FIP1晶體結構及其與FIN219/JAR1協同活性之研究

Abstract

光訊息對於植物生長與發育佔有重要的地位，光型態發生與暗型態發生是植物為了適應光照環境產生的生理機制，包含了植物感應光的強度、週期、向光性、避蔭反應以及概日韻律等現象。茉莉酸是植物重要的荷爾蒙，於植物受到創傷或是病原菌感染時大量產生並產生防禦反應，近年來的研究證明茉莉酸會影響根部的形成、花器的發育以及果實的成熟，對於生殖生理有重要的影響。 FIN219 (Far-red insensitive 219)又稱為JAR1 (Jasmonate Resistant 1)，參與植物遠紅外光訊息路徑，同時具有催化茉莉酸與異白胺酸形成JA-Ile的酵素活性分子的功能。此外，利用酵母菌雙雜合系統篩選到的FIP1 (FIN219-Interacting Protein 1)同樣也參與在遠紅外光訊息路徑，為阿拉伯芥AtGSTU的成員，擁有GST酵素活性，由先前研究已知FIN219能與FIP1產生交互作用，而且FIP1能穩定FIN219並調控FIN219的酵素活性。 本論文為探討FIN219與FIP1的蛋白質結構、功能與酵素活性，利用大腸桿菌系統表現蛋白質以及利用膠體過濾層析法純化FIN219與FIP1。純化過程發現穩定性提升的FIN219能夠與表現載體pGEX 4T-1表達的SjGST蛋白質結合，並影響GST結合至親和性管柱的能力，並在膠體過濾層析中發現FIN219能與SjGST產生蛋白質複合體。利用石英晶體微天平分析證實FIN219與FIP1以及FIN219與SjGST的交互作用，分析FIN219酵素活性發現FIP1可以提升FIN219的最大速率以及催化常數。最後我們篩選出FIN219-GST、FIN219-FIP1以及FIP1晶體的結晶條件，並解出FIP1結構為對稱性的蛋白質雙體並帶有兩個活性區域，活性區中G-site帶有高度保守性的Glu以及Ser殘基，而H-site擁有比GmGSTU4-4更大的結合位，顯示GST tau類型對於植物代謝毒素以及殺草劑類化合物的重要性。

Light signal plays an important role in plant growth and development. Photomorphogenesis and skotomorphogenesis are plant physiological mechanisms developed for adapting to light, including light fluence, photoperiodism, phototropism, shade avoidance and circadian rhythms. Jasmonate is a crucial phytohormone. It can be accumulated in the wounded or infected tissue and triggered defense response. Recent study confirms that jasmonate regulates root formation, flower development and fruit maturation, it affects deeply the reproductive physiology. FIN219 (Far-red insensitive 219), also known as JAR1 (Jasmonate Resistant 1), involved in far-red light signaling pathway and possessed catalytic activity in conjugating jasmonic acid to isoleucine. In addition, FIP1 (FIN219-interacting protein 1) screened from yeast two-hybrid system also participated in far-red light signaling pathway. It belongs to the member of Arabidopsis thaliana AtGSTU family and possesses GST enzyme activity, previous study identified that FIN219 interacts with FIP1, then FIP1 improves FIN219 stability and regulates FIN219 enzyme activity. This study aimed to resolve FIN219 and FIP1 protein structure, function and enzyme activity. FIN219 and FIP1 proteins were expressed in Escherichia coli system and further purified using gel filtration chromatography. Interestingly, FIN219 could interacts with SjGST, a GST protein expressed from vector pGEX 4T-1, and interfered the ability of SjGST binding to GST affinity column. The FIN219-SjGST complex could be observed in gel filtration chromatography. The binding affinities and enzymatic kinetics between FIN219 and FIP1, and between FIN219 and SjGST were determined by Quartz Crystal microbalance (QCM). The results of kinetics of FIP1 demonstrated that FIP1 would promote maxmium velocity (Vmax) and catalytic constant (Kcat) of FIN219. Finally we screened FIN219-GST, FIN219-FIP1 and FIP1 crystallization condition. Only the crystals of FIP1 were obtained. The resolved structure of FIP1 is a dimer and possesses two active sites, with highly conserved residues Glu and Ser in G-site. In addition, FIP1 contained a larger H-site in comparison to that of GmGSTU4-4. It exhibited the importance of GST tau class in detoxification and in metabolism of herbicide compounds.