Bid蛋白在傳遞細胞凋亡的過程中扮演重要的角色，當細胞接收到凋亡訊息後，Bid蛋白會被Caspase 8蛋白酶切斷成cBid，並轉移至粒線體外膜上與其他Bcl-2家族蛋白作用。近年來，大多數研究著重於解析Bid蛋白在膜上的結構以及了解Bid如何藉由與Bcl-2家族蛋白作用來調控細胞凋亡的機制，然而其結構穩定性與其生物活性之關聯性卻未曾被深入探討。本研究利用圓二色光譜(CD)以及溫度解析連續波型電子自旋共振光譜(cw-ESR)探討Bid蛋白整體與局部結構之熱穩定性，進而了解Bid結構穩定性是如何影響其生物功能。利用圓二色光譜技術我們發現Bid蛋白在高溫(350 K以上)仍保有部分二級結構，並會產生分子間聚集。此外，我們利用GdnHCl降低其結構的熱穩定性，在3 M GdnHCl溶液中，圓二色光譜的結果顯示Bid蛋白的結構約於320 K即開始瓦解。於此研究中，我們利用ESR光譜技術，解析Bid蛋白局部的結構與其生物功能的關聯性。我們首先確認在3 M GdnHCl的環境中，於300 K時的Bid蛋白能保持其原先構形。隨後我們在Bid蛋白不同的螺旋區域，挑出30個位點並搜集其300 K至345 K的cw-ESR光譜，藉此勾勒出Bid蛋白局部結構的熱穩定性。實驗結果顯示Bid蛋白在加熱後會從螺旋3、6和8的交界面開始攤開，此交界面正是包覆BH3 domain的交界面，表示BH3 domain會在升溫的過程中最先從結構中暴露。本研究也發現，Bid蛋白在加熱至95 ℃(368 K)後，依然能正常傳遞細胞凋亡訊息，顯示即便Bid蛋白的結構已受到高溫破壞， Bid蛋白仍保持其生物功能。

BH3-interacting-domain death agonist (Bid) protein plays a key role in the induction of mitochondria-mediated apoptosis. Upon receipt of apoptotic stimuli, Bid interacts with apoptotic Bcl-2 family proteins and consequently triggers the activation of the apoptotic proteins, leading to the cell death. The structure of Bid was determined more than a decade ago, but its structure-function relation remains largely unexplored. In this study, we investigated the thermal stability of Bid protein and explored how the death-promoting function of Bid is affected by thermally-induced unfolding. First, we show by circular dichroism (CD) spectroscopy that Bid remains partially unfolded at high temperature (350-368 K). The thermal denaturation of Bid is irreversible and accompanied with intermolecular associations leading to protein aggregation. No clear melting temperature Tm is observed. We demonstrated by cw-ESR that, in 3 M GdnHCl the structure of Bid is largely preserved, but the protein stability is affected such that a clear melting temperature of Bid is determined. More than 30 single-labeled Bid mutants are studied using ESR in the temperature range of 300−345 K. The ESR results provide site-specific information about temperature dependence of local structure in Bid, thus enabling the discrimination between the onsets of unfolding and aggregation for respective sites. We map out the local thermal stability over Bid structure and reveal that the interface of helices 3, 6 and 8 is the beginning of structural unfolding. We also investigated the apoptotic activity of the thermally-induced Bid aggregates and showed that Bid retains the death-promoting function even when unfolded and aggregated.

第一章 緒論 6
1.1 細胞凋亡 6
1.2 Bcl-2家族蛋白 6
1.3 BH3-interacting domain death agonist (Bid) 8
1.4 研究動機與目的 9
第二章 儀器介紹與原理 11
2.1 電子自旋共振光譜儀 11
2.1.1儀器裝置 11
2.1.2 連續波型電子自旋共振光譜 13
2.1.3 定位自旋標記 14
2.1.4 ESR光譜線形及標記物之運動相關性 14
2.2 圓二色光譜儀 16
2.3 蛋白質沈澱與濁度 17
2.4 Thermofluor assay 18
第三章 樣品製備與儀器測量方法 20
3.1 Bid蛋白樣品製備 20
3.1.1 Bid蛋白的定點突變 20
3.1.2 Bid蛋白的表現 20
3.1.3 Bid蛋白的純化 21
3.2 ESR的樣品製備、量測和分析 22
3.2.1 ESR的樣品製備 22
3.2.2 cw-ESR的量測 23
3.2.3 ESR的數據分析 24
3.3 圓二色光譜量測 25
3.4 Thermofluor assay 25
3.5 濁度量測 26
3.6 Cytochrome c release assay 26
第四章 結果與討論 28
4.1 Bid蛋白具有高度的熱穩定性 28
4.2 利用3M GdnHCl降低Bid蛋白的熱穩定性 30
4.3 加入3M GdnHCl並不會明顯影響Bid蛋白的整體構形 32
4.4 利用SDSL-ESR對Bid蛋白進一步做位點局部區域的解析 36
4.5 分析不同區域Bid蛋白的升溫cw-ESR光譜線形 43
4.5.1 α1與α2的熱穩定性探討 43
4.5.2 Loop與α3的局部熱穩定性較低 45
4.5.3 α4與α5具有較高的熱穩定性 49
4.5.4 α6尾端與α7的具有較高的局部熱穩定性 52
4.5.5 α8的局部熱穩定性較低 56
4.6 Bid加熱過程中BH3 domain會先從結構中暴露 58
4.7 加熱後的Bid蛋白仍具有傳遞細胞凋亡訊息的功能 60
4.8 Bid和Bax蛋白因兩者功能的差異造成兩者局部結構上熱穩定性的不同 62
第五章 結論 64
第六章 參考文獻 65

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