人類細胞中的肝癌衍生生長因子(hHDGF)，不論在細胞膜內外皆具有刺激細胞增殖的能力，細胞核中作為轉錄因子與DNA結合，細胞膜外扮演生長因子的角色與細胞膜上的受體鍵結。HDGF是經由位於序列N端的 PWWP/HATH domain鍵結上細胞表面的硫酸乙醯肝素(heparan sulfate)進而進入到細胞當中。PWWP/HATH domains 是HDGF相關蛋白質家族(HDGF-related proteins, HRPs)中，一段具有高度保留性的區域(序列相似度大於70%)，而這段區域皆包含特殊的PWWP motif。在HRPs序列中的PWWP motif，由第一個殘基的不同可區分為兩種類型，第一種序列為PHWP (Pro-His-Trp-Pro)，另一種為AHWP。為了解PWWP motif第一個殘基對蛋白質功能的影響，選用hHDGF為研究對象，將hHDGF PWWP motif第一個殘基Pro變異成Ala，將變異後的蛋白質簡稱為HATH P24A，進一步觀測變異蛋白與原始蛋白在穩定度與肝素(heparin)鍵結的改變。表面電漿共振(SPR)與恆溫滴定微卡計(ITC)皆顯示殘基對肝素鍵結能力(KD ≈ 1×10-6 M)影響不大，然而由核磁共振(NMR)氫氘交換實驗與圓二色光譜儀(CD)測得的耐受溫度(Tm)發現，殘基變異大幅降低 HATH domain的穩定度。此外，HATH P24A特徵也與先前觀察HRPs的AHWP類型相似，蛋白質溶液有嚴重的聚集(aggregation)現象產生，最後我們使用NMR得到蛋白質動力學(dynamic)參數，探討殘基變異對於蛋白質動態所造成的影響。總結，利用單點突變以及核磁共振的技術，本研究證明PWWP motif第一個殘基，對於HATH domain的穩定性扮演重要角色。

Hepatoma-derived growth factor (hHDGF) stimulates cell proliferation on both sides of plasma membrane by either binding to membrane receptor as a growth factor or binding to DNA in nucleus as a transcriptional factor. Secreted hHDGF recognizes cell surface heparan sulfate to promote its internalization and the N-terminal PWWP/HATH domain have been proved to be responsible for the heparan sulfate binding. The PWWP/HATH domains are highly conserved among the HDGF-related proteins (HRPs) with high identity > 70% and all domains contain the characteristic structural motif, PWWP motif. The PWWP motifs in HRPs can be classified into two types due to the difference of the first residue. One is with sequence of PHWP (Pro-His-Trp-Pro) and the other is AHWP. In order to realize the significance of the first residue of PWWP motif in mediating protein function and structure, we chose HDGF as models and examined the protein stability and heparin binding by replacing the Pro residue to Ala (mutation P24A). As shown by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), the binding affinity of heparin (KD ≈ 1×10-6 M) was less related to the residue. However, the substitution significantly reduced PWWP/HATH domain stability as reflected in NMR H/D exchange experiment and circular dichroism (CD) melting temperature measurement. In addition, we also detected NMR dynamic parameters on the local region to illustrate the dynamic differences derived from the mutation. Nevertheless, the phenomenon well corresponds to the severe aggregation tendency previously observed in AHWP-type HRPs. In summary, using site-direct mutagenesis and solution NMR method, we identified that the first residue of PWWP motif plays the key role in mediating PWWP domain stability.

目錄
英文摘要 I
中文摘要 III
縮寫(Abbreviations) V
第一章. 前言 1
1.1 HDGF的介紹 1
1.2 hHDGF HATH domain的功能 2
1.3 hHDGF HATH domains的結構 3
1.4 PWWP motif 4
1.5 hHDGF HATH domains殘基對肝素鍵結的貢獻 5
1.6 研究動機 6
第二章. 材料與方法 10
2.1 Cloning: hHDGF與HRP-4單點突變(Site-Directed Mutagenesis) 10
2.2 蛋白質表現與純化 11
2.3 圓二色光譜儀(Circular Dichroism, CD) 13
2.4 核磁共振(Nuclear magnetic resonance, NMR) 15
2.4.1 NMR樣品製備 15
2.4.2 HATH P24A backbone assignment 15
2.4.3 蛋白質二級結構預測 16
2.4.4 蛋白質化學位移擾動(chemical shift perturbation) 16
2.4.5 核磁共振的氫/氘交換實驗(NMR H/D exchange) 17
2.5 蛋白質動力學(dynamic)分析 19
2.5.1 核磁共振自旋弛豫理論(NMR spin relaxation) 19
2.5.2 Nuclear Overhauser Effect (NOE)43 20
2.5.3 無模型法則(Model-free formalism) 21
2.6 表面電漿共振(surface plasmon resonance, SPR) 24
2.6.1 SPR 樣品製備 25
2.6.2 肝素Biochip的製備 25
2.6.3 SPR實驗參數設定 25
2.6.4 Scatchard plot分析數據 26
2.6.5 競爭反應 27
2.7 恆溫滴定微卡計(Isothermal Titration Calorimetry, ITC) 28
2.7.1 ITC樣品製備 29
2.7.2實驗操作流程 29
第三章. 結果 34
3.1 CD圖譜比較原始與變異蛋白在二級結構上的差異 34
3.2 化學位移擾動 35
3.3 HATH P24A 二級結構分析 36
3.4 HATHs的氫氘交換實驗分析 36
3.5 HATHs的動力學分析 38
3.6 HATHs與肝素結合能力的分析 40
第四章. 討論 42
參考文獻 59

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