以螢光共振能量轉移揭示蛋白質PGB1分子內摺疊機制

The Folding Mechanism of Protein G B1 Domain Revealed by Fluorescence Resonance Energy Transfer

鍾偉賢

指導教授 ： 張家靖

國立交通大學/生物科技學院/生物科技學系暨研究所/碩士(2011年)

https://doi.org/10.6842/NCTU.2011.00946

摘要

蛋白質如何摺疊成特殊的結構，為目前學界不斷探討的重點。為了要能夠理解蛋白質摺疊機制，研究單功能區蛋白質（single-domain protein）變成了一個很重要的指標。本研究中，我們以Protein G B1 Domain（PGB1）作為研究之標的蛋白質，利用本實驗室所開發之蛋白質摺疊方法，Over-critical Folding Process，將PGB1由未摺疊態摺疊至自然態並觀察摺疊中間的結構變化。PGB1是一個小型蛋白質只有56個胺基酸，是Protein G的IgG-binding domain。藉由觀察PGB1自身的Trp43螢光光譜和acrylamide焠熄光譜，可發現PGB1之摺疊是一個二狀態反應（two-state reaction）的摺疊過程々而以動態光散射（dynamic light scattering）觀察分子大小的變化認為PGB1摺疊過程應具有另一個中間態存在。藉由螢光共振能量轉移（Fluorescence Resonance Energy Transfer，FRET）分析各主要二級結構之間在摺疊過程中的變化，發現PGB1的β-hairpin 2與α-helix在摺疊初期就已互相靠近，但其β-hairpin 1顯然較前者晚形成々綜合以上結果顯示蛋白質PGB1的摺疊是一個多狀態反應（multi-state reaction）。本研究藉由FRET方法為PGB1摺疊研究開啟新的一頁。

關鍵字

蛋白質摺疊 ； 螢光共振能量轉移 ； 螢光焠滅

並列摘要

―How does a protein fold?‖ This has been questioned for long period. Studying on a single-domain protein can be a remark to understand the protein folding mechanism in general. Recently, we folded the Protein G B1 Domain (PGB1) by an over-critical folding process, which was developed by our lab, and studied the conformational changes in each folding state. PGB1 is a small protein with 56 residues, and is an IgG-binding domain of protein G. Because PGB1 contains the basic folding elements in a short sequence of amino acid without disulfide bonds, making it an excellent model for protein folding studies. The intrinsic Trp43 fluorescence of PGB1 and the acrylamide-quenching fluorescence showed that the PGB1 folding is a two-state reaction. However, the molecular diameter changes during the folding process indicated a folding intermediate exists in the PGB1 folding process. The FRET experiment, with Trp/IAEDANS as fluorophore pairs, showed that the β-hairpin 2 attaches to the α-helix long before the β-hairpin 1 formation. All the results together suggest the PGB1 folding process is a multi-state reaction. Overall, we were successful to reveal the folding process of PGB1 by FRET analysis.

並列關鍵字

Protein G B1 Domain ； Protein Folding ； FRET ； Fluorescence Quenching

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