果蠅三磷酸鳥苷蛋白，Beck，為表現在果蠅的腦部中蕈狀體的蛋白質，分子量22千道耳吞(22kDa)，胺基酸數量為185個。Beck是屬於 Rab (在腦部中發現與Ras相似蛋白)家族的蛋白質，且與之Rab同源蛋白有36%~78%的相似度，Rab 家族蛋白在生物體中專司於調節細胞的囊泡的形成，以及胞器間蛋白質的運輸。而Rab 家族也被報導發現與神經退化性疾病有關，例如: RAB7L1被報導與帕金森氏症相關缺陷疾病有關。本論文中，我們提出Beck 會因為溫度誘導使其由α- helice變成β-strand，並進一步形成類澱粉聚集。此現象我們經由會與amyloid專一性結合的染劑：硫代黄素-T及剛果紅和利用與蔗糖高速離心的方式，最後再用穿透式電子顯微鏡來確認Beck會形成類澱粉聚集。另外，Beck的非結合受質構型會形成較大的寡聚物，而結合受質構型為單體而穩定。本論文藉由研究Beck 蛋白質形成類澱粉聚集來探討該蛋白可能與神經退化性疾病有關。

Drosophila melanogaster GTPase protein, Beck, was a 22kDa protein with 185 amino acids which was found in the mushroom of body brain. The Beck protein belongs to Rab family (Ras like protein in brain) and haves approximately 35%~78% sequence identities with other Rab family members. The Rab family proteins were reported to participate in cellular processes, such as facilitating vesicle formation and protein vesicle trafficking. Rab family protein were associated with neurodegenerative disease, for example, Parkinson’s disease-associated defects in Rab family protein, RAB7L1. In this study, we observed that Beck went through α-helical conformational to β-strand induced by thermal process and further form the amyloid-like β-aggregate which bind to specific dye, thioflavin-T and Congo red. The amyloid-like fibrils were centrifuged with sucrose and studied by using transmission electron microscopy. In addition, the free form of Beck shows protein aggregation and assembly into higher order, while GTP-bound form of Beck enhanced the correct folding and stability of monomer. Our studies of the Beck protein conformational change and formation of amyloid-like β-aggregate formation may contribute to the understanding of neurodegenerative diseases in Rab family proteins.

Abbreviations IV
Abstract 1
中文摘要 2
謝誌 3
Chapter 1. Introduction 4
1.1 GTPase family 4
1.2 Ras superfamily 4
1.3 Rab family 5
1.4 Introduction of Beck 6
1.5 Septin family 6
1.6 Amyloid and amorphous aggregates formation related disease 7
1.7 The object of this thesis 8
Figure 1.1 The cycle of GTPase active and inactive form 9
Chapter 2.Materials and Methods 10
2.1 Construction of Beck 10
2.2 Agarose electrophoresis 10
2.3 Expression and Purification of Beck 10
2.4 Tricine SDS-PAGE 12
2.5 Mass analysis (MALDI-TOF) 12
2.6 Quantification of protein concentration 13
2.7 Analytical Ultracentrifugation (AUC) 13
2.8 Circular Dichrosim (CD) 13
2.8.1 Thermal induce conformational change 14
2.8.2 Concentration induce conformational change 14
2.9 GTPase activity assay 15
2.10 Thioflavin T (ThT) spectroscopic assay 15
2.11 Congo red assay 16
2.12 Sedimentation assay 16
2.13 Transmission electron microscopy (TEM) 17
2.14 Modeling and docking of Beck 17
2.15 Table and Figure 19
Table 2.1 Primer sequence 19
Table 2.2 PCR mixture solution for truncated Beck cDNA amplification 19
Table 2.3 The PCR program for truncated Beck cDNA amplification 20
Table 2.4 The program of gel filtration 20
Table 2.5 Preparation of GTPase activity assay 21
Figure 2.1 pET21b-Beck vector map 21
Figure 2.2 ThT. 22
Figure 2.3 Congo red 22
Figure 2.4 GTP 23
Chapter 3. Result and Discussion 24
3.1 Multiple sequence alignment and truncated Beck 24
3.2 Construction of Beck 24
3.3 Expression test of Beck protein 25
3.4 Purification of Beck protein and resolve of oligomer in the absence of GTP 25
3.5 Comparison of free and bound form enzyme activity 26
3.6 Secondary structure of Beck 27
3.6.1 Thermal induce conformational change 27
3.6.2 Concentration induce conformational change 27
3.6.3 Concentration induce Beck form oligomer 28
3.7 Identification of amyloid-like β-aggregate 28
3.8 Modeling and docking of Beck structure 30
3.9 Table and Figure 32
Table1. Beck information 32
Figure 3.1 Multiple Sequence Alignment of Beck homologous protein 34
Figure 3.2 cloning of the Beck expression plasmids 35
Figure 3.3 Expression test of Beck protein 36
Figure 3.4 Purification of Beck with Ni2+ column 37
Figure 3.5 MALDI-TOF mass spectra of Beck 38
Figure 3.6 Purification of Beck by gel filtration. 39
Figure 3.7 Analytical Ultracentrifugation (AUC) of Beck protein 40
Figure 3.8 Standard curve for quantifying phosphate ion concentration 41
Figure 3.9 Free and bound form enzyme activity comparison 42
Figure 3.10 Far-UV CD spectra of Beck 43
Figure 3.11 Thermal conformational change of Beck. 44
Figure 3.12 Far-UV CD spectra of Beck at various temperatures 45
Figure 3.13 Far-UV CD spectra of Beck at different protein concentration. 46
Figure 3.14 Far-UV CD spectra of Beck in concentration dependent 47
Figure 3.15 Beck in different concentration were analyzed by native gel and cross-link test in SDS-PAGE. 48
Figure 3.16 Thioflavin T (ThT) spectroscopic assay at various temperature 49
Figure 3.17 Congo red UV- spectroscopic assay at various temperature 50
Figure 3.18 Sedimentation assay 51
Figure 3.19 Transmission electron microscopy 52
Figure 3.20 Modeling structure of Beck 53
Figure 3.21 Molecular docking of Beck structure 54
Figure 3.22 Various interactions between GTP and Beck, as detected by Ligplot, are shown schematically. 55
Chapter 4. Conclusion 56
Appendix. 1 Principle of GTPase activity assay 58
Appendix. 2 Septin-4 observed by transmission electron microscopy 59
Reference 60

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