碩士 / 國立中興大學 / 生物化學研究所 / 105 / 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.
| Identifer | oai:union.ndltd.org:TW/105NCHU5107014 |
| Date | January 2017 |
| Creators | Ciou-Ting Wang, 王秋婷 |
| Contributors | 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 | 53 |
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