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標題: Xanthomonas campestris pv. campestris Hrp調控蛋白FhrR 與人類先天免疫系統dsRNA受體蛋白DDX1-DDX21之複合體研究
Studies of the Xcc Hrp regulatory protein FhrR and human innate immunity dsRNA receptor protein DDX1-DDX21 complex
作者: 游雅婷
Ya-Ting Yu
關鍵字: dsRNA受體蛋白;黑腐病菌;調控蛋白;DDX1;DDX21;FhrR;dsRNA receptor
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本論文主要分為兩個部分:第一部分為研究植物黑腐病菌Xanthomonas campestris pv. campestris (Xcc) hrp致病基因調控蛋白XccFhrR的結構。FhrR是屬於TetRs轉譯因子家族,可以調控鞭毛的形成、核醣體蛋白和Hrp蛋白的合成。我們已得到XccFhrR的結晶,大小約為0.2 x 0.5 x 0.3 mm,並利用X-ray晶體繞射技術得到一組解析度為為2.9 A的繞射數據。我們嘗試以轉譯調控蛋白Sco0520蛋白(相似度35%)為參考模板,利用分子置換法進行相位角計算,但是無法成功計算出相位角。因此進一步以Selenomethionine 標定XccFhrR蛋白質,藉以計算其相位角。然而所得的晶體太小而無法收集數據,目前正在微調中,以期能長出品質更好的晶體。第二部份則是研究人類先天免疫系統dsRNA受體蛋白DDX1與DDX21的複合體結構。先天免疫系統主要藉由細胞中的受體辨識病毒的核酸,而RNA解螺旋酶DDX1與DDX21是受體蛋白其中重要的成員。此一辨識機制主要由DDX1結合病毒dsRNA再與DDX21、DHX36交互作用,藉由TRIF的路徑向下游傳遞訊息,引發第一型的干擾素的大量產生以抵抗病毒入侵。然而全長的DDX1與DDX21的可溶性不佳,因此我們構築了數個不同長度片段的蛋白,並獲得可溶性極佳的DDX11-440與DDX211-571蛋白片段,克服了蛋白溶解度問題,但目前尚未篩選到適合的蛋白結晶條件。

The study contains two parts: the first part is determing the structure of protein XccFhrR, which regulates causative hrp genes, in Xanthomonas campestris pv. campestris (Xcc). FhrR transcription factor belongs to the TetR family, and regulates the expression of genes that encode flagella, Hrp and ribosomal proteins. We have obtained the native crystals of XccFhrR that diffracted to a resolution of 2.9 A. We tried to get the phase by the molecular replacement approach using the most similar protein Sco0520, but were not able to successfully extract the phase information. To solve the problem, we have prepared the Se-Met labeled protein. We have obtained preliminary Se-Met labeled crystals, but they are too small for X-ray diffrection. More efforts are required to grow bigger crystals. The second part is to study the complex structure of the dsRNA receptor protein DDX1 and DDX21 in the mammalian innate immune system. The full-lenth DDX1 and DDX21 are very difficult to obtain in soluble form, and we finally found the DDX11-440 and DDX211-571 fragements are very soluble. We have tried to obtain their co-crystal by using a variety of crystallization conditions, yet no crystal could be obtained so far.
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