FIP1之結構模擬與功能性分析及其與FIN219間的協同活性之研究

Abstract

FIN219 (Far-red insensitive 219)又稱為Jasmonate Resistant 1 (JAR1)，在光型態發生中扮演重要角色，並且具有將茉莉酸(Jasmonic acid)和Isoleucine (Ile)結合成JA-Ile的酵素活性，FIN219的蛋白質結構和其分子機制至今仍是未知。然而FIP1 (FIN219 Interaction Protein1)具有GST的活性，與FIN219蛋白發生交互作用時可減少FIN219的降解。本實驗中試著將GST-FIN219和FIP1蛋白質進行結晶，並將晶體進行X-ray繞射的分析，但得到的繞射數據並不清晰。由於FIP1是屬於Tau Glutathione S-transferases (GSTs)家族成員之一，並且有許多的Tau GST蛋白質的結構已被解析，這些蛋白質其中之一的fluorodifen-inducible GST (GmGSTU4-4, PDB ID: 2VO4)和FIP1間的胺基酸序列相似性高達64%。利用modeller9v8軟體，進行FIP1蛋白的結構模擬(homology modeling)，經由FIP1 model推測FIP1會形成雙體(dimer)的結構，並進一步地分析FIP1和GST間其dimeric interface與活性部位。本實驗利用數種的生化分析方法，如膠體過濾色層分析法(gel filtration chromatography)，native PAGE，和glutaraldehyde (GA) cross-linking等實驗，來證實FIP1確實以雙體存在。而先前在純化FIN219的過程中，我們發現到FIP1不只會與FIN219結合亦會和GST發生交互作用，利用GST親和性管柱和pull-down分析的方法，證實了FIP1和GST，FIN219和GST，及FIN219和FIP1，蛋白質和蛋白質間具有交互作用的存在。最後我們使用CDNB分析來檢測FIP1的GST酵素活性，並發現到FIN219和FIP1間的酵素活性是一個協同活性的關係。

FIN219 (Far-red insensitive 219), also known as JAR1 (Jasmonate Resistant 1), plays an important role to mediate photomorphogenesis and possesses the enzymatic activity of conjugating JA to Isoleucine. The overall structure of FIN219 and its molecular mechanism are still unknown. However, a protein with GST activity, named FIP1, has been identified to interact with FIN219 and might reduce the decay of FIN219. Therefore, we tried to crystallize the GST-FIN219 and FIP1 only, and got some crystals of them. From preliminary results, those crystals got poor X-ray diffraction pattern. Since FIP1 is a member of Tau Glutathione-S-Transferases (GSTs), several structures of Tau GSTs have been resolved. One of them, fluorodifen-inducible GST (GmGSTU4-4, PDB ID: 2VO4) shares highly sequence identities (64%) with FIP1. The structural model of FIP1 was built using modeller9v8, a program for homology modeling. Based on the FIP1 model, the dimeric interface and the active site of GST were explored. We also performed several biochemical assays such as the gel-filtration chromatography, the native PAGE and glutaraldehyde (GA) cross-linking experiments to confirm the dimerization of FIP1. From previous purification of FIN219, we found that FIP1 not only interacts with FIN219, but also interacts with GST. Protein-protein interactions between FIP1 and GST, FIN219 and GST, and FIP1 and FIN219 were also performed by GST affinity column, pull-down assay. Finally, the GST activity of FIP1 was tested by CDNB assay, and the relationship of enzymatic activity between FIN219 and FIP1 showed a cooperative activity.