碩士 / 國立中興大學 / 生物化學研究所 / 103 / 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.
| Identifer | oai:union.ndltd.org:TW/103NCHU5107074 |
| Date | January 2015 |
| Creators | Xing-Han Sun, 孫倖涵 |
| Contributors | Nien-Jen Hu, 胡念仁 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 67 |
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