果蠅脂肪結合蛋白結構和脂肪結合蛋白家族具高度相似性，主要由十個β-平板和兩個α-螺旋組成，其涉及調控睡眠以及長期記憶相關的基因表現。在實驗室先前的研究中，已知其對長鏈不飽和脂肪酸有專一性，並驗證Tyr127及Arg125為脂肪酸重要結合位置之一。從蛋白結構的表面電性可以觀察到，α-螺旋上有正電荷群聚的現象，而此蓋狀區域被認為與調控脂肪酸的出入有關。因此，我們將此區域的鹼性胺基酸個別點突變使之不帶電性 (K21A、R30A及K31A)，探討其結構與功能上的變化。從旋光儀實驗結果中可得知，突變後仍維持與原生型相同的二級結構，但在蛋白熱降解實驗中，卻觀察到Tm大幅增加至20℃的情形。顯示消除α-螺旋上任一正電排斥力，皆可使熱穩定性變好。等溫滴定量熱儀 (ITC) 偵測脂質結合能力，突變對油酸 (OA) 的結合能力與原生型相似，但位於α-Ⅱ上的R30A及K31A對二十二碳六烯酸 (DHA) 結合能力卻較原生型差。由此推知，突變雖增加蛋白的熱穩定性，使結構變的更緊密，但相對的，可能因此降低了蛋白的彈性，進而影響脂肪酸的進入，尤其是位於入口上方的α-Ⅱ突變-R30A及K31A。綜合以上，原生型α-螺旋區域上，這些不穩定的正電聚集現象，是提供彈性的主要因子，調控脂肪結合蛋白的脂肪酸結合入口區域。搭配模型與晶體結構上的觀察與測量，可以和此現象相互佐證。此外，從熱穩定和限制酵素剪切實驗，我們也發現脂質結合可以增強原生型的結構穩定性。而從螺旋型蛋白結構區域間作用力上，我們還發現D17其負電荷與R30、K31及N34皆有氫键交互作用力，除了可降低的排斥力外，其也可能作為鞏固兩螺旋型蛋白結構之間的橋樑，此部分值得更進一步的探討。

Drosophila melanogaster Fatty acid-binding protein (dFABP) comprises ten antiparallel β-strands and two α-helixes which is the typical structural feature of FABP family. dFABP was found to play an important role in long-term memory formation recently. In our previous studies, we have identified the critical residue-Y127 and R125 of binding sites in the β-barrel region. The helical region of FABP is referred to as the ligand entering site. Basic residues in this region are considered to be involved in membrane interaction and also act as nuclear location signal. In order to investigate the role of these basic amino acids, we replaced residues K21A, R30A and K31A with alanine using site-directed mutagenesis. The secondary structures of mutants are similar to wild-type dFABP. However, the melting temperature measured by circular dichorism shows a significant increase for all of three mutants. The enhancement of thermal stability may due to the elimination of repulsive force of positive charges on the helical region. Furthermore, isothermal titration calorimetry was employed to examine the binding ability toward fatty acids. Although the binding constant for oleic acid (OA) are similar, R30A and K31A, both on α-II helix, shows a ten-fold decrease in binding affinity to docosahexaenoic acid (DHA) compared to the wild-type dFABP. Our results indicate that basic residues in α-II region play the important role in modulating entrance for ligand binding. The binding affinity of dFABP is carefully balanced between structural stability and flexibility.

中文摘要 I
Abstract II
Acknowledgement III
Contents IV
List of Figures VII
Abbreviations 1
Keywords 1
Chapter 1. Introduction 2
1.1 Classification of fatty acid binding protein family 2
1.2 Structure-related function of FABP family 3
1.3 Function and structure analysis of Drosophila FABP 5
1.4 Motivation 6
Chapter 2. Materials and Methods 7
2.1 Protein construction, recombinant and mutagenesis of dFABP 7
2.2 Protein expression and purification 7
2.2.1 Protein expression 7
2.2.2 Protein purification 8
2.3 Identification of dFABP purity 9
2.3.1 Tricine SDS-PAGE 9
2.3.2 MALDI-TOF MS analysis 10
2.4 Quantification of protein concentration 10
2.5 Nuclear magnetic resonance spectroscopy 10
2.6 Circular dichroism spectroscopy 11
2.7 Isothermal titration calorimetry 12
2.8 Limited proteolysis assay 13
2.9 Molecular modeling and entrance calculation 13
Chapter 3. Results and Discussions 15
3.1 Improvement of purification procedure 15
3.2 Biophysical properties of holo-form dFABP 16
3.2.1 Isothermal titration calorimetry binding assay of wild-type dFABP 16
3.2.2 Thermostability of holo-form dFABP 17
3.2.3 Limited proteolysis resistance 17
3.3 Mutating basic residues in helical region 18
3.3.1 Analysis helical region of dFABP by molecular modeling 18
3.3.2 Construction of mutant dFABP – K21A, R30A, K31A 19
3.3.3 Secondary structure comparison of mutants with wild-type dFABP 19
3.3.4 Thermostability of apo-form mutants 20
3.3.5 Thermostability of holo-form mutants 21
3.3.6 Isothermal titration calorimetry binding assay of mutants 21
3.4 Crystal structure of dFABP analysis 22
3.4.1 Comparison of x-ray structure with model structure 22
3.4.2 B-factor of dFABP x-ray structure 22
3.4.3 Local interaction forces in helical region 22
3.5 Entrance hypothesis 23
3.6 Entrance measurement 23
Chapter 4. Conclusion 25
Figures 26
Tables 58
Table 1: Human fatty acid binding protein genes 58
Table 2: Holo-form crystal structures of FABP 59
Table 3: Binding constants and Gibbs free energy changes of fatty acids binding to dFABP and mutants 60
Table 4: Melting temperature of dFABP and Mutants 61
Reference 62
Appendixes 68
Appendix 1: The PCR materials and program for site-directed mutagenesis 68
Appendix 2: The programs for purification by using AKTA Prime 69

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