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標題: | 表皮葡萄球菌TcaR及TcaR-水楊酸複合體之晶體結構 Structural study of TcaR and its complex with salicylic acid form Staphylococcus epidermidis |
作者: | Yao-Jen Yeh 葉耀仁 |
指導教授: | 王惠鈞(Andrew H.-J. Wang) |
關鍵字: | 表皮葡萄球菌,生物膜,水楊酸,翼狀螺旋,ica啟動子,TcaR,MarR家族, Staphylococcus epidermidis,biofilm,salicylate,winged helix,ica promoter,TcaR,MarR family, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | TcaR是ica操縱子的阻抑蛋白,此操縱子會影響表皮葡萄球菌生物膜的形成。在此論文我們針對TcaR以及TcaR-水楊酸-二甲砷酸複合體的晶體結構做研究,其解析度分別為2.3 Å以及2.5 Å。結構顯示出TcaR是以二聚體的形式存在,並且含有翼狀螺旋DNA結合模體。重要的是我們發現水楊酸以及二甲砷酸(含有重金屬砷)與TcaR之間產生交互作用。對於一些MarR家族的蛋白質來說,水楊酸扮演著抑制劑的角色,抑制蛋白質與DNA結合,且此種抑制的關係對於TcaR與二甲砷酸之間作用力的關係也許是相同的。我們比較TcaR本身蛋白質的結構以及複合體蛋白質的結構發現兩者之間的結構有變化,也就是說TcaR與DNA之間交互作用的調控機制也許與TcaR結構中DNA結合性結構域的結構變化有關聯。為了證實TcaR與ica啟動子之間有交互作用,我們使用純化後的TcaR與184-bp的探針來作EMSA實驗,此184-bp探針代表完整野生型表皮葡萄球菌RP62A的ica啟動子。結果顯示確實TcaR會與ica啟動子結合。我們進一步的預測三段可能會與TcaR結合的33-mer DNA片段,這些片段皆包含在ica啟動子中且具有部分迴紋序列,並且執行EMSA實驗來證實TcaR與此三片段DNA是否會有交互作用。一系列的試驗顯示,兩個TcaR二聚體會與33-mer探針結合且不具有協同性。總結來說,我們的研究目標是致力於確認TcaR蛋白的調控機制以及找出可能會調控TcaR蛋白的小分子。 TcaR is a repressor of ica operon that affects biofilm formation in Staphylococcus epidermidis. Here we describe the crystal structure of the apo form TcaR and TcaR-salicylate-cacodylate complex that are determined at the resolution of 2.3 Å and 2.5 Å, respectively. The structure shows TcaR as a dimer with the winged-helix DNA binding motif. It is important that we find the salicylic acid and cacodylate (containing heavy metal As) which can interact with TcaR. Salicylic acid, which inhibits DNA binding, is a known inactivator for some MarR family proteins and the relationship between cacodylate and TcaR may be the same. We compare the structure of apo form TcaR with complex TcaR, revealing that the mechanism of regulation maybe involves a conformational change in the DNA binding domain. In order to confirm TcaR binds specifically to the ica promoter, we combined purified TcaR with the 184-bp probe representing the sequence of the entire wild-type ica promoter from S. epidermidis RP62A in electrophoretic mobility shift assay (EMSA). The results indicate that TcaR indeed bind to the ica promoter. We further predict three fragments of 33-mer DNA that contain partial palindrome sequence within ica operon, which may interact with TcaR, and perform EMSA experiment to confirm the interaction. A series of EMSA experiments revealed that two TcaR dimers bind to 33-mer probe without cooperativity. In summary, our study advances efforts aimed at identifying the regulation mechanisms and finding out small molecules that could modulate the function of the TcaR protein. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40789 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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