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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81990完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 周信宏(Hsin-Hung David Chou) | |
| dc.contributor.author | Wen-Yu Hsieh | en |
| dc.contributor.author | 謝文瑜 | zh_TW |
| dc.date.accessioned | 2022-11-25T05:33:41Z | - |
| dc.date.available | 2026-09-03 | |
| dc.date.copyright | 2021-11-12 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-09-09 | |
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Kao YL: Characterization of the metal specificity of the CueR metal-binding domain by saturation mutagenesis. Master Thesis 2018:1-62. 2. He MY, Lin YJ, Kao YL, Kuo P, Grauffel C, Lim C, Cheng YS, Chou HD: Sensitive and specific cadmium biosensor developed by reconfiguring metal transport and leveraging natural gene repositories. ACS Sensors 2021, 6(3):995-1002. 3. Kuo ST, Jahn RL, Cheng YJ, Chen YL, Lee YJ, Hollfelder F, Wen JD, Chou HD: Global fitness landscapes of the Shine-Dalgarno sequence. Genome Res 2020, 30(5):711-723. 4. Korostelev YD, Zharov IA, Mironov AA, Rakhmaininova AB, Gelfand MS: Identification of position-specific correlations between DNA-binding domains and their binding sites. Application to the MerR family of transcription factors. PLoS One 2016, 11(9):e0162681. 5. Pei J, Grishin NV: PROMALS3D: multiple protein sequence alignment enhanced with evolutionary and three-dimensional structural information. Methods in molecular biology 2014, 1079:263-271. 6. 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Ibáñez MM, Cerminati S, Checa SK, Soncini FC: Dissecting the metal selectivity of MerR monovalent metal ion sensors in Salmonella. Journal of Bacteriology 2013, 195(13):3084. 13. Changela A, Chen K, Xue Y, Holschen J, Outten CE, Halloran TV, Mondragón A: Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 2003, 301(5638):1383. 14. Giachino A, Waldron KJ: Copper tolerance in bacteria requires the activation of multiple accessory pathways. Molecular Microbiology 2020, 114(3):377-390. 15. Saulou-Bérion C, Gonzalez I, Enjalbert B, Audinot JN, Fourquaux I, Jamme F, Cocaign-Bousquet M, Mercier-Bonin M, Girbal L: Escherichia coli under ionic silver stress: An integrative approach to explore transcriptional, physiological and biochemical responses. PLoS One 2015, 10(12):e0145748. 16. Kowalsky CA, Klesmith JR, Stapleton JA, Kelly V, Reichkitzer N, Whitehead TA: High-resolution sequence-function mapping of full-length proteins. PLoS One 2015, 10(3):e0118193. 17. Philips SJ, Canalizo-Hernandez M, Yildirim L, Schatz GC, Mondragón A, O'Halloran TV: Allosteric transcriptional regulation via changes in the overall topology of the core promoter. Science 2015, 349(6250):877-881. 18. Lorimer D, Raymond A, Walchli J, Mixon M, Barrow A, Wallace E, Grice R, Burgin A, Stewart L: Gene composer: database software for protein construct design, codon engineering, and gene synthesis. BMC Biotechnology 2009, 9:36. 19. Einav T, Phillips R: How the avidity of polymerase binding to the –35/–10 promoter sites affects gene expression. Proceedings of the National Academy of Sciences 2019, 116(27):13340. ChapterIII. 1. Dudev T, Lim C: Competition among metal ions for protein binding sites: determinants of metal ion selectivity in proteins. Chemical Reviews 2014, 114(1):538-556. 2. Kuo ST, Jahn RL, Cheng YJ, Chen YL, Lee YJ, Hollfelder F, Wen JD, Chou HD: Global fitness landscapes of the Shine-Dalgarno sequence. Genome Res 2020, 30(5):711-723. 3. Xu H, Xu DC, Wang Y: Natural indices for the chemical hardness/softness of metal cations and ligands. ACS Omega 2017, 2(10):7185-7193. 4. Changela A, Chen K, Xue Y, Holschen J, Outten CE, Halloran TV, Mondragón A: Molecular basis of metal-Ion selectivity and zeptomolar sensitivity by CueR. Science 2003, 301(5638):1383. 5. Starr TN, Thornton JW: Epistasis in protein evolution. Protein Science 2016, 25(7):1204-1218. 6. Stoyanov JV, Hobman JL, Brown NL: CueR (YbbI) of Escherichia coli is a MerR family regulator controlling expression of the copper exporter CopA. Molecular Microbiology 2001, 39(2):502-511. 7. Saulou-Bérion C, Gonzalez I, Enjalbert B, Audinot JN, Fourquaux I, Jamme F, Cocaign-Bousquet M, Mercier-Bonin M, Girbal L: Escherichia coli under ionic silver stress: An integrative approach to explore transcriptional, physiological and biochemical responses. PLoS One 2015, 10(12):e0145748. 8. Waldron KJ, Robinson NJ: How do bacterial cells ensure that metalloproteins get the correct metal? Nature Reviews Microbiology 2009, 7(1):25-35. 9. Mazmanian K, Sargsyan K, Lim C: How the local environment of functional sites regulates protein function. Journal of the American Chemical Society 2020, 142(22):9861-9871. 10. Van Rossum G, Drake FL: Python 3 Reference Manual. Scotts Valley, CA: CreateSpace 2009. 11. Brown NL, Stoyanov JV, Kidd SP, Hobman JL: The MerR family of transcriptional regulators. FEMS Microbiology Reviews 2003, 27(2-3):145-163. 12. Korostelev YD, Zharov IA, Mironov AA, Rakhmaininova AB, Gelfand MS: Identification of position-specific correlations between DNA-binding domains and their binding sites. Application to the MerR family of transcription factors. PLoS One 2016, 11(9):e0162681. 13. Coordinators NR: Database resources of the National Center for Biotechnology Information. Nucleic acids research 2016, 44(D1):D7-D19. 14. Pei J, Grishin NV: PROMALS3D: multiple protein sequence alignment enhanced with evolutionary and three-dimensional structural information. Methods in molecular biology 2014, 1079:263-271. 15. Crooks GE, Hon G, Chandonia JM, Brenner SE: WebLogo: a sequence logo generator. Genome Res 2004, 14(6):1188-1190. 16. Parkhill J, Lawley B, Hobman JL, Brown NL: Selection and characterization of mercury- independent activation mutants of the Tn501 transcriptional regulator, MerR. Microbiology 1998, 144(10):2855-2864. 17. Philips SJ, Canalizo-Hernandez M, Yildirim L, Schatz GC, Mondragón A, O'Halloran TV: Allosteric transcriptional regulation via changes in the overall topology of the core promoter. Science 2015, 349(6250):877-881. 18. Ibáñez MM, Checa SK, Soncini FC: A single serine residue determines selectivity to monovalent metal Ions in metalloregulators of the MerR family. Journal of Bacteriology 2015, 197(9):1606. 19. Liu XC, Hu QY, Yang JM, Huang SQ, Wei TB, Chen WZ, He YF, Wang D, Liu ZJ, Wang K, Gan JH, Chen H.: Selective cadmium regulation mediated by a cooperative binding mechanism in CadR. Proceedings of the National Academy of Sciences 2019, 116(41):20398. 20. Stoyanov JV, Brown NL: The Escherichia coli copper-responsive copA promoter is activated by gold. Journal of Biological Chemistry 2003, 278(3):1407-1410. 21. Checa SK, Espariz M, Audero ME, Botta PE, Spinelli SV, Soncini FC: Bacterial sensing of and resistance to gold salts. Molecular Microbiology 2007, 63(5):1307-1318. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81990 | - |
| dc.description.abstract | 細胞中負責感應並調控金屬恆定的蛋白對特定金屬應具高度專一性。然而，前人研究顯示這些感應蛋白常非專一地與多種金屬離子反應。為探究蛋白質對金屬選擇性的原理，我選用大腸桿菌的CueR轉錄因子作為模式系統。CueR蛋白可與一價銅、銀和金離子結合來開啟下游基因表現，我以大規模平行分析法建構十六萬種 CueR金屬結合域變異株並量測它們對三種離子的反應。結果顯示CueR蛋白的反應性主要由金屬結合域的單一胺基酸決定，而涉及脯胺酸的雙位點交互作用則在一價銅與金離子的選擇性上扮演主角。此外，如金屬結合域出現色胺酸會減損CueR蛋白的反應性，使CueR變成不受金屬調控的轉錄活化蛋白，此不良效應為MerR家族的金屬結合域皆缺乏色胺酸提供一項合理的解釋。總結，我的研究顯示演化如何塑造CueR蛋白的金屬結合域及其金屬離子選擇性的設計原理。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T05:33:41Z (GMT). No. of bitstreams: 1 U0001-0309202112463700.pdf: 5528235 bytes, checksum: cf85e3ce1a19b8ad055f187f48a7b43c (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 口試委員審定書 i 致謝 ii 摘要 iii Abstract iv Contents v Figures viii Tables x Chapter I. Introduction 1 References 3 Chapter II. Mapping the CueR MBD sequence-function relationship by massive parallel assays. 6 Introduction 6 Materials and Methods 8 Results 22 2.1 Selecting four phylogenetically diverse residues in CueR MBD for saturation mutagenesis 22 2.2 Determining a suitable metal concentration for massive parallel assays 24 2.3 Fluorescence-activated cell sorting (FACS) successfully partitioned the variant library into multiple ranks in terms of GFP fluorescence 26 2.4 Comparison of massive parallel assays (MPA) revealed the superior variant coverage of the GenScript library 33 2.5 Reliability and reproducibility of MPA of the GenScript library 35 Discussion 38 References 40 Chapter III. Revealing the molecular basis of metal ion reactivity and selectivity through computational analysis 43 Introduction 43 Materials and Methods 44 Results 46 3-1 Comparison of transcriptional activation of each CueR variant under the baseline and metal induction conditions 46 3-2 MBD sequence features contributing to transcriptional activation under the baseline condition 47 3-3 Sequence features associated with low transcriptional activation under metal induction conditions 49 3-4 Metal reactivity of variants under different metal inductions 51 3-5 Sequences features associated with metal reactivity 53 3-6 Correlation between tryptophan content in MBD and metal reactivity 55 3-7 Motif summation method and the explanatory power of order contribution 57 3-8 Investigating the similarity of order 1 effects among metal conditions 59 3-9 Investigating the role of order 2 effects among metal conditions 61 3-10 Impact of copper- and gold-selective pairwise residue combination on metal reactivity 66 Discussion 68 References 74 Chapter IV. Conclusions and future perspectives 78 | |
| dc.language.iso | en | |
| dc.subject | MerR 蛋白家族 | zh_TW |
| dc.subject | 基因合成 | zh_TW |
| dc.subject | 螢光細胞分選 | zh_TW |
| dc.subject | 金屬離子反應性 | zh_TW |
| dc.subject | CueR | zh_TW |
| dc.subject | 金屬蛋白 | zh_TW |
| dc.subject | 次世代定序 | zh_TW |
| dc.subject | 金屬離子選擇性 | zh_TW |
| dc.subject | MerR family | en |
| dc.subject | next-generation sequencing | en |
| dc.subject | fluorescence-activated cell sorting | en |
| dc.subject | gene synthesis | en |
| dc.subject | metal reactivity | en |
| dc.subject | metal selectivity | en |
| dc.subject | metalloprotein | en |
| dc.subject | CueR | en |
| dc.title | 以大規模平行分析揭露 CueR 金屬感應蛋白決定金屬選擇性及反應性之設計原理 | zh_TW |
| dc.title | Massive parallel assays elucidate principles governing the selectivity and reactivity of the CueR metallosensor | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄭貽生(Hsin-Tsai Liu),詹迺立(Chih-Yang Tseng),林小喬 | |
| dc.subject.keyword | 金屬蛋白,CueR,MerR 蛋白家族,金屬離子選擇性,金屬離子反應性,基因合成,螢光細胞分選,次世代定序, | zh_TW |
| dc.subject.keyword | metalloprotein,CueR,MerR family,metal selectivity,metal reactivity,gene synthesis,fluorescence-activated cell sorting,next-generation sequencing, | en |
| dc.relation.page | 79 | |
| dc.identifier.doi | 10.6342/NTU202102973 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-09-10 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生命科學系 | zh_TW |
| dc.date.embargo-lift | 2026-09-06 | - |
| 顯示於系所單位： | 生命科學系 | |
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