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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 趙玲(Ling Chao) | |
| dc.contributor.author | Ching-Chun Huang | en |
| dc.contributor.author | 黃靖淳 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:51:38Z | - |
| dc.date.available | 2025-08-17 | |
| dc.date.copyright | 2020-08-28 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-18 | |
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Tsay, CHL1 Is a Dual-Affinity Nitrate Transporter of Arabidopsis Involved in Multiple Phases of Nitrate Uptake. The Plant Cell, 1999. 11(5): p. 865. 31. Liu, K.-H. and Y.-F. Tsay, Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation. The EMBO journal, 2003. 22(5): p. 1005-1013. 32. Ho, C.-H., et al., CHL1 Functions as a Nitrate Sensor in Plants. Cell, 2009. 138(6): p. 1184-1194. 33. Sun, J., et al., Crystal structure of the plant dual-affinity nitrate transporter NRT1.1. Nature, 2014. 507(7490): p. 73-77. 34. Harris, D.C., Quantitative Chemical Analysis. 2010, New York: W. H. Freeman. 35. Rashid, M., et al., Adaptive Regulation of Nitrate Transceptor NRT1.1 in Fluctuating Soil Nitrate Conditions. iScience, 2018. 2: p. 41-50. 36. Rashid, M., et al., Feedforward Control of Plant Nitrate Transporter NRT1.1 Biphasic Adaptive Activity. Biophysical Journal, 2020. 118(4): p. 898-908. 37. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78332 | - |
| dc.description.abstract | 作為溝通細胞內外的媒介，膜蛋白參與了許多細胞的生理作用如物質傳輸、受 體結合與訊息傳遞。為了研究膜蛋白的功能，解析膜蛋白結構便成為了首要目標。 目前常用於研究蛋白結構的方法包含X-ray晶體學、蛋白質核磁共振 (NMR) 以及 冷凍式電子顯微鏡 (cryo-EM) 等技術。然而，X-ray晶體學及冷凍式電子顯微鏡無 法在水溶液環境中研究蛋白質的動態變化，而核磁共振雖可達成前述研究，其可分 析的蛋白質大小卻受限於 25kDa 以下。為了突破這些困境，我們利用了共軛焦拉 曼光譜儀來研究被保存在支撐式細胞膜中的膜蛋白，並且在水溶液的環境下檢測 膜蛋白的動態結構變化。我們使用化學發泡技術來從具有大量表達硝酸鹽運輸蛋 白 (CHL1) 的青蛙卵細胞中取得含有該蛋白的巨大細胞囊泡，並將其在基材上形 成支撐式細胞膜。同時，我們也運用了金奈米增強來自膜蛋白的微弱拉曼光譜訊號， 並觀察到硝酸鹽運輸蛋白在不同磷酸鹽濃度環境中 amide I 和 amide III 區域的 特徵峰變化，發現其與硝酸鹽運輸蛋白的磷酸化相關。透過曲線擬合 (curve-fitting) 分析，我們進一步將這些特徵峰變化與蛋白結構變化之間的關聯做出解讀。這些結 果顯示我們所發展的技術不僅提供了非侵入、無標記式研究生物材料的方法，也在 研究膜蛋白結構變化方面具有巨大潛力。 | zh_TW |
| dc.description.abstract | Membrane proteins involve in several cellular processes such as substrate binding, signal transduction and ion transport. However, it is difficult to directly study membrane proteins in native cells due to the complexity of the cell membrane. Although there are some well-developed techniques, such as X-ray crystallography and cryo-electron microscopy (cryo-EM), for studying membrane protein structure, it is still challenging to study the protein dynamic changes after stimulation. In this study, we used confocal Raman spectroscopy to study membrane proteins embedded in supported cell membranes and examined the dynamic changes of protein structures. In order to study the conformational changes of a nitrate transporter, CHL1, we first derived giant plasma membrane vesicles from CHL1-injected Xenopus oocytes and formed supported cell membrane platforms. We also used gold nanoparticles to enhance the weak Raman signals originated from our samples and observed the protein characteristic peak changes in amide I and amide III regions. We found the correlation between the Raman peak changes and the CHL1 phosphorylated state, and interpreted how the peak changes might be correlated to the protein conformational changes. The results show that this technique provides a non-invasive and label-free way to study biomaterials in the native environment and has a great potential in studying conformational changes of membrane proteins. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:51:38Z (GMT). No. of bitstreams: 1 U0001-0308202021213500.pdf: 4888762 bytes, checksum: a0d3781632ce4af8437a53497f6598e6 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員審定書.................................................................... i 誌謝............................................................................ ii 摘要........................................................................... iii Abstract........................................................................ iv Table of Content................................................................ vi Figure Captions.................................................................. x Table Captions.................................................................. xx Chapter 1 Introduction........................................................... 1 1.1 Overview..................................................................... 1 1.2 Giant Plasma Membrane Vesicles (GPMVs)....................................... 4 1.3 Raman Spectroscopy........................................................... 5 1.3.1 Basic theory for Raman scattering.......................................... 6 1.3.2 Molecular vibration........................................................ 7 1.3.3 Characteristic vibrational modes of protein secondary structures.......... 10 1.3.4 Inter- and Intra-molecular Hydrogen Bonds Affect the Amide Vibrations..... 13 1.3.5 Surface-enhanced Raman Spectroscopy (SERS)................................ 14 1.4 CHL1 Membrane Protein Functions as A Dual-Affinity Nitrate Transporter Regulated by Phosphorylation.............................................................. 16 Chapter 2 Materials and Method.................................................. 28 2.1 Materials................................................................... 28 2.2 Apparatus................................................................... 30 2.3 Preparation of Giant Plasma Membrane Vesicles (GPMVs) from Cells............ 32 2.4 Preparation of Large Unilamellar Vesicles for Supported Lipid Bilayer Formation........................................................................32 2.5 Preparation of Polydimethylsiloxane (PDMS) Wells with Flow Channels......... 33 2.6 Preparation of Gold-Nanoparticle-Coated Substrates.......................... 34 2.6.1 Gold nanoparticle synthesis [37].......................................... 34 2.6.2 Pretreatment of glass coverslips.......................................... 35 2.6.3 Gold nanoparticle deposition onto glass coverslips........................ 36 2.7 Formation of Supported Plasma Membrane Patches.............................. 36 2.7.1 Preparation of Solid Substrates........................................... 36 2.7.2 Deposition of GPMVs....................................................... 37 2.7.3 Deposition of Large Unilamellar Lipid Vesicles............................ 37 2.8 Microscopy.................................................................. 38 2.9 Raman Microscopy............................................................ 38 2.10 Raman Data Analysis........................................................ 39 2.10.1 Background subtraction................................................... 40 2.10.2 Curve-fitting............................................................ 41 Chapter 3 Results and Discussion................................................ 42 3.1 Preparation of Supported Cell Membrane Patches ............................. 42 3.1.1 Preparation of Giant Plasma Membrane Vesicles (GPMVs)..................... 42 3.1.2 GPMVs and Fast-DiO Labelled DOPC Deposition............................... 43 3.2 Structural Changes of CHL1 Examined by Raman Spectroscopy................... 44 3.2.1 Observation of the CHL1 Protein Structural Changes During Phosphorylation. 44 3.2.2 Raman spectra of Phosphorylation-mimicking Mutant CHL1-T101D and Phosphorylation-Defective Mutant CHL1-T101A in the Low and High Nitrate Concentration Conditions...................................................................... 47 3.3 Localized Surface-enhanced Raman Spectra Obtained from Non-injected, CHL1-injected and T101A-injected Oocyte membranes.................................... 53 3.4 Curve-fitting Results of Raman Spectra at Amide III and Amide I Region...... 57 3.5 Interpretation of the Observed Raman Spectra................................ 65 3.5.1 The correlation between the observed peak shifts and the phosphorylation of CHL1............................................................................ 65 3.5.2 Change in different states of CHL1 at the low nitrate concentration....... 67 3.5.3 Observation of nitrate transport.......................................... 72 Chapter 4 Conclusion............................................................ 77 References...................................................................... 78 Appendix A...................................................................... 81 Appendix B...................................................................... 84 | |
| dc.language.iso | en | |
| dc.subject | 金奈米 | zh_TW |
| dc.subject | 支撐式細胞膜平台 | zh_TW |
| dc.subject | 膜蛋白 | zh_TW |
| dc.subject | 拉曼光譜學 | zh_TW |
| dc.subject | 無標記檢測 | zh_TW |
| dc.subject | label-free | en |
| dc.subject | membrane protein | en |
| dc.subject | supported cell membrane platform | en |
| dc.subject | gold nanoparticle | en |
| dc.subject | Raman spectroscopy | en |
| dc.title | 利用拉曼光譜檢測支撐式細胞膜中膜蛋白之結構變化 | zh_TW |
| dc.title | Using Raman Spectroscopy to Detect Membrane Protein Structural Changes in Supported Cell Membranes | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蔡宜芳(Yi-Fang Tsay),謝之真(Chih-Chen Hsieh) | |
| dc.subject.keyword | 拉曼光譜學,金奈米,支撐式細胞膜平台,膜蛋白,無標記檢測, | zh_TW |
| dc.subject.keyword | Raman spectroscopy,gold nanoparticle,supported cell membrane platform,membrane protein,label-free, | en |
| dc.relation.page | 86 | |
| dc.identifier.doi | 10.6342/NTU202002319 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-19 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 化學工程學研究所 | zh_TW |
| dc.date.embargo-lift | 2025-08-17 | - |
| 顯示於系所單位： | 化學工程學系 | |
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