2015-01-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657964摘要：利用核磁共振光譜與蛋白質結晶學，已可為我們進行蛋白質結構研究，獲得原子層級的觀察。而目前蓬勃發展的冷凍低溫電子顯微鏡，以直接觀察與單顆粒重建的技術，可讓我們獲得許多上述兩種方法無法獲得的資訊。因此結合此多項利器成為整合性結構生物學的研究，已是未來結構系統生物學的趨勢。本計劃將利用整合性結構生物學方法與其他生化及細胞生物學研究，探討與蛋白質合成與品質控管有關的兩個龐大複合體架構與功能作用機制。 在蛋白質合成工廠核醣體的組裝與運輸過程，需要許多其他輔助因子的協助與嚴密調控，已確保此重要生命機制的運作。目前我們解析出一BCP1蛋白之晶體結構，也了解其與核醣體RPL23蛋白有交互作用，而BCP1如何協助核醣體運輸則有待與進一步生化功能與複合體結構生物的探討。另一議題為蛋白質品質控管的Lon蛋白&#37238;，Lon是一種 ATP依賴型蛋白&#37238;，普遍地存在於各種原核生物及真核生物粒線體中。同時Lon在演化上也具有高度的保留性，由此可見其在生物體內的重要。在功能方面，它能夠分解細胞內不正常累積的蛋白質，或是將折疊錯誤的蛋白質回復成正常構形。我們已利用核磁共振方法與蛋白質結晶學獲得alpha區塊的結構及與DNA結合的模式，進一步將配合低溫冷凍電顯了解Lon與不同輔助因子結合時所造成的構型變化，並解釋其變化所造成的分子功能。透過此次與北京清華大學的合作與交流，除了能使這些重要生物議題得到更詳盡的分子機制解釋，同時幫助臺灣大學發展與建立更完善的整合性結構生物學設施，以面對未來結構系統生物學的研究。 <br> Abstract: NMR spectroscopy and protein crystallography both are the important tools to determine protein structure on atomic level. Currently in developing, cryo-electron microscopy (cryo-EM) can acquire molecular image directly, and subsequently the sub-atomic structure could be obtained by single particle reconstruction technique. In addition, cryo-EM may allows us to get more structural information than the above two methods. Hence, combining these tools could come out an integrated structural biology, which is the trend of structure-based systems biology. In this proposal, the integrative structural biology approach as well as other biochemical and biophysical tools will be utilized to study the protein complexes involved in protein synthesis and quality control. In the process of protein synthesis, the transport and assembly of ribosome need the tight regulation with the help of many cofactors. We have determined the crystal structure of a protein BCP1, which could interact with ribosomal RPL23. However, how BCP1 facilitates the transport and assembly of ribosome are poorly understood. Another issue is Lon protease involved in the protein quality control. Lon is an ATP-dependent protease, commonly found and evolutionary highly conserved in various prokaryotes and eukaryotes mitochondria. It plays an important role in intracellular protein degradation and selectively degrades damaged and abnormal proteins and several short-lived transcription factors that control cell division, synthesis of capsular oligosaccharides, pathogenesis, or formation of biofilms. Recently, we solved the structure and understood the DNA binding mode of Bt-Lon alpha domain by utilizing protein crystallography and NMR technique. For further understanding of conformational changes of Lon significantly induced by cofactor binding, cryo-EM would be applied to explore the structural and functional relationship of the huge complex. Through this cooperation with Tsinghua University in Beijing, these important biological issues can get a more detailed explanation of the molecular mechanism. The co-operation also will help National Taiwan University for the development of integrated structural biology facilities and the studies of structure-based systems biology.核醣體Lon蛋白&#37238低溫冷凍電子顯微鏡結構生物學蛋白質交互作用ribosomeLon proteasecryo-electron microscopystructural biologyprotein interaction