MTGFP融合蛋白摺疊過程中分子內螢光能量轉移研究

Intramolecular Fluorescence Energy Transfer Study of MTGFP fusion protein during protein folding process

姜芳馨(Fang-Hsing Chiang)

指導教授 ： 張家靖

國立交通大學/生物科技學院/分子醫學與生物工程研究所/碩士(2009年)

https://doi.org/10.6842/NCTU.2009.00652

摘要

為能即時觀測蛋白質摺疊的過程中做蛋白質摺疊狀態的變化，在這個研究中，我們設計MTGFP (金屬硫蛋白融合綠色螢光蛋白) 蛋白質分子內的螢光共振能量系統(FRET)，可用來偵測金屬結合在金屬硫蛋白結合上。利用過臨界點摺疊路徑進行蛋白質的摺疊，發現MT蛋白質在摺疊過程中同時有FRET效應的產生。這是個新的技術利用螢光能量共振轉移做蛋白質摺疊的即時偵測。金屬硫蛋白具有61個胺基酸內有20個硫基，並具有2個金屬鍵結環。利用波長395 nm 激發綠色螢光蛋白(GFP)，可在508 nm 放出螢光，提供螢光環做為FRET的系統。利用準靜態熱平衡透析法(TED)摺疊MTGFP蛋白質，利用動態光散射儀(DLS)、圓二色光譜儀(CD)去證明蛋白質結構摺疊回自然態.利用感應耦合電漿原子發射光譜法（ICP-AES)偵測到金屬離子已經摺疊到MTGFP蛋白質的內部，證明MTGFP具有結合金屬的能力。然而MTGFP蛋白質在摺疊過程中，508nm螢光波長的強度逐步降低。發現MT可以做為一個粹熄子(quencher)會將GFP的螢光衰減掉。換句話說，MT及GFP蛋白之間彼此有螢光共振能量轉移(FRET)現象也有粹熄(quenching)的現象。這是一個新的方式可以用來研究蛋白質摺疊的過程。

關鍵字

金屬硫蛋白 ； HASH(0xaad9a20) ； 綠色螢光蛋白

並列摘要

Protein folding status indicator is highly desired for revealing the folding process in real time. Therefore, ultra sensitive detection system in molecular scale is required. In this study, we designed an intramolecular fluorescence resonance energy transfer (FRET) system to monitor the folding status of metal binding protein, metallothionein (MT) by liking with green fluorescence protein (GFP). Both FRET and quench effect took place when the protein folding followed the over-critical reaction path. This new perspectives may provide significant strategy for investigation of protein folding process in real time.Metallothionein (MT) is a 61 amino-acids protein which contains 20 cysteine residues and forms two metal binding clusters (α-, β-domains). Green fluorescent protein (GFP), which emits green fluorescent light (λ max = 508 nm) by exciting with ultraviolet light (395 nm), can be used as a fluorophore in fluorescence resonance energy transfer (FRET) system. A fusion protein of MTGFP was cloned and refolded to native form by stepwise thermal equilibrium dialysis (TED). Both dynamic light scattering (DLS) and circular dichroism (CD) spectra indicate that the fusion protein has been refolded to its native state. The inductively couple plasma-mass (ICP-mass) spectrum also indicates that the refolded-MTGFP has restored its metal binding capability However, the fluorescence intensity of MTGFP at 508 nm declines during the refolding process. Therefore the MT seems acting as a quencher of GFP when it folds to native state. Namely, there is intramolecular fluorescence energy transfer taking place between MT and GFP. This new perspectives may provide significant strategy for investigation of protein folding process.

並列關鍵字

Metallothionein ； HASH(0xaad9f6c) ； Green Fluorescence Protein

參考文獻

159. 張家靖: 蛋白質摺疊與聚集. 物理雙月刊 2006, 28, 668-675.

32. 鄧昌蔚: 化學發光. 科學教育月刊 民91, 246期.

110. Campbell MK: The three-dimensional structure of proteins. In in Biochemistry. Edited by: Saunders College Publishing, Philadelphia.; 1995:86-188.

2. Edelmann J, Denton M: The uniqueness of biological self-organization: challenging the Darwinian paradigm. Biology and Philosophy 2007, 22, 579-601.

3. Bühlera RHO, Kägi JHR: Human hepatic metallothioneins. FEBS Letters 1974, 39, 229-234.