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研究生:陳婷鈺
論文名稱:鏈激酉每突變種(SKβK180A)的蛋白結晶與鏈激酉每/人類血纖維蛋白溶酉每/酉每原作用結構模型之建立
論文名稱(外文):Crystallization of SKBK180A and three-dimensional modeling of SK-plasmin-plasminogen complex
指導教授:樓國隆
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:口腔生物科學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:鏈激酉每人類血纖維蛋白溶酉每
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人類的止血作用主要依靠血液凝固系統與血纖維蛋白溶解系統相互制衡來發揮正常作用。 血纖維蛋白溶解系統主要是因人類血纖維蛋白溶酉每原 (human plasminogen, HPlg) 被活化成具酵素水解活性的人類血纖維蛋白溶酉每 (human plasmin, HPlm),進而水解血纖維蛋白 (fibrin),達到溶解血栓的目的。人類血纖維蛋白溶酉每原活化成為人類血纖維蛋白溶酉每的過程中,分子主要於Arg68-Met69、Lys77-Lys78、Lys78-Val79與Arg561-Val562四處被切割;其中Arg561-Val562的切割只可由人類血纖維蛋白溶酉每原的活化因子來執行。
鏈激酉每 ( Streptokinase),為人類血纖維蛋白溶酉每原的活化因子之一,用於臨床上治療血栓。鏈激酉每本身並不具蛋白酵素活性,而是經由特殊機制活化人類血纖維蛋白溶酉每原;雖可用於促進血栓的溶解,然而至今其與人類血纖維蛋白溶酉每原作用的機制卻尚未完全了解。本研究企圖以形成鏈激酉每β domain點突變 (SKβK180A) 蛋白結晶作為開始,進一步了解片段點突變與正常鏈激酉每β domain結構功能上的差異,並配合HPlg分子片段之共結晶 (co-crystallization) 來分析鏈激酉每β domain和HPlg kringle domains 在活化過程中擔負之角色。另一方面,為了進一步了解鏈激酉每與人類血纖維蛋白溶酉每原反應產生人類血纖維蛋白溶酉每的作用模式,本研究也以三度空間結構模型之建立(3-D modeling),配合電腦軟體的運算模擬,來提出合理可能的嵌合 (docking) 模式和結構細節分析。
本研究大致找出形成鏈激酉每β domain點突變 (SKβK180A) 蛋白結晶之條件方向。該結晶是刀狀,長度可達0.3 mm。初步X-光結晶繞射分析顯示晶體解析度約8Å,目前正朝更佳結果邁進。經由電腦模擬,鏈激酉每與人類血纖維蛋白溶酉每原作用產生人類血纖維蛋白溶酉每的鍵結模式,已有合理結果,由此可推得於相當程度上三個分子間作用機制的結構功能詮釋
Abstract
In human blood, the coagulation and the thrombolytic systems strongly associate with each other and the equilibrium between them can be therefore dynamically achieved. Among this, the fibirnolysis fuctions as the consequence of the activation of plasminogen (Plg) to plasmin (Plm), the latter then proteolyzes fibrin to remove blood clot. During the activation, the plasminogen polypeptide has to be cleaved in four places: Arg68-Met69, Lys77-Lys78, Lys78-Val79and Arg561-Val562. The last one can be accomplished only by specific plasminogen activators.
Streptokinase (SK), a plasminogen activator, is widely used in treating blood-clotting disorders. However, SK is not a protease. It forms a stoichiometric complex with either Plg or Plm to activate other Plg molecules. The mechanism is until present not fully understood, for only limited structural information is thus far available. Our study starts with trials for crystallization of a molecule containg onlyβ subunit of SK with Lys-180 mutated to alanine, namely SKβK180A. In combination with the co-crystallization of plasminogen fragments in the future, our goal is to investigate the variation between wild type and mutant SK both structurally and functionally, which may lead to the understanding of the roles of SK-β domain and HPlg kringles in the HPlg activation. In addition, to further realize the interaction between SK-Plm and Plg, we have finished the 3-D modeling of such complex. Upon utilization
of molecular simulation, reasonable docking orientations and structural details can be available and analyzed.
The condition for crystallizing SKβK180A has been obtained. The crystals are in a long, thin, knife-like shape with a diameter up to 0.3 mm Preliminary X-ray diffration suggested 8Å resolution for our crystals. Of course more efforts will be done to produce better results. Nevertheless, the simulation results have provided fresh insights into the structural-functional interpretation for the docking between SK, plasmin and plasminogen to a certain extent.
目錄
摘要 -------------------------------------------------- I
Abstrct------------------------------------------------II
緒論
凝血過程和藥物作用------------------------------------------------1
人類血纖維蛋白溶酉每原與人類血纖維蛋白溶酉每簡介----------------------1
鏈激酉每簡介--------------------------------------------------------2
鏈激酉每活化人類血纖維蛋白溶酉每原的機轉------------------------------3
鏈激酉每各區域與微小型人類血纖維蛋白溶酉每/酉每原( μPm/μPg)的關聯-------5
目的---------------------------------------------------------------7
實驗方法:
一、SKβK180A融合蛋白質的純化-------------------------------------8
二、蛋白質純度分析------------------------------------------------8
三、蛋白質濃度測定------------------------------------------------9
四、蛋白質穩定性測定----------------------------------------------9
五、蛋白質結晶形成------------------------------------------------10
1.初步篩選
2.進一步條件測試: pH, PEG濃度及溫度
六、X光結晶繞射分析----------------------------------------------11
七、三度空間結構模型建立-----------------------------------------12
結果:
一、SKβK180A融合蛋白質的純度測定-------------------------------14
二、蛋白質穩定性測定--------------------------------------------14
三、蛋白質濃度測定----------------------------------------------14
四、SKβK180A之結晶形成-----------------------------------------14
1.初步篩選
2.蛋白結晶與pH、PEG之關係
3.蛋白結晶與溫度之關係
五、初步X光結晶繞射分析結果------------------------------------16
六、基本結構模型與不同嵌合方式之能量比較-------------------------17
討論:
一、不同條件對SKβK180A蛋白形成晶體的意義----------------------19
二、透過三度空間結構模型所提供之結構功能-分析-------------------21
三、未來方向----------------------------------------------------22
圖--------------------------------------------------------------23~36
表-------------------------------------------------------------37~38
附圖-----------------------------------------------------------39~42
附錄
參考文獻
參考文獻
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張麗琴 人類血纖維溶酉每原與突變種鏈激酉每之交互反應 國立成功大學生物化學研究所碩士論文(1997)
高雅靜 醫用藥理學 中華民國85年
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