探討螺旋區域上鹼性殘基對果蠅脂肪酸結合蛋白熱穩定性及脂肪酸結合之影響

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研究生：	黃韻芳 Huang, Yun-Fang
論文名稱：	探討螺旋區域上鹼性殘基對果蠅脂肪酸結合蛋白熱穩定性及脂肪酸結合之影響 Basic residues in helical region affect protein stability and ligand binding in Drosophila Fatty Acid Binding Protein
指導教授：	呂平江 Lyu, Ping-Chiang
口試委員:	徐尚德 Shang-Te Danny Hsu 楊立威 Lee-Wei Yang
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2014
畢業學年度：	102
語文別：	中文
論文頁數：	69
中文關鍵詞：	果蠅蛋白、點突變、熱穩定性、蛋白二級結構、序列比對、螺旋區域、正電荷排斥、配體入口
外文關鍵詞：	Drosophila Proteins, Mutagenesis, Site-Directed, Thermostability, Protein Secondary Structure, Sequence Alignment, Helical region, Positive charge repulsion, Ligand entrance
相關次數：	點閱：2 下載：0
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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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