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Title: | CueR蛋白質金屬結合域之金屬選擇性 Characterization of the metal specificity of the CueR metal-binding domain by saturation mutagenesis |
Authors: | Yi-Ling Kao 高宜鈴 |
Advisor: | 周信宏(Hsin-Hung Chou) |
Keyword: | 金屬蛋白,金屬蛋白結合親和性,螢光流式細胞分選儀,次世代定序,轉錄因子,CueR,MerR蛋白質家族, metalloprotein,protein-metal binding affinity,fluorescence-activated cell sorting,next generation sequencing,CueR,transcription factor,MerR family, |
Publication Year : | 2018 |
Degree: | 碩士 |
Abstract: | 在生物體中約三成的蛋白質需要與金屬結合才能完整運作,但蛋白質和金
屬結合的化學特性卻從來沒有被完整而深入的研究過。為了探討蛋白質序列對於 金屬的選擇性和親和性,我們結合了突變誘發、細胞分選和次世代定序三種技 術,分析在 CueR 轉錄因子金屬結合域中各個位置的氨基酸變化如何影響其對 銅、銀、金三種金屬的結合性,進而影響其對於下游基因轉錄的能力。我們發現 只要單一的突變,就可以使原本非專一性的 CueR 轉變為對金專一的個體,或是 持續表現不受金屬影響的個體。在未來,我們會探索更多位子的突變,有很大的 可能找到對其他種金屬專一反應的個體或是對金屬更為敏感的個體,並將其運用 在金屬污染的探測之上。 About forty percent of proteins require metals for function, but the chemical basis of protein-metal coordination has not been fully elucidated. To study how the metal affinity and selectivity of a protein were affected by its sequence composition, I applied saturation mutagenesis, fluorescence-activated cell sorting, and next generation sequencing to characterize how residue changes in the metal binding domain of the CueR transcription factor altered its ability to activate gene expression in response to three monovalent metals, Cu+, Ag+ , and Au+ at four different concentrations. I found that a single mutation in the CueR metal-binding domain was sufficient to turn a promiscuous protein into either a Au+-sensitive or a constitutive transcriptional activator. My study shows the great potential of transforming CueR into a highly specific and sensitive metal biosensor valuable to the mining industry or monitoring heavy metals pollution. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21940 |
DOI: | 10.6342/NTU201803632 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 生命科學系 |
Files in This Item:
File | Size | Format | |
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ntu-107-1.pdf Restricted Access | 9.72 MB | Adobe PDF |
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