2011-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/703189摘要：目前人類基因體已完全解碼，人類已進入後基因時代，但對於這些基因所表現出的蛋白質卻仍然未能有充分的瞭解，蛋白質在細胞內扮演非常重要的角色，舉凡細胞分裂、代謝反應、訊息傳遞、基因的複製與修復、養分的運送等，都需要蛋白質分子參與反應，蛋白質分子之間透過彼此交互作，用形成複雜的蛋白質網絡，而只要其中某一蛋白質表現失衡，即有可能使蛋白質網絡受到破壞，連帶造成細胞的病變，因此為了研究蛋白質與疾病之關係且從分子尺度來解釋蛋白質的功能與交互作用，勢必要從最基本的蛋白質三維結構出發，而從結構生物學的觀點著手不僅可以驗證我們假設的正確性，另外亦讓我們能從實際建構的三維分子影像歸納出合理的推測與解釋，此將加速我們對功能性蛋白質之瞭解，也將會在製藥、疾病防治、基因複製與修復上提供重要的資訊。 傳統結構生物學現今賴以使用解生物大分子的技術仍是以X光結晶繞射為主，但其困難性在於使高純度蛋白質大分子在特定環境下形成具有規&#63960;堆疊的蛋白質晶體，且此技術被人詬病的一點，就是所解出的結晶結構其實並非蛋白質分子在生理環境下之原型。所幸科學家們目前已經有辦法製備能在電子顯微鏡高真空環境下維持完全水合之原型蛋白質試片，此一新穎之技術稱為冷凍電子顯微術（Cryo-EM），即利用急速冷凍之處理，將蛋白質分子包埋在非結晶態之冰當中，此技術克服了蛋白質X光結晶繞射技術之限制，但Cryo-EM的缺點是尚未具有原子級的解析能力，而限制冷凍電顯解析度最根本的問題是在於輻射傷害，因為在非結晶態冰中並無自由電子，故不具導電性，在電子束照射下會產生永久性無法修復的輻射傷害。在本計畫中，我們將發展一種冷凍試片帶電技術(CSE)使冷凍生物試片在電子束照射下其所受之輻射傷害能進一步降低，本方法乃藉由對冷凍試片施以電解質離子摻雜並且充電，使得摻雜充電過後的非結晶態冰，變成有接近導體的行為，故當有輻射傷害產生時，試片當中的自由電子能迅速返還修復因輻射傷害而失去電子的離子、蛋白質帶電分子片段與水分子的自由基，進而使得電子束對生物試片與非晶態冰所造成之輻射傷害能夠大幅降低，故能夠給予足夠之電子劑量而能夠清楚觀察生物材料的原型，此外，我們將自製一冷凍充電之試片治具用於CSE實驗以配合本計畫之進行。 本計畫將分三年進行實施，所將進行的研究課題主要包含: (1) 血紅蛋白、GroEL蛋白質與HSV病毒外膜之結構重建與解析度改善; (2)細胞分裂調控蛋白FtsK之高解析結構重建與其功能性之探討; (3) XerC與XerD蛋白質之完整結構表現與重建以及其生物功能性之研究; (4) FtsK、XerC/D與DNA複合體之結構重建以及此複合體運作機制的研究; (5) 細胞分裂調控蛋白SpoIIIE之高解析結構重建與其功能性分析; 以及(6) 以細胞分裂調控蛋白結構與功能為基礎之抑制細胞分裂藥物的開發。本計畫諸多與細胞分裂有關的研究課題將有助於相關疾病與癌症的防治，未來亦將與生物與生化學家以及臨床醫生合作使用Cryo-EM與CSE技術進行更多有趣並且重要的研究課題，我們相信本研究計畫整合跨領域的研究資源的潛力無限，也將開啟整合物理、生化與醫學應用研究新的里程碑。 <br> Abstract: Important cellular mechanisms are carried out through the formation of large macromolecular complexes. These biological macromolecules are responsible for key processes such as cell signaling and reproduction, as well as being crtical in diseases like cancer and viral infections. Understanding of the molecular structure of these macromolecules is not only essential for the comprehension of their function and mechanism, but can also provide clues for the developing therapeutics related to health and disease. Nevertheless, the structures of only a small number of macromolecular complexes have successfully been determined at atomic resolution using x-ray diffraction (XRD), because crystallization of proteins and these macromolecular complexes remains a major hurdle to structural analysis with XRD. Cryo-electron microscopy (Cryo-EM) is a technique to freeze a hydrated sample and derive the 3D structures of the biological macromolecules using an electron microscope. In Cryo-EM, the samples do not need to be crystallized as in x-ray crystallography. However, Cryo-EM technique still lacks the resolution to determine the atomic structure of biological macromolecules&#8213;due to the radiation damage which limits the resolution in most biological materials. In this project, we develop a new technique, the cryo-specimen electrification (CSE) technique, which reduces radiation damage to biological samples during electron beam irradiation. A unique cryo-charging specimen holder will be developed for CSE experiments. The ion doped cryo-specimen after charging can not only trap and store charge in equilibrium with the free and mobile charges, but also has a conductivity level close to that of metals. Hence it can promptly return electrons to ionized atoms and fragments in the frozen sample to efficiently repair the radiation damage, and thus greatly increase the electron dosage tolerance of the sample under electron beam exposure. Therefore, the CSE technique accompanied with Cryo-EM has opened up the possibility of studying the frozen biological sample at higher resolution. This project is planned for three years. We are going to conduct and develop following research topics, including: (1) resolution improvement and structure reconstruction of hemoglobin, GroEL protein, and HSV virus; (2) high-resolution 3D reconstruction of FtsK division protein and its function analysis; (3) complete structure expression and reconstruction of XerC and XerD proteins and the study of their biological functions; (4) structure reconstruction of FtsK, XerC/D, and DNA complex and the research of this complex mechanism; (5) high-resolution structure reconstruction of SpoIIIE division protein and its function analysis; and (6) drug development based on the structural and functional study of above-mentioned cell division proteins. These research topics related to the cell division in this project should help in diseases and cancer control. In the future, we will also collaborate with biologists, biochemists, and medical doctors to conduct many other interesting research topics using Cryo-EM. We believe that the future application potential of the Cryo-EM plus the merit of the CSE technique should be beyond our expectation.電子顯微鏡冷凍電子顯微術細胞分裂調控蛋白結構生物學electron microscopecryo-electron microscopycell division proteinsstructural biology