具有42個胺基酸的乙型類澱粉樣蛋白42(amyloid beta 42, Aβ42)係造成阿茲海默症的主要物質。近期研究(Angew. Chem. Int. Ed. 2021, 60, 4014–4017)指出Aβ42基原纖維與脂多糖(lipopolysaccharide, LPS)形成之共組裝過程可造成Aβ42降解，以阻止神經細胞凋亡，為有機會治療阿茲海默症之新方法，但對於此過程中Aβ42於分子層次之構形變化尚未被討論。此外，亦未有研究利用Aβ42自身酪胺酸作為螢光探針，以定量其成核構象轉換(nucleated conformational conversion)聚集時之構形改變能量變化。因此吾人以酪胺酸螢光溫度躍升系統觀測濃度約90 μM Aβ42於pH = 7.4下，於25°C培養不同時間之Aβ42由25°C躍升至35–51°C，及添加LPS後之Aβ42由25°C躍升至44°C之熱致構形改變動態過程。吾人以兩態模型(two-state model)分析上述動力學過程，並利用單指數函數擬合各熱致構形改變時間側寫D(t)，獲得其構形改變速率常數k，並以阿瑞尼斯法分析培養不同時間之Aβ42構形改變表觀活化能。當培養時間較長時，其表觀活化能較小，歸因於聚集程度較高的Aβ42之構形變化較為一致所導致。而添加LPS後之Aβ42構形改變速率則變慢，歸因為LPS阻礙其構形改變所造成。由上述結果推測Aβ42聚集過程與其調整構形之能力相關。當Aβ42間結構相似，容易調整其構形造成後續之聚集，也就造成較小的表觀活化能；而構形改變速率變慢則表示Aβ42較不容易調整其構形進行聚集現象。故與Aβ42相關疾病需於其尚未產生嚴重聚集之發病初期進行治療，且治療之藥物可選擇能使構形改變速率延緩的分子進行測試。

Among the various amyloid beta peptide isoforms, amyloid beta 42 (Aβ42), which is composed of 42 residues, is majorly responsible for the Alzheimer's disease (AD). The recent study (Angew. Chem. Int. Ed. 2021, 60, 4014–4017) has shown that the lipopolysaccharides (LPS) trigger the non-equilibrium co-assembly and clearance of Aβ42 to inhibit apoptosis. The aforementioned study could be regarded as a new method for AD therapeutics. However, the conformational changes of Aβ42 have not been studied at the molecular level. In addition, no investigations have been performed using the intrinsic fluorescence of the tyrosine residue in the Aβ42 as a fluorescent probe to quantify the energetics of conformational change during nucleated conformational conversion aggregation. In this work, a fluorescent temperature jump apparatus was employed to investigate the thermally-induced dynamic process of Aβ42 incubated at 25°C for different periods and mixed with LPS. The Aβ42 solution was prepared at a concentration of ca. 90 μM and pH was controlled at 7.4 for all of our experiments. The protein dynamics of Aβ42 were observed upon jumping the temperature from 25°C to 35–51°C for different incubation time at 25°C and to 44°C upon adding LPS. A two-state model was proposed to extract the kinetics of the dynamic structural changes of Aβ42 from the protein dynamic functions (D(t)) fitted by a single exponential function in terms of a rate constant k. The Arrhenius plot was utilized to extract the apparent activation energy of conformational changes about Aβ42 incubated at 25°C for different periods. The observed apparent activation energy was decreased as increasing the incubation time, attributed to the increased aggregation of Aβ42 which persisted the conformation for further aggregation. Besides, the dynamic process of Aβ42 was retarded in the presence of LPS because the attachment of LPS hampered the conformational change of Aβ42. From those results, the aggregation process of Aβ42 might be related to its capability of adjusting conformation. When the conformations of the constituent Aβ42 were similar, the conformation change required less energy, hence promoting the subsequent aggregation. The slower protein dynamic of Aβ42 probably indicates the less flexibility in adjusting the conformation for aggregation. Therefore, Aβ42-related diseases should been dosed at the initial stage of the disease before serious aggregation.

第一章 緒論 1
1.1 溫度躍升法 1
1.2 乙型類澱粉樣蛋白 2
1.2.1 Aβ42之聚集過程 3
1.2.2 Aβ42聚集與濃度之關係 3
1.2.3 Aβ42聚集與溫度之關係 4
1.2.4 與Aβ42形成共組裝應用於治療阿茲海默症之方法 4
1.3 酪胺酸之螢光性質 5
1.3.1 酪胺酸之螢光機制 6
1.3.2 酪胺酸螢光強度之溫度相依姓 6
1.3.3 蛋白質中酪胺酸之螢光性質 7
1.4 共軛焦成像技術 8
1.5 實驗動機及目的 8
參考文獻 26
第二章 實驗儀器原理 32
2.1 穩態紫外可見吸收光譜(steady-state UV/vis absorption spectroscopy) 32
2.2 遠紫外光圓二色光譜(Far-UV circular dichroism spectroscopy) 34
2.3 靜態螢光光譜(steady-state fluorescence spectroscopy) 36
參考文獻 47
第三章 樣品配製和實驗裝置與步驟 49
3.1 樣品製備流程 49
3.1.1 Aβ42於25°C培養第1天及第3天後用於熱致構形改變之實驗 49
3.1.2 觀測添加LPS之Aβ42熱致構形改變 49
3.2 樣品配製 50
3.2.1共軛焦螢光擷取系統偵測位置優化之樣品 50
3.2.1.1 10 mM pH = 7.4磷酸鹽緩衝溶液(phosphate buffer) 50
3.2.1.2 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之367.1 μM酪胺酸溶液 50
3.2.1.3 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之80.7 μM酪胺酸溶液 51
3.2.2比較Aβ42-1 day及Aβ42-3 day熱致構形改變之樣品 51
3.2.2.1 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之98.0 μM酪胺酸溶液 51
3.2.2.2 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之86.2 μM Aβ42溶液 51
3.2.3 觀測添加LPS之Aβ42熱致構形改變之樣品 51
3.2.3.1 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之113.2 μM酪胺酸溶液 52
3.2.3.2 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之159.9 μM Aβ42溶液 52
3.2.3.3 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之400 nM LPS溶液 52
3.2.3.4 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之95.9 μM Aβ42溶液 52
3.2.3.5 溶於10 mM pH = 7.4磷酸鹽緩衝溶液而含有160 nM LPS之95.9 μM Aβ42溶液 53
3.2.3.6 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之160 nM LPS溶液 53
3.3穩態紫外吸收光譜(steady-state ultraviolet absorption spectroscopy) 53
3.4 圓二色光譜(circular dichroism spectroscopy, CD) 54
3.4.1比較Aβ42-0 h、Aβ42-1 day和Aβ42-3 day之構形改變實驗 54
3.4.2 觀測添加LPS之Aβ42構形改變之實驗 54
3.5 靜態螢光光譜(steady-state fluorescence spectroscopy) 54
3.5.1 酪胺酸之變溫靜態螢光光譜偵測 55
3.5.2 各Aβ42樣品之定溫靜態螢光光譜偵測 55
3.6 溫度躍升螢光偵測系統之建立及優化 55
3.6.1 溫度躍升螢光光譜之偵測 57
3.6.2 利用酪胺酸作為螢光溫度計 57
3.6.3 共軛焦螢光擷取系統偵測位置之優化 59
參考文獻 82
第四章 實驗結果與討論 83
4.1 各樣品定溫吸收光譜及定量樣品濃度 83
4.2 比較Aβ42於25°C培養第1天及第3天之熱致構形改變 84
4.2.1 穩態光譜定性 84
4.2.1.1 定溫遠紫外光圓二色光譜 84
4.2.1.2 定溫靜態螢光光譜 85
4.2.2 Aβ42-1 day及Aβ42-3 day之溫度躍升實驗 85
4.2.2.1 Aβ42隨溫度演進之螢光強度變化 85
4.2.2.2 以兩態模型分析Aβ42之熱致構形改變過程 87
4.2.2.3 Aβ42-1 day及Aβ42-3 day於各躍升溫度下的動力學擬合及兩態模型中之表觀活化能 88
4.3 觀測添加LPS之Aβ42熱致構形改變之實驗 89
4.3.1 穩態光譜定性 89
4.3.1.1 定溫遠紫外光圓二色光譜 90
4.3.1.2 定溫靜態螢光光譜 90
4.3.2 觀測加入LPS之Aβ42其熱致構形改變的溫度躍升實驗 91
4.3.2.1 Aβ42隨溫度演進之螢光強度變化 91
4.3.2.2 以兩態模型分析Aβ42及包含LPS的Aβ42之熱致構形改變過程 92
4.3.2.3 Aβ42及含有LPS之Aβ42熱致構形改變過程的動力學擬合 92
參考文獻 114
第五章 結論 116
附錄A 117
A.1 以兩態模型描述Aβ42-1 day熱致構形改變過程之推導 117
A.2 以兩態模型描述Aβ42-3 day熱致構形改變過程之推導 118
A.3 以兩態模型描述Aβ42熱致構形改變過程之推導 120
A.4 以兩態模型描述包含LPS的Aβ42熱致構形改變過程推導 120
附錄B 123
B.1 Aβ42和含有LPS之Aβ42變溫靜態螢光光譜實驗 123
B.1.1 Aβ42和含有LPS之Aβ42變溫靜態螢光光譜的樣品製備流程 123
B.1.2 樣品配製 124
B.1.2.1 10 mM pH = 7.4磷酸鹽緩衝溶液(phosphate buffer) 124
B.1.2.2 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之156.0 μM Aβ42溶液 124
B.1.2.3 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之400 nM LPS溶液 125
B.1.2.4 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之93.6 μM Aβ42溶液 125
B.1.2.5 溶於10 mM pH = 7.4磷酸鹽緩衝溶液而含有160 nM LPS之93.6 μM Aβ42溶液 125
B.1.2.6 溶於10 mM pH = 7.4磷酸鹽緩衝溶液之160 nM LPS溶液 125
B.1.3 實驗裝置與步驟 125
B.1.3.1穩態紫外吸收光譜(steady-state ultraviolet absorption spectroscopy) 125
B.1.3.2 靜態螢光光譜(steady-state fluorescence spectroscopy) 126
B.1.4 實驗結果與討論 126
B.1.4.1 定溫吸收光譜及定量樣品濃度 126
B.1.4.2 Aβ42和含有LPS之Aβ42變溫靜態螢光光譜 127
B.2 Aβ42和含有LPS之Aβ42之溫度躍升實驗 128
B.2.1 Aβ42和含有LPS之Aβ42隨溫度演進之螢光強度變化 128
B.2.2 以兩態模型說明Aβ42及含有LPS之Aβ42的熱致構形改變過程 129
參考文獻 142
附錄C 143
C.1 以兩態模型描述Aβ42熱致構形改變過程之推導 143
C.2 以兩態模型描述含有LPS的Aβ42熱致構形改變過程推導 144

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第二章
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6. 王珮云，用溫度躍升後色胺酸螢光強度之變化區分蛋白質動態過程：牛血清白蛋白及人血清白蛋白之比較，2021，國立清華大學。
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第三章
1. Fu, Z.; Aucoin, D.; Davis, J.; Van Nostrand, W. E.; Smith, S. O. Mechanism of Nucleated Conformational Conversion of Aβ42. Biochemistry 2015, 54, 4197–4207.
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第四章
1. Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed., Springer: Baltimore, Maryland, USA, 2006, pp. 532.
2. Das, S.; Das, S.; Roy, A.; Pal, U.; Maiti, N. C. Orientation of Tyrosine Side Chain in Neurotoxic Aβ Differs in Two Different Secondary Structures of the Peptide. Royal Soc. Open Sci. 2016, 3, 160112.
3. Szabo, A. G.; Lynn, K. R.; Krajcarski, D. T.; Rayner, D. M. Tyrosinate Fluorescence Maxima at 345 nm in Proteins Lacking Tryptophan at pH 7. FEBS Lett. 1978, 94, 249–252.
4. Whitmore, L.; Wallace, B. A. Protein Secondary Structure Analyses from Circular Dichroism Spectroscopy: Methods and Reference Databases. Biopolymers 2008, 89, 392–400.
5. Goldfarb, A. R.; Saidel, L. J.; Mosovich, E. The Ultraviolet Absorption Spectra of Proteins. J. Biol. Chem. 1951, 193, 397–404.
6. Edelhoch, H. Spectroscopic Determination of Tryptophan and Tyrosine in Proteins. Biochemistry 1967, 6, 1948–1954.
7. Bhagavan, N. V. Medical Biochemistry. Three-Dimensional Structure of Proteins. Academic Press: San Diego, 2002, pp. 51–65.
8. Terol, P. A.; Kumita, J. R.; Hook, S. C.; Dobson, C. M.; Esbjörner, E. K. Solvent Exposure of Tyr10 as a Probe of Structural Differences between Monomeric and Aggregated Forms of the Amyloid-β Peptide. Biochem. Biophys. Res. Commun. 2015, 468, 696–701.
9. Fu, Z.; Aucoin, D.; Davis, J.; Van Nostrand, W. E.; Smith, S. O. Mechanism of Nucleated Conformational Conversion of Aβ42. Biochemistry 2015, 54, 4197–4207.
10. Vattepu, R.; Klausmeyer, R. A.; Ayella, A.; Yadav, R.; Dille, J. T.; Saiz, S. T.; Beck, M. R. Conserved Tryptophan Mutation Disrupts Structure and Function of Immunoglobulin Domain Revealing Unusual Tyrosine Fluorescence. Protein Sci. 2020, 29, 2062–2074.
11. Cornog, J. L., Jr.; Adams, W. R. The Fluorescence of Tyrosine in Alkaline Solution. Biochim. Biophys. Acta 1963, 66, 356–365.
12. Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed., Springer: Baltimore, Maryland, USA, 2006, pp. 530–531.
13. Yang, Y.; Arseni, D.; Zhang, W.; Huang, M.; Lövestam, S.; Schweighauser, M.; Kotecha, A.; Murzin, A. G.; Peak-Chew, S. Y.; Macdonald, J.; Lavenir, I.; Garringer, H. J.; Gelpi, E.; Newell, K. L.; Kovacs, G. G.; Vidal, R.; Ghetti, B.; Ryskeldi-Falcon, B.; Scheres, S. H. W.; Goedert, M. Cryo-EM Structures of Amyloid-β 42 Filaments from Human Brains. Science 2022, 375, 167–172.
14. Gremer, L.; Schölzel, D.; Schenk, C.; Reinartz, E.; Labahn, J.; Ravelli, R. B. G.; Tusche, M.; Lopez-Iglesias, C.; Hoyer, W.; Heise, H.; Willbold, D.; Schröder, G. F. Fibril Structure of Amyloid-β(1–42) by Cryo–Electron Microscopy. Science 2017, 358, 116–119.
15. Wu, T.-H.; Lai, R.-H.; Yao, C.-N.; Juang, J.-L.; Lin, S.-Y. Supramolecular Bait to Trigger Non-Equilibrium Co-Assembly and Clearance of Aβ42. Angew. Chem. Int. Ed. 2021, 60, 4014–4017.
16. Ibrahim, M.; Alaam, M.; El-Haes, H.; Jalbout, A. F.; Leon, A. D. Analysis of the Structure and Vibrational Spectra of Glucose and Fructose. Eclet. Quím. 2006, 31, 15–21.
附錄B
1. Fu, Z.; Aucoin, D.; Davis, J.; Van Nostrand, W. E.; Smith, S. O. Mechanism of Nucleated Conformational Conversion of Aβ42. Biochemistry 2015, 54, 4197–4207.
2. Das, S.; Das, S.; Roy, A.; Pal, U.; Maiti, N. C. Orientation of Tyrosine Side Chain in Neurotoxic Aβ Differs in Two Different Secondary Structures of the Peptide. Royal Soc. Open Sci. 2016, 3, 160112.
3. Szabo, A. G.; Lynn, K. R.; Krajcarski, D. T.; Rayner, D. M. Tyrosinate Fluorescence Maxima at 345 nm in Proteins Lacking Tryptophan at pH 7. FEBS Lett. 1978, 94, 249–252.
4. Whitmore, L.; Wallace, B. A. Protein Secondary Structure Analyses from Circular Dichroism Spectroscopy: Methods and Reference Databases. Biopolymers 2008, 89, 392–400.
5. Goldfarb, A. R.; Saidel, L. J.; Mosovich, E. The Ultraviolet Absorption Spectra of Proteins. J. Biol. Chem. 1951, 193, 397–404.