Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92211
標題: 陽離子/質子交換運輸蛋白CpaA與環形雙單磷酸腺苷的作用功能與結構分析
The functional and structural characterizations of Cation/Proton Antiporter CpaA and c-di-AMP
作者: 孫倖涵
Xing-Han Sun
關鍵字: 膜蛋白;環形雙單磷酸腺苷;陽離子/質子交換運輸蛋白;金黃色葡萄球菌;Membrane Protein;c-di-AMP;Cation/Proton Antiporter;Staphylococcus aureus
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摘要: 
二級訊號分子cyclic bis-(3',5')- dimeric adenosine monophosphate (c-di-AMP)為近幾年的研究中被發現廣泛存在於細菌和古生菌中,微生物會利用這類的二級訊號分子去反應外界的刺激以調控各種反應 。在過去的文獻中得知Staphylococcus aureus 中的CpaA (cation proton antiporter A)的C端 RCK (Regulator of Conductance of K+) domain為能辨識c-di-AMP ,而根據其在不同菌種裡的homologous protein NapA, NhaA 推測主要功能為調控細胞內之Na+濃度及pH值,但目前並不清楚c-di-AMP 如何調控 CpaA 的功能。 目前已知CpaA為細菌細胞膜上的Na+/H+陽離子反向通道蛋白,N端為穿膜區域,而C端的RCK domain又分為RCK_N與RCK_C兩個區域,其中的RCK_C是與 c-di-AMP 結合的區域。在本篇實驗中,我們以全長之CpaA 跟 c-di-AMP 結合後所產生的功能和結構構形變化為目標,將建構膜蛋白的C端接上GFP(Green fluorescent protein) 和His-Tag,讓目標蛋白 CpaA 跟GFP一起被表現,便可以從whole cell 的狀況下觀測GFP的螢光強度去估計 CpaA 的表現狀況。我們更換了數種 E. coli 菌株和更營養的培養基,成功塞選出適合的培養方式。之後將 純化的CpaA 蛋白reconstitute到liposome裏面模擬蛋白質在細胞膜上的狀況進行功能性測試,並使用Pyranine 這種對pH 敏感的螢光染劑,將染劑包埋在 liposome 內部,在鈉或是鉀濃度梯度存在的狀況下去監控pH的變化。我們發現在 c-di-AMP 存在的情況下,CapA 轉運Na與K+的效率會有所提升。根據FSEC (Fluorescence Size Exclusion Chromatography)篩選能讓CpaA最能穩定存在的detergent為DM ( n-Decyl-β-D –Maltopyranoside),期望之後能以此條件得到蛋白質晶體,提高以x-ray繞射得到高解析度之晶體結構的機會。

Cyclic-di-AMP, a new type of second messenger, has been found abundant in bacteria and archaea in recent years. Microbes utilize the signaling molecule to mediate physiological function in response to external stimuli. A previous study shows that CpaA (cation proton antiporter A) in Staphylococcus aureus contains a C-terminal RCK (regulator of Conductance of K+) domain which can recognize c-di-AMP. RCK domain is composed of RCK_N and RCK_C subdomains and it has been confirmed that it is the RCK_C subdomain specifically interacts with c-di-AMP. The N-terminal domain of CpaA is a transmembrane region which is homologous to cation proton antiporter family, such as NapA and NhaA. Sequence analysis shows that Asp171 of CpaA, which is highly conserved in Caton Proton Antiporter (CPA) family, is believed to be involved in antiporter activity. It is thus assumed that CpaA can receive the signal of c-di-AMP and thus mediate cytosolic sodium concentration and pH values. However, the signaling mechanism is not well characterized. In this study, we aim to study the functional and structural mechanism of full-length CpaA. We constructed an expression vector with GFP fused at the C-terminus of target protein. This GFP-fusion improves the throughput of overexpression condition screening by measuring the whole cell fluorescence counts of GFP. We have successfully purified CpaA using DM detergent. We then reconstituted purified CpaA into liposomes containing a pH-sensitive fluorescence dye, pyranine, and characterized the mechanism of sodium or potassium concentration gradient regulated cation/proton translocation. We found that CpaA can transport sodium/potassium and protons in antiparallel fashion, and the exchange activity is further activated in the presence of c-di-AMP.
URI: http://hdl.handle.net/11455/92211
Rights: 同意授權瀏覽/列印電子全文服務,2018-08-26起公開。
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