臺灣博碩士論文加值系統

CpaA (cation proton antiporter A)，為金黃色葡萄球菌中參與陽離子和質子交換的蛋白，被認為是會結合c-di-AMP的受體蛋白，其中c-di-AMP是新型發現的二級訊號分子，幫助細胞調控生理反應以因應外界刺激。分析其CpaA的胺基酸序列比對，CpaA的N端為穿膜區鑲嵌於細胞膜上，同源蛋白為鈉離子/質子反向轉運蛋白，如NhaA、NapA、NhaP和鉀/質子反轉錄酶KefB。CpaA的C端屬於RCK domain (Regulator of Conductance of K+)，可以進一步分為RCK_N和RCK_C subdomains。先前文獻利用生物化學實驗及晶體結構證實，c-di-AMP會專一性結合於CpaA RCK_C domain的高保守性胺基酸。然而，目前對於CpaA的功能仍是未知的是： (i) CpaA可以運輸那些陽離子進而與質子交換？ (ii) CpaA RCK_C domain結合c-di-AMP是如何調控陽離子/質子反向轉運活性？ (iii) 突變c-di-AMP結合CpaA RCK_C domain的胺基酸是否會影響其功能？在本研究中，我們已經成功地純化全長CpaA包覆於DDM micelles中。我們的目標是使用螢光光度法來研究結合c-di-AMP是如何調節CpaA的陽離子/質子交換活性。利用pH值敏感的螢光染劑(pyranine)被包裹在含有CpaA的蛋白脂質體(proteoliposome)中，並且透過觀察螢光下降監測質子流出。我們發現CpaA運輸鉀離子活性最好，其次是運輸鈉離子和鋰離子，而正二價的鈣離子之運轉效率最差。有趣的是，當加入c-di-AMP時，螢光會進一步地下降，顯示CpaA結合c-di-AMP後，會運送更多的鉀離子進入和質子流出proteoliposome。這項結果指出，在c-di-AMP存在下，會促進CapA運輸效率。但是在CpaA c-di-AMP結合位的突變株R560A和H583A中卻不明顯。未來，我們將嘗試獲得全長CpaA N端結合受質和有著c-di-AMP結合RCK_C domain的複合體高解析度結構。這項結構研究將有助於我們了解，c-di-AMP調控CpaA對細菌離子滲透壓的作用。

CpaA (cation proton antiporter A) is involved in the exchange of cation and proton in Staphylococcus aureus, and CpaA has been implicated as a receptor protein for c-di-AMP, a novel second messenger that relays the external signals into the cells. Sequence analysis of CpaA suggests that the N-terminus of CpaA is a transmembrane domain, which is similar to sodium/proton antiporters, such as NhaA, NapA, NhaP and potassium/proton antiporter KefB. The C-terminus of CpaA belongs to a RCK (Regulator of Conductance of K+) domain which can be further divided into RCK_N and RCK_C subdomains. Previous biochemical and crystallographic structure of CpaA RCK_C subdomain have shown that the c-di-AMP binding site is mapped on the RCK_C subdomain interacting with highly conserved residues. Nevertheless, three intriguing questions remain unanswered. (i) Which cation can be transported by CpaA in exchange with proton? (ii) How binding of CpaA RCK domain to c-di-AMP regulates cation transports activity? (iii) Whether mutation of c-di-AMP-binding amino acids affects the function of CpaA? In this study, we have successfully purified full-length CpaA in detergent (using DDM) micelles. We characterized the c-di-AMP-mediated cation/proton exchange activity of CpaA using a fluorophotometric approach. The pH-sensitive fluorescence dye, pyranine, was encapsulated in proteoliposomes containing CpaA and the H+ efflux was monitored by the changes of fluorescence intensity. We found that CpaA showed the best acitivity of transportation for K+, followed by Na+ and Li+, but poor acitivity for Ca2+. Interestingly, adding c-di-AMP resulted in further prominent changes in the fluorescence intensities, suggesting, in the presence of c-di-AMP, CapA transport efficiency was ehhanced. But, no obvious changes were observed in CpaA mutants R560A and H583A, which is c-di-AMP-binding residues in CpaA RCK_C domain. In the future, we will try to obtain high resolution structure of full-length CpaA N-terminal binding substrate and RCK_C domain with c-di-AMP complex. This structural studies will enlighten us the c-di-AMP regulatory role of CpaA on ion homeostasis in bacteria.

摘要 i
Abstract ii
目次 iii
圖目次 vi
第一章 前言 1
一、 新型的二級訊號分子c-di-AMP 1
二、 結合c-di-AMP的受體蛋白 1
三、 KtrA RCK_ CTD結合c-di-AMP的結構 2
四、 CpaA RCK_ CTD結合c-di-AMP的結構 3
五、 比較CpaA與KtrA之RCK_CTD結合c-di-AMP的複合體結構 3
六、 陽離子/質子反向轉運蛋白家族(CPA)中的CpaA 4
七、 本論文研究目的 5
第二章 材料與方法 6
一、 構築去除CpaA-GFP其中 GFP基因的質體 6
(一) 設計引子 6
(二) 聚合酶連鎖反應(Polymerase chain reaction, PCR) 6
(三) Colony PCR 7
(四) DNA膠體電泳 7
(五) 抽質體DNA 8
二、 CpaA突變株 8
(一) 單點突變CpaA 8
(二) 位點導向基因突變(Site-Directed Mutagenesis) 8
三、 大量表現與純化CpaA WT及突變株 10
(一) 製備勝任細胞 10
(二) 轉殖作用(Transformation) 10
(三) 大量表現全長CpaA 10
(四) 破菌取得細胞膜 10
(五) 自細胞膜上萃取膜蛋白 11
(六) Ni-NTA親和性管柱純化 11
(七) TEV蛋白酶去除His-tag 11
(八) 膠體過濾純化 12
(九) 蛋白質濃度測定 12
四、 大量表現及純化TEV蛋白酶 12
(一) 轉殖作用(Transformation) 12
(二) 大量表現TEV蛋白酶 13
(三) 破菌 13
(四) Ni-NTA親和性管柱純化 13
五、 純化VcDncV合成c-di-AMP 14
(一) 大量表現VcDncV 14
(二) 純化VcDncV 14
(三) 合成c-di-AMP 14
(四) 計算c-di-AMP濃度 15
六、 製備Proteioliposome及CpaA功能性測定 15
(一) 掃描pyranine激發波長 15
(二) 製備empty liposome 15
(三) 重建目標蛋白至liposome形成proteoliposome 15
(四) 包埋螢光染劑pyranine於proteoliposome內部 16
(五) 銀染確認CpaA proteoliposome 16
(六) 空liposome控制組測試 17
(七) 空liposome加入不同濃度c-di-AMP測試 17
(八) CpaA Proteoliposome稀釋倍率 17
(九) CpaA Proteoliposome運輸功能測定 17
第三章 結果 19
一、 CpaA序列比對的結果 19
二、 構築不含GFP的全長CpaA質體及其突變株 19
三、 純化TEV蛋白酶 20
四、 純化全長CpaA 21
五、 純化VcDncV及合成c-di-AMP的結果 22
六、 Pyranine在不含蛋白的liposome之螢光結果測試 22
(一) Pyranine的螢光測試 23
(二) 空liposome控制組測試 23
(三) 空liposome加入不同濃度c-di-AMP的影響 23
七、 含有全長CpaA的proteoliposome之功能測定 24
(一) 構築包含CpaA之proteoliposome 24
(二) Proteoliposome的稀釋倍率 25
(三) 全長之CpaA wt構築至proteoliposome運輸功能測定 25
(四) CpaA突變株的運輸功能測定 26
第四章 討論 28
一、 CpaA N端之序列分析比對 28
二、 c-di-AMP對於螢光染劑pyranine之影響 28
三、 c-di-AMP對於CpaA轉運效率的影響 29
四、 Future work 30
五、 總結 30
參考文獻 32

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