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標題: | 偵測快速摺疊動力學:藉由對光不穩定的包覆策略和雷射光解裝置來研究抗凍蛋白RD1的折疊動力學 Probing the Fast Folding Kinetics:Folding Studies of an Antifreeze Protein RD1 by Using Photolabile Caging Strategy and Laser Flash Photolysis |
作者: | Chun-Chieh Hsu 許淳傑 |
指導教授: | 陳佩燁 |
關鍵字: | 摺疊,RD1,包覆策略,聲波熱卡度計,動力學, folding,RD1,cage,photolabile,photoacoustic calorimetry,kinetics, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | 為了進一步了解蛋白質摺疊,所以我們必須要對於摺疊的過程進行系統性的研究。我們利用蛋白質包覆策略(caging-strategy),並結合時間解析的聲波熱卡度計(PAC,photoacoustic calorimetry)研究RD1蛋白質(分子量7 kDa)的摺疊過程──包含摺疊的動力學、焓的變化及體積的變化。在蛋白質包覆策略策略中,為了在RD1序列中創造適合結合的取代基,我們利用定點突變技術將第七號胺基酸(丙氨酸;Ala,alanine)突變為半胱氨酸(Cys,cysteine)(突變後蛋白質縮寫:RD1-A7C),並且利用易受光分解的分子(4-(bromomethyl)-6,7-imethoxycoumarin,BrDMC)與半胱氨酸殘基結合(RD1-A7C-DMC),而破壞蛋白質的摺疊。此外,蛋白質的結構則利用核磁共振(NMR,Nuclear Magnetic Resonance)及圓二色光譜(CD,circular dichroism)技術分析。聲波熱卡度計以一道紫外光雷射(約10-9秒)打斷RD1-A7C-DMC中蛋白質與DMC之間的鍵結,同時引發蛋白質摺疊。由聲波熱卡度計的實驗結果發現,RD1-A7C的摺疊具有兩個過程:第一步為快速的體積收縮(volume contraction),摺疊的時間為20奈秒,並伴隨著-9.7 mL / mol的體積變化;接著為結構上的重新排列(comformational rearrangement),摺疊的時間約為470奈秒,同時伴隨著-1.4 mL / mol的體積變化。 In order to understand the intrinsic principle of protein folding, events of the fold-ing process have to be systematically explored. In this work, the folding information of a small protein (RD1, about 7 kDa) including kinetic, enthalpy and volume change were reported by combining the photo-triggered caging-strategy and time-resolved photo-acoustic calorimetry. This strategy required the mutation with Ala-7 to Cys (designated RD1-A7C) that was introduced to incorporate a photolabile cage group, 4-(bromomethyl)-6,7-dimethoxycoumarin, to unfold the protein. The structural proper-ties of the caged protein were analyzed by nuclear magnetic resonance spectroscopy (NMR) and circular dichroism spectroscopy (CD). A pulse UV laser (~10-9 s) was used to break the photolabile cage and two events were observed in the refolding of RD1-A7C toward its native state by using photoacoustic calorimetry (PAC). The fast event, which has a folding time of 20 ns and a volume change of -9.7 mL/mol, was ex-plained as the result of rapid volume contraction. This event was followed by a con-formational rearrangement, which has a folding time ~ 470 ns and small volume change (-1.4 mL/mol). |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44261 |
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顯示於系所單位: | 生化科學研究所 |
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