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標題: Xanthomonas campestris pv.campestris未知功能蛋白XC1692/XC1691複合物的晶體結構及功能分析
Structural determination and functional analysis of unknown function protein complex XC1692/XC1691 in Xanthomonas campestris pv. campestris
作者: 黃照煒
Huang, Chao-Wei
關鍵字: Xanthomonase campestris pv. campestris;十字花科黑腐病病原菌;complex;unknown function protein;複合物;未知功能蛋白
出版社: 生命科學系所
本論文研究的菌株為Xanthomonase campestris pv. campestris,屬於革蘭氏陰性菌,為兼具學術性及應用性之菌種。X. campestris能分泌多種胞外蛋白,為研究格蘭氏陰性菌蛋白分泌模式之標準系統之ㄧ,並為感染十字花科造成黑腐病之植物病原菌,屬世界性之病害。藉由基因體解碼下,此一病原菌被預測的基因約有4100個。本論文利用X-ray繞射法研究X. campestris str. 17菌株之11個目標蛋白質,其中XC1692由141個胺基酸組成,功能未知。比對已知物種基因資料庫後發現,XC1692 在不同原核生物體皆有相似序列,且同譯註為未知功能的蛋白。直至目前為止,沒有任何與XC1692序列相似(>20%)蛋白之三級結構被解析出來。我們已經得到高解析度的XC1692晶體(1.45 Å),但為利用多波長異常繞射數據解決其相位問題時, 無法獲得Se-Met 標定蛋白俱高解析度的晶體。為了解決此一問題, 我們藉由突變將Met轉成Ala, 減少Met個數,以降低XC1692晶体可能因Se-Met 標定造成的結構不穩定。藉此我們順利地得到結晶,並成功地解決相位的問題。由於XC1692為未知功能蛋白,所以我們利用結構比對得到XC1692相似結構為核醣核酸水解酶之抑制物。搜尋X. campestris基因庫發現XC1691之功能為核醣核酸水解酶,且XC1691位於XC1692上游,並由同一啟動子所調控。這暗示XC1692可能為核醣核酸水解酶抑制物。利用pull down assay實驗,證明XC1691與XC1692之間有交互作用力的存在,初步確定了XC1692的功能為核醣核酸水解酶抑制物。目前我們正在進行XC1691及XC1692複合物的結晶實驗,希望可以獲得不錯的數據,進一步從結構的角度來證實XC1692的功能的確為核醣核酸水解酶抑制物。

The bacterial strain studied in this thesis is Xanthomonase campestris pv. campestris, which is a gram-negative bacterium and an important pathovar both academically and industrially. X. campestris can secrete many kinds of extracellular proteins, and is a model system for studying protein secretion of gram-negative bacteria. It is phytopathogenic to cruciferous plants and can cause worldwide agricultural loss. From the sequenced genome, about 4100 genes were predicted. This thesis tries to study 11 of them by using X-ray crystallography technique. XC1692 is one of them containing 141 amino acids with no identified function. However many sequences with high similarity score were detected by a BLAST search. Until now, there is also no similar tertiary protein structure determined. However, although we have obtained high resolution crystals of native XC1692 (diffracted to 1.45 Å), we couldn't solve its phase using multiwavelength anomalous diffraction (MAD) method, due to the poor resolution data of Se-substituted crystals. To solve the problem, we have used mutagenesis method to change Met to Ala to reduce the number of Met to possibly increase the crystal stability. Using this approach, we have successfully solved the phase problem and got a preliminary structure of XC1692 using the MAD approach. XC1692 was then found to adopt a similar architecture with a ribonuclease inhibitor through a structure alignment. Also, a gene (XC1691) annotated as a ribonuclease was found to be upstream of XC1692 and controlled by the same promoter. Altogether, these data imply that XC1692 may serve as a ribonuclease inhibitor, which was further confirmed by a pull-down assay. We are currently crystallizing the XC1691/XC1692 complex to determine its functions from a structure perspective.
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