RUF)
fluorescent protein
staphylococcus aureus protein A
IgG binding domains
FRET efficiency
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摘要: 以蛋白質工程學的原理為基礎來設計功能性蛋白質已被廣泛應用在許多研究領域之中,而在許多蛋白質設計的策略裏蛋白質開關(protein-switch) 於近幾年更有許多成功的實際應用。此種蛋白質開關的設計原理主要為利用蛋白質分子因「熱力學穩定度」的變化而能在兩種構型之間來回切換,用以執行不同的功能。有趣的是,有別於熱力學穩定度的應用,過去並無文獻使用蛋白質「摺疊動力學」來設計蛋白質開關,而本研究即在探討將折疊動動力學性質應用在蛋白質設計的可能性。金黃色葡萄球菌(Staphylococcus aureus)表面蛋白質¬¬protein A有五個具rapidly unfolding / folding (RUF) 特性的IgG binding domains,因為具有過渡態(transition state)自由能偏低特色,可以從摺疊態(folded state)自行經歷過渡態到不具有構型的解摺疊態(unfolded state),再回到摺疊態,本實驗即使用上述的其中兩個domain,做為以蛋白質摺疊動力學為基礎設計蛋白質開關的模型。於本研究中,我們成功的將螢光蛋白CyPet和YPet構築在選定的domain兩端,搭配螢光共振能量轉移(Förster resonance energy transfer,FRET)的技術進行蛋白質折疊動力學的定量觀測。比較一系列設計的融合蛋白之能量轉移效率(FRET efficiency),我們發現FRET efficiency會隨CyPet以及YPet之間domain的折疊動力學性質而有明顯的變化。另外,我們也觀察到不同的domain串連個數也會對FRET efficiency有明顯的改變。此研究的結果或可作為將來蛋白質功能設計上的新策略以及依據。
Protein engineering is widely used in various research fields and industrial applications. Among different engineering strategies, protein switch gets more and more attention in recent years. The basic principle of designing protein-switch is to alter the conformation of proteins according to the difference of 'thermodynamic stability'. Protein switch is a powerful tool used in designing biosensor, biomaterial or controllable-enzyme. Interestingly, folding kinetics is another characteristic of protein conformation, based on our literature search there is no application of this characteristic on designing protein-switch. Therefore we seek to demonstrate the possibility of applying protein-folding kinetics on the design of protein-switch. Protein A is a surface protein of staphylococcus aureus, which has five IgG binding domains on its N-termius. These domains were selected as our test model because of their special property with rapidly unfolding and folding (RUF) kinetics. In order to test this RUF kinetics effect on the protein engineering, the fluorescent proteins CyPet and YPet were fused on the two sides of designated domains. We then observed the kinetic effect of these domains by Förster Resonance Energy Transfer (FRET). Our results indicate that RUF kinetics effect can still be observed by fusion with flanked proteins. Meanwhile, the FRET efficiency of designed constructs was changed according to the difference of RUF characteristic. This identification can potentially be used as a new tool in the strategies of designing protein function.
文章公開時間: 10000-01-01
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