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研究生:許家豪
研究生(外文):Jia-Hau Shiu
論文名稱:含RGD序列蛋白在辨識組合蛋白上的結構與功能關係之研究
論文名稱(外文):Structure-Function Relationships of RGD-containing proteins in Integrin Recognition
指導教授:莊偉哲
指導教授(外文):Woi-Jer Chuang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:94
中文關鍵詞:去組合蛋白馬來蝮蛇蛇毒蛋白gamma-雨傘節蛇毒蛋白核磁共振光譜蛋白質結構蛋白質動力學
外文關鍵詞:disintegrinrhodostomingamma-bungarotoxinNMRprotein structureprotein dynamics
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組合蛋白(integrin)是一群位於細胞表面的膜蛋白,調控細胞外的間質與細胞間的交互作用。而這種作用控制細胞的生長、分化與遷移,也牽涉到許多疾病的發生。因此,組合蛋白被視為有潛力發展藥物設計的目標物。而各種不同的組合蛋白有其生理功能且會辨識特殊的結合配體(ligand),結合後產生生理反應,過去的研究中發現有較多數的組合蛋白會與含RGD(Arg-Gly-Asp)序列的蛋白結合,主要包含的組合蛋白有integrin <FONT FACE="symbol">a</font>IIbb3、avb3和a5b1,近年的研究也發現由蛇毒中發現的去組合蛋白在RGD周圍不同的序列下會針對不同的組合蛋白有選擇性,可是目前不知道這些序列如何影響RGD對組合蛋白的辨識能力。因此,為了瞭解RGD motif 如何受到不同序列影響,而改變其辨識組合蛋白的能力,我們利用NMR研究去組合蛋白rhodostomin含不同序列的突變蛋白與一個含RGD序列具有較弱抑制血小板凝集能力的神經性蛇毒g-bungarotoxin之結構與動力性質。目前,實驗室利用細胞粘合測試研究去組合蛋白rhodostomin對不同RGD序列辨識組合蛋白能力的結果發現,有三個突變株對組合蛋白有不同辨識能力,分別是A48R49G50D51W52N53、AKGDWN和ARGDDL:ARGDWN對integrin aIIbb3和avb3有較好的親和性﹔當R突變成K(AKGDWN)時則對avb3親和性下降達約750倍,進而特異性的選擇integrin aIIbb3﹔ARGDDL中則特異性的選擇integrin avb3。因此,我們利用NMR來分析這三個去結合蛋白3D立體結構上的不同,而說明功能上的差異。目前由結構上發現在ARGDWN與AKGDWN中並沒有太大的差異,僅側鏈官能基的不同,而含W52的蛋白與野生株(PRGDM52P)比較,發現W52會與C端H68有作用,拉近C端與RGD motif的距離,過去研究顯示含W52的去組合蛋白與integrin b1 親和性較差,因此我們認為W52會去調控C端而辨識不同integrin b次單元。ARGDDL則發現其RGD motif與A(R/K)GDWN和野生株(PRGDMP)在D51位向上有不同的方向,或許這便是造成在ARGDDL功能上的不同特異性。我們利用NMR決定出的g-bungarotoxin 3D結構中,包含兩股與三股的b-摺版及三個環圈,而RGD序列則位於第二的環圈中形成一個reverse turn的結構,這與我們在去組合蛋白上RGD的區域結構類似,然而測量其平均R33至G36之間的Ca原子距離為6.02 Å,比起其他去組合蛋白相同位置的距離約在6.52~7.46 Å來得較短。同時,我們也利用在NMR下可觀察蛋白質動力學的特性,計算NMR描述蛋白質動力學的參數來說明蛋白質在水溶液中運動的情況,結果發現RGD motif與N端、C端是去結合蛋白中最具有彈性的區域,進一步計算其內部動力學參數(S2),RGD motif具有低於0.5的S2數值。由以上結果證實RGD周圍的序列會影響RGD的結構與動力性質﹔而RGD motif的動力性質對其辨識組合蛋白與親和力大小扮演重要的角色。未來,利用以上結果可針對不同組合蛋白進行藥物設計之研究。
Integrins are member of the large family of cell surface receptors that mediate both cell-matrix and cell-cell interactions. These interactions control the adhesion and migration of cells, as well as the transduction of signals that regulate cell growth and differentiation. It is known that integrins play important roles in the initiation and progression of many common diseases. Therefore, antagonism of integrins provides an approach for the treatment and prevention of these diseases. The RGD sequence is the recognition site for at least nine integrins, including integrin aIIbb3, avb3 and a5b1. Recent studies have showed that the amino acid residues flanking the RGD motif of snake venom disintegrins control their specificities. However, little is known about structural information how the amino acid residues franking the RGD motif of disintegrins control their interactions to integrins. Therefore, we study the structural and dynamical properties of g-bungarotoxin, an RGD-containing neurotoxin, and the rhodostomin mutants by NMR to explore the structure and function relationship of RGD-containing proteins. Based on the results of platelet aggregation and cell adhesion assays, we identified three mutants (A48R49G50D51W52N53、AKGDWN、and ARGDDL) containing different amino-acid residues flanking the RGD motif that have different selectivity to integrins. The ARGDWN mutant exhibits high affinity to both integrins aIIbb3 and avb3. In contrast, the mutation of the R residue to K decreases its affinity to integrin avb3. The ARGDDL mutant selectively inhibited integrin avb3. Based on our NMR studies, the conformations of the RGD loops of the ARGDWN and AKGDWN mutants are similar, and the only difference is in the functional groups of R and K. Interestingly, the residues W52 could bring the C-terminus closer to the RGD motif that may abolish its binding to integrin b1. The orientation of the D51 residue at the RGD motif of ARGDDL mutant, an integrin avb3-specific disintegrin, is opposite to that of rhodostomin and other mutants. In addition, the solution structure of g-bungarotoxin has been determined by NMR. It is similar to that of the short chain neurotoxins that contains three loops extending from a disulfide-bridged core. The RGD sequence is located at the apex of the flexible loop II that is similar to that of the RGD-containing proteins. The average Ca to Ca distance for R33 to G36 of g-bungarotoxin is 6.02 Å that is shorter than that of the RGD-containing proteins ranging from 6.52 to 7.46 Å. We also used 15N nuclear relaxation to probe the dynamics of the protein backbone amide moieties of rhodostomin and its mutants on ps-ns and ms-ms timescales. In our preliminary studies, we showed that the RGD motif is as flexible as the N-terminal and C-terminal regions of rhodostomin that have order parameters less than 0.5. These results demonstrate that the residues flanking the RGD motif of RGD-containing proteins regulate its function-structure relationships. The dynamical properties of the RGD loop may play an important role in recognizing and changing the affinity of disintegrins to different integrins. These studies on structure and function relationships of RGD-containing proteins could shed some light on how RGD-containing proteins interact with integrins. Based on their structural and dynamic properties, we will design the potent integrin-specific inhibitors.
中文摘要 I
英文摘要 III
致謝 V
目錄 VI
圖目錄 VIII
表目錄 XI
縮寫檢索表 XII
儀器 XIII
第一章 緒論
1-1 結合蛋白 (Integrin) 之介紹 1
1-2 含RGD序列蛋白之介紹 3
1-3 馬來蝮蛇去結合蛋白 (Rhodostomin) 之介紹 5
1-4 雨傘節神經性蛇毒(g-Bungarotoxin)之介紹 6
1-5核磁共振(NMR)決定蛋白質三維結構與動力學之介紹 6
1-6 論文研究動機與內容之簡介 8
第二章 材料與方法
2-1含RGD序列蛋白之純化
2-1-1 g-Bungarotoxin蛇毒蛋白的取得 10
2-1-2 Rhodostomin突變蛋白的誘發 10
2-2 NMR蛋白質樣品的製備 18
2-3 以核磁共振(NMR)研究含RGD序列蛋白之三維結構
2-3-1原理 19
2-3-2 NMR光譜的判定 19
2-3-3限制條件的找尋 21
2-3-4 計算蛋白分子的三度空間結構 23
2-4 以NMR研究rhodostomin與其突變蛋白之骨架動態行為
2-4-1 15N弛緩速率的測量 27
2-4-2 Model Free 計算 28
2-4-3利用tensor2計算蛋白質骨架Model free 動力參數 29
第三章 實驗結果
3-1含RGD序列蛋白之NMR圖譜分析
3-1-1 NMR圖譜之循序判斷 31
3-1-2 限制條件的獲得 33
3-2 含RGD序列蛋白之三維結構計算
3-2-1 g-Bungarotoxin蛇毒蛋白 35
3-2-2 Rhodostomin突變蛋白(ARGDWN;AKGDWN;ARGDDL) 36
3-3 Rhodostomin與其突變蛋白骨架動力學之分析
3-3-1 15N標定R1、R2、NOE弛緩實驗 37
3-3-2 Rhodostomin Model free 之計算 38
第四章 討論
4-1 g-Bungarotoxin蛇毒蛋白的部分 39
4-2 Rhodostomin突變蛋白部分 41
4-3 Rhodostomin與突變蛋白骨架動力學探討 44
第五章 結論 45
參考文獻 46
圖 51
表 83
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