Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92186
標題: Cyclic di-AMP 受體蛋白 S. aureus CpaA 和 M. smegmatis DarR 的結構與功能分析
Structure and function studies of S. aureus CpaA and M. smegmatis DarR in complex with cyclic di-AMP
作者: 梁峻銘
Chun-Ming Liang
關鍵字: 受體蛋白
Cyclic di-AMP
S. aureus CpaA
M. smegmatis DarR
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摘要: 原核生物因應環境的改變,會藉由二級傳訊分子傳遞訊號,幫助原核生物做出適當反應。Cyclic-di-AMP (CDA) 是近幾年新發現的二級傳訊分子,其廣泛存在細菌與古生菌中,負責調控許多重要的生理功能,例如: 孢子形成、偵測 DNA 的損傷、滲透壓的調節、細胞壁代謝、細胞膜上脂質的動態平衡等。目前已知 CDA 傳遞訊號的方式與 c-di-GMP (CDG) 類似,其合成由 diadenylyl cyclases (DACs) 將兩分子的 ATP 合成為一分子的 CDA,而 phosphodiesterases (PDEs) 可將 CDA 分解為 pApA,藉由這兩種酵素調控細菌體內 CDA 的濃度,影響其受體蛋白的活化或抑制,改變細菌生理活性。為了進一步探討 CDA 的傳訊機制,本篇論文選擇 CDA 的受體蛋白進行結構與功能的分析。過去文獻指出 Staphylococcus aureus 中的 cation proton antiporter A (CpaA) 蛋白為 CDA 的受體,主要調控細胞內鈉鉀離子濃度,並也證實 CDA 是結合在 CpaA 蛋白C端的RCK domain上。而 Mycobacterium smegmatis 中的 c-di-AMP receptor regulator (DarR) 蛋白是另一個 CDA 的受體,可以調控細胞膜上脂質的動態平衡。本篇論文建構了不同片段的 CpaA RCK domain 與 DarR 蛋白,嘗試解析出與 CDA 複合體結構。經由 X-ray 晶體繞射分析方法,CpaA native 和 SeMet-labeled 蛋白晶體解析度分別可達到3.5 A和3.0 A,但在解析結構的過程中,發現晶體有孿晶 (twinning) 的現象,因此無法順利解析其相位角。而 DarR 蛋白晶體收到 X-ray 繞射資料解析度只有6 A,無法解析其結構,尚待調整晶體培養條件,以期找到解析度更好的晶體。此外,目前也研擬構築不同長度的 CpaA和 DarR 蛋白片段,期待獲得品質較好的單晶晶體可供結構解析。
Cyclic diadenosine monophosphate (c-di-AMP) is a novel signaling molecule that has been recently discovered in bacteria. It is a second messenger moleule that can help bacteria adapt to fast-changing environment. C-di-AMP is produced from two molecules of ATP by the diadenylyl cyclase (DAC) enzymes and is degraded to pApA by the phosphodiesterase (PDE) enzymes. By using these two enzymes to regulate the c-di-AMP concentration, bacteria can either active or inhibit downstream gene expression. To futher understand how c-di-AMP is involved in bacterial signal transduction, we study the structure and function of S. aureus CpaA_RCK domain and M. smegmatis DarR, which have been confirmed as the c-di-AMP receptors in previous strudies. To date, we have obtained both the native and SeMet-labeled co-crystals of S. aureus CpaA_RCK and M. smegmatis DarR with c-di-AMP. The X-ray diffraction data of SeMet-labeled CpaA_RCK domain crystals were collected to a resolution of 3.0 A. In the process of solving the CpaA_RCK structure phase, we found that the crystals suffer from twinning problem, leading to structure determination impossible. The X-ray diffraction data of DarR crystals were collected only to a resolution of 6 A, which is also insuffient to solve its structure. No further improvement in resolution could be achieved by manual fine-tuning of screening condition. It may be necessary to study S. aureus CpaA and M. smegmatis DarR proteins of different lengths to get better crystals quality to determine their structures.
URI: http://hdl.handle.net/11455/92186
文章公開時間: 2017-10-20
Appears in Collections:生物化學研究所

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