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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳平 | |
dc.contributor.author | Hsien-Chen Chang | en |
dc.contributor.author | 張嫺楨 | zh_TW |
dc.date.accessioned | 2021-06-16T23:36:22Z | - |
dc.date.available | 2017-08-07 | |
dc.date.copyright | 2012-08-07 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-07-26 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65320 | - |
dc.description.abstract | 靜電作用力對於穩定蛋白質結構扮演相當重要的角色,主要由帶相反電荷的胺基酸所提供。然而,帶電荷胺基酸雖具有不同側鍊長度之類似物但卻不為自然界生物所利用。為研究側鍊長短對於β-hairpin結構穩定之影響,遂利用固相合成法合成出一系列帶有不同類似物(Agb, Agp及Dap)的胜肽,並以二維核磁共振光譜(TOCSY, ROESY及 DQF-COSY)找出胜肽鏈中各殘基氫原子之化學位移。由各殘基α氫原子化學位移之偏移,3JHNα偶合常數及NOE效應,可確認胜肽分子所形成之β-hairpin結構。比較所合成之胜肽與控制組胜肽中各殘基α氫原子化學位移差異可計算出胜肽分子之摺疊比例與自由能。β-hairpin跨股側向作用力之能量大小以雙突變循環分析,結果顯示:離子對Asp-Agb與Asp-Agp有相近的能量,而離子對Asp-Dap較Asp-Agp能量高。側鍊長短對於離子對Arg-Asp作用力沒有影響,而無胍基之胜肽具有較強之離子對作用力。
Tat蛋白與TAR RNA之結合對於人類免疫缺陷病毒基因組複製是不可或缺的。Tat蛋白上47-57位置是兩者專一性結合的區域,此區域上具有六個Arg,對於TAR RNA辨認相當重要。將此六個Arg分別取代成Lys,利用電泳偏移分析Tat衍生物對TAR RNA之結合能力與專一性,結果顯示,將Arg取代成Lys時,Tat與TAR RNA之結合能力降低。而Tat -Lys53在polydIdC或tRNA存在下,專一性較正常態大為降低,顯示Arg 53對於Tat蛋白與TAR RNA之專一性結合影響重大。 | zh_TW |
dc.description.abstract | Electrostatics is important for protein structure stability. The interaction can form between oppositely charged residues. Interestingly, the natural charged amino acids, Arg, Lys, Glu, and Asp all have analogs with alternative side chain lengths. However, these analogs are not used in natural proteins. In order to study the effect of side chain length on β-hairpin stability, peptides HPTAspXaa, containing different charged amino acid analogs Agb, Agp and Dap, were synthesized by solid phase methods. The sequence specific assignments were based on TOCSY, ROESY, and DQF-COSY spectra. The hairpin structures were confirmed by chemical shift deviation, 3JHNα coupling constants and NOE signals. The fraction folded and delta Gfold of peptides were derived by comparing the chemical shifts with the folded and unfolded reference peptides. The double mutant cycles were applied to analyze the cross strand lateral Asp-Xaa interaction energy. The Asp-Agb and Asp-Agp have similar interactions energy in β-hairpins, suggesting the different side chain lengths of Arg analogs do not affect ion-pairing interactions with Asp. The Asp-Dap pair was more stabilizing than the Asp-Agp pair in β-hairpins, which may be due to the charge dispersion of the guanidinium group on Agp.
HIV Tat protein and TAR RNA binding is an essential step for HIV-1 virus genome replication. A short region (residues 47-57) in the Tat protein binds to the TAR RNA specifically. This region contains six Arg residues, which are important for TAR RNA recognition. The Arg in Tat peptide was individually replaced with Lys to study the effect of the guanidinium group on TAR RNA recognition. The binding affinity of all Tat-derived peptides decreased, when introducing Lys. The KD of Tat-Lys53 increased in the presence of polydIdC and tRNA showing that the guanidinium group at Arg53 is particularly important for RNA recognition. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T23:36:22Z (GMT). No. of bitstreams: 1 ntu-101-R99223211-1.pdf: 2451367 bytes, checksum: f2359a959c2881da7c412693f39d1d2d (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 中文摘要 iii Abstract iv Table of Contents vi List of Figures x List of Tables xiii Abbreviations xv Chapter 1. Introduction 1 1-1 Proteins 1 1-2 Protein Structure and Function 2 1-3 Four Levels of Protein Structure 3 1-4 Dominant Forces for Protein Folding 7 1-5 Ion Pairing Interaction in Hyperthermophilic Proteins 9 1-6 Charged Amino Acids 10 1-7 Electrostatic Interactions in RNA Recognition 12 1-8 Thesis Overview 15 1-9 References 17 Chapter 2. 23 2-1 Introduction 23 β-Sheet 23 β-Hairpin 24 β-Turn 27 β-Sheet Propensity 30 Cross Strand Interactions 32 Effect of Side Chain Length on Cross Strand Electrostatics 36 Quantitative Methods 37 Double Mutant Cycle 39 2-2 Results and Discussion 40 Peptide Sequence Design 40 Synthesis of Fmoc-Agp(Boc)2-OH 41 Peptide Synthesis 43 Peptide Structure Analysis by 2D-NMR Spectrometry 45 Sequence Specific Assignment 46 Chemical Shift Deviation 51 3JHNα Coupling Constant 53 Nuclear Overhauser Effect (NOE) 57 Hairpin Stability 62 Interaction Energy 64 2-3 Discussion 65 2-4 Conclusions 66 2-5 Acknowledgement 67 2-6 Experimental Section 67 General Materials and Methods 67 Peptide Synthesis 71 NMR Spectrometry Analysis 80 Chemical Shift Deviation 91 3J HNα Coupling Constant 91 NOE Signal Integration for Distance Determination 91 Fraction Folded and delta G fold of the Hairpin Peptides 92 Interaction Energy of the Hairpin Peptides 93 2-7 References 94 Chapter 3. 96 3-1 Introduction 96 Ribonucleic acid (RNA) 96 Human Immunodeficiency Virus (HIV) 98 Trans-Activation Response Element (TAR) RNA 99 Trans-Activator of Transcription (Tat) Protein 100 Tat-Activated Transcription 101 Inhibition of Tat-TAR Interactions 103 Charged Amino Acids in Tat-TAR RNA Interactions 103 3-2 Results and Discussion 106 Peptide Design and Synthesis 106 Gel Shift Assay in the Absence of Poly-dIdC 108 Gel Shift Assays in the Presence of Poly-dIdC 113 Gel Shift Assays in the Presence of Bulk E. coli tRNA 119 Tat-Derived Peptide Binding Affinity and Specificity Towards TAR RNA 125 3-3 Conclusion 129 3-4 Acknowledgement 130 3-5 Experimental Section 131 Peptide Synthesis 131 Ultraviolet-Visible (UV-vis) Spectroscopy 135 Gel Shift Assay 136 3-6 References 139 | |
dc.language.iso | en | |
dc.title | 精胺酸側鏈長短與胍基對β-Hairpin結構穩定度及核糖核酸辨認之影響 | zh_TW |
dc.title | Effect of Arginine Side Chain Length and Guanidinium Group on β-Hairpin Stability and RNA Recognition | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳佩燁,黃人則 | |
dc.subject.keyword | 靜電作用力,帶電荷胺基酸,β-sheet,β-hairpin,RNA辨認, | zh_TW |
dc.subject.keyword | electrostatics,charged amino-acids,β-sheet,β-hairpin,RNA recognition, | en |
dc.relation.page | 142 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-07-26 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 化學研究所 | zh_TW |
顯示於系所單位: | 化學系 |
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