Bcl-2 associated X protein(簡稱BAX蛋白)，隸屬於Bcl-2家族蛋白中的促凋亡蛋白，與細胞凋亡相關。BAX平時存在於在細胞質中，一旦接收到死亡訊息，包含與BH3-only蛋白上的BH3 domain作用等，BAX會受到活化而轉移到粒線體外膜上，接著BAX寡聚化後使粒線體外膜通透(MOMP)，最終造成細胞死亡。本研究選用與Bim蛋白的BH3 domain序列相同的26個胺基酸，稱作BimBH3胜肽，來探討BAX活化的這一連串過程，並結合time-resolved ESR(TR-ESR)及DEER技術進行量測。TR-ESR可以隨時間偵測BimBH3胜肽及BAX間作用的改變，藉由觀測自旋標記BAX的TR-ESR光譜可以觀測到BAX寡聚化的過程，而觀測自旋標記BimBH3胜肽的TR-ESR光譜可以研究BimBH3胜肽連接於BAX的過程。研究結果顯示，BimBH3平時以無規則的型態存在，與BAX反應後，BimBH3胜肽可以連接於BAX上的兩個活化位置，促使BAX結構改變並生成寡聚體，而此活化過程會遵循hit and run模型及induced-fit的過程。由DEER的結果顯示，當BimBH3胜肽連接於BAX上時，BimBH3胜肽的α螺旋比例有升高的現象，而隨著反應時間增長，BimBH3胜肽從BAX上離開，α螺旋的比例會逐漸降低。關於BimBH3胜肽連接以及離開BAX的訊息可以透過分析TR-ESR光譜而得到，用於提供hit and run模型的動力學參數。又由BAX突變體的結果顯示，BAX的兩個位置對於正常活化BAX的過程是必須的。綜合以上結果，可以得知BimBH3胜肽透過induced fit及hit and run的過程來活化BAX，使BAX經一連串構形改變後形成寡聚體，以利細胞凋亡的進行。

Bcl-2 associated X (BAX) protein is a pro-apoptotic Bcl-2 family member that functions as a critical gateway to mitochondrial apoptosis. BAX is a cytosolic protein in healthy cells. Upon receipt of apoptotic signaling, BAX is activated to form a mitochondria-associated complex, leading mitochondrial outer membrane permeabilization (MOMP) and then cell death. A diversity of stimuli have been implicated in initiating BAX activation, including direct involvement by select BH3 domains and BH3-only proteins. To explore the elusive initiating event for BAX activation, here we study the interaction of BAX with a 26-residue-long BH3-derived peptide, BimBH3, which we previously demonstrated to be effective to initiate the BAX oligomerization and MOMP, using ESR techniques including time-resolved (TR) ESR and double electron-electron resonance (DEER) techniques. Basically, TR-ESR is carried out to monitor the time-dependent interactions between BimBH3 and BAX protein. The process of BAX oligomerization is monitored through the spectral changes of spin-labeled BAX proteins, while the binding kinetics of BimBH3 is obtained by observing the changes in the spectral intensity of spin-labeled BimBH3 peptides over the incubation time. We show that the BAX activation to form oligomers can be induced by BimBH3 peptide through a hit-and-run mechanism, which is coupled with an induced-fit transformation concerning the BimBH3 secondary structure. BimBH3 is structurally disordered prior to the engagement with BAX. Upon incubation with inactive BAX monomers, BimBH3 binds to BAX at two different binding sites to initiate the conformational changes of BAX, which in turn promotes the formation of BAX oligomer. Our DEER results show that the helical propensity of the disordered BimBH3 is clearly increased upon binding to BAX, but is reduced after being released from the activated BAX due to the subsequent conformational changes of BAX that are necessary for the BimBH3-induced BAX oligomerization. The release and binding rates between the BimBH3 and BAX are quantitatively depicted by the spectra from TR-ESR measurements, providing kinetic insights into the hit-and-run process. Our results suggest that the engagements of BimBH3 to both of the binding sites on BAX are required for the oligomerization to proceed in a regular manner. Disruption by mutation in either site would largely reduce the rate of the oligomerization. Taken together, this study shows that BAX activation is a highly regulated, multi-step process involving an interaction-triggered conformational change from the BAX side, an induced-fit conformational change from the BimBH3 activator side, mitochondrial translocation, and oligomerization that ultimately leads to mitochondrial dysfunction and apoptosis.

謝誌………………………………………………………………………I
Abstract…………………………………………………………………..III
摘要…………………………………………………………………….....V
總目錄…………………………………………………………………....VI
圖目錄………………………………………………………………….... IX
表目錄…………………………………………………………………...XIV

第一章　緒論………………………………………………………………………...1
1.1細胞凋亡(Apoptosis)……………………………………………………………..1
1.2 Bcl-2(B-cell lymphoma)家族蛋白………………………………………………..1
1.3 Bim………………………………………………………………………………...3
1.4 Bcl-2-associated X protein(BAX)………………………………………………...4
1.4.1 BAX活化位置 ………………………………………………………………5
1.4.2 BAX蛋白之兩種構形……………………………………………………….6
1.5 研究目的與動機………………………………………………………………….8
第二章　儀器介紹與原理…………………………………………………………..10
2.1 電子自旋共振光譜儀…………………………………………………………...10
2.1.1 歷史發展……………………………………………………………………10
2.1.2 儀器裝置……………………………………………………………………10
2.1.3. 連續波型電子自旋共振光譜原理簡介(CW-ESR)……………………….12
2.1.3.1 賽曼效應(Zeeman interaction)………………………………………...12
2.1.3.2 超微細偶合作用(Hyperfine interaction)……………………………....14
2.1.3.3 g值及A值的各向異性(anisotropy)…………………………………15
2.1.3.4 定位自旋標記(Site-Directed Spin Labeling，SDSL)…………………16
2.1.3.5　ESR光譜線型及自旋標記物之轉動相關性時間 …………………..17
2.1.3.6 CW-ESR光譜於距離量測之應用……………………………………20
2.1.4. 雙重電子-電子光譜共振光譜簡介(Double Electron-Electron Resonance，DEER)……………………………………………………………………………..23
2.2 圓二色旋光光譜儀(Circular Dichroism，CD)…………………………………27
第三章 樣品製備與儀器測量方法…………………………………………………29
3.1實驗流程………………………………………………………………………....29
3.1.1 time-resolved ESR實驗流程………………………………………………..29
3.1.2 DEER實驗流程……………………………………………………………..30
3.2 樣品製備 ………………………………………………………………………..30
3.2.1 BAX蛋白純化 ……………………………………………………………..30
3.2.2 BAX寡聚體純化……………………………………………………………32
3.2.3 定位自旋標記樣品製備……………………………………………………33
3.3 儀器測量方法…………………………………………………………………...33
3.3.1 連續波型-電子自旋共振光譜(CW-ESR)量測…………………………….33
3.3.2 雙重電子-電子共振光譜(DEER)量測…………………………………….34
3.3.3 圓二色旋光光譜儀(Circular Dichroism,CD)量測………………………...35
3.4 藥品與儀器……………………………………………………………………...36
3.4.1 藥品…………………………………………………………………………36
3.4.2 儀器…………………………………………………………………………37
第四章 結果與討論…………………………………………………………………38
4.1 126/164C影響WT於UM及UM′間平衡之研究……………………………….38
4.1.1 WT-R1及126/164-R1與BimBH3胜肽之time-resolved ESR光譜………..39
4.1.2 WT-R1：126/164C= 1：1之time-resolved ESR光譜……………………...42
4.2 以time-resolved ESR研究BimBH3-R1於BAX活化過程之運動變化……….45
4.2.1 BimBH3胜肽之圓二色旋光光譜儀及ESR光譜…………………………..45
4.2.2 BimBH3-R1於time-resolved ESR系統中之穩定性測試…………………46
4.2.3 BimBH3-R1胜肽活化126/164C之time-resolved ESR光譜……………..48
4.2.4 126/164C與BimBH3-C之time-resolved FPLC……………………………58
4.2.5 BimBH3-R1胜肽與126/164C寡聚體之time-resolved ESR光譜……….60
4.2.6 BimBH3-R1胜肽與WT之time-resolved ESR光譜………………………63
4.2.7 BimBH3-R1胜肽與BAX K21E之time-resolved ESR光譜 ……………..66
4.2.8 BimBH3-R1胜肽與Bcl-2ΔC22之time-resolved ESR光譜 ……………..69
4.2.9 BAX與BimBH3-R1之反應速率常數……………………………………...70
4.3 以DEER量測於不同反應時間下BimBH3-2R1之距離……………………..74
4.3.1 BimBH3-2R1之CD光譜…………………………………………………...74
4.3.2 BimBH3-2R1與126/164C於不同反應時間下之DEER光譜……………75
4.3.3 BimBH3-2R1與WT於不同反應時間下之DEER光譜變化…………….79
　4.3.4 BimBH3活化BAX之模型………………………………………………….82
4.4 以DEER推測BimBH3-R1於BAX上的活化位置…………………………..83
第五章　結論…………………………………………………………………………90
參考文獻………………………………………………………………………………92

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