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標題: | 正負電荷側鏈長度對β-hairpin 的影響及精胺酸側鏈電荷對辨認DNA 的影響 The Effect of Side Chain Length on the Cross-Strand Ion Pairing Interactions in β-Hairpins and Arginine Side Chain Charge on DNA Recognition |
作者: | Min-Fan Tang 楊敏汎 |
指導教授: | 陳平 |
關鍵字: | 摺板結構,髮夾結構,正負電荷作用力,DNA 辨認, β-sheet,β-hairpin,electrostatic interaction,DNA recognition, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 摺板結構為其中一個主要的蛋白質二級結構,主要由strand
間藉由許多穩定的作用力所形成。因此,探討摺板結構的穩定性有助於了解蛋白質的結構。髮夾結構是最常用來探討摺板結構的模型,因為其為最簡單的摺板結構,由兩條β-strand所構成。然而在許多的作用力之中,帶電荷胺基酸彼此之間的靜電作用力,對整 個構形的穩定度有很大的貢獻。在自然界裡面帶電荷的胺基酸,其側鏈長短都有 所不一,因此我們藉由髮夾結構去探討帶電荷胺基酸的側鏈長短,對於兩條strand 之間,正負吸引力的影響以及對於整個髮夾結構的影響。根據2D-NMR 實驗的實 驗顯示,具有較長側鏈的胺基酸可以提供較好正負電荷之間的吸引力還有較高的 結構穩定性。由於正負電荷之間的吸引力不只對蛋白質本身結構的穩定性很重要, 對於辨識DNA 也扮演很重要的角色。因此,我們用真核生物轉錄活化蛋白質GCN4 其上面基本區域(basicregion)的精氨酸側鏈電荷來探討GCN4 上個別精氨酸對於CRE DNA 還有AP-1 DNA 辨識能力的影響•.本研究利用瓜氨酸取代精氨酸來探討電荷對於結合能力及選擇性影響,並且利用電泳技術分析合成之胜肽對於特定DNA 之間作用關係•.本研究顯示WtArgGCN4ss和MinArg11Ass以及MinArg4Ass作為對照組與其不同實驗組對照發現,WtArgGCN4ss 實驗組顯示相對 MinArg11Ass較高的結合能力•.除外,WtCit18GCN4ss表現出相對於對照組以及其他實驗組較差的專一性;WtGCN4Cit24ss 擁有比WtArgGCN4ss 更好的專一性 Min11ACit2ss 表現出相對於對照組(MinArg11Ass)以及其他實驗組較好的專一性•. 本研究利用理論計算的方式模擬合成出的實驗組胜肽與DNA之間的結合情形。 β-Sheet is a very common secondary structure in proteins. A β-sheet consists of twoor more β-strands linked and stabilized by hydrogen bonds between adjacent strands.Moreover, ion pairing interactions between β-strands can stabilize the β-sheetconformation. Interestingly, the natural charged amino acids, which form ion pairs, havedifferent side chain lengths. To investigate how the side chain length affects stability,β-hairpin was used as a model system, because β-hairpins represent the simplest form ofantiparallel β-sheets. Three lateral cross strand ion pairs were investigated: Aad-Orn, Glu-Orn, and Aad-Lys. Peptides with different glutamate and lysine analogs were synthesized by solid phase peptide synthesis using Fmoc-based chemistry: HPTAadOrn,HPTAadLys, and HPTGluOrn, and all purified to at least 95% purity. We alsosynthesized fully folded peptides and unfolded peptides as references. The structure ofthe fraction folded of β-hairpin were analyzed based on the NMR TOCSY, DQF-COSY,NOESY, and ROESY spectra. After assigning the chemical shifts, the secondarystructure of the peptides were confirmed. The folding percentage followed the trend HPTAadLys ≥HPTAadOrn > HPTGluOrn. There was no significant difference between peptides with different lysine analogs. However, the longer side chain gave higherstability for the peptides with different glutamate analogs. This trend revealed that properside chain length of charged residues could promote of β-hairpin formation. Interestingly,the lateral cross strand interaction followed the trend: Aad-Lys > Aad-Orn > Glu-Orn.These two trends are apparently complimentary to one another. The residues with longerside chain length contribute more to the interaction to favor β-hairpin formation. Arg-bearing proteins play important roles in DNA recognition. To study how thearginine side chain charge affects DNA recognition, peptides derived from the GCN4basic region with each individual arginine replaced with a neutral amino acid citrulline(Cit) were investigated. These peptides were synthesized by solid phase peptidesynthesis using Fmoc-based chemistry. Gel shift assays were used to determine thebinding affinity and specificity of the peptides with activator proein-1 (AP-1). TheWtArgGCN4ss mutants showed higher affinity and specificity compared to theMinArg11Ass and MinArg4Ass mutants. WtGCN4Cit24ss showed higher affinity andspecificity compared to WtArgGCN4ss. Molecular mechanic calculations wereperformed to explain the gel shift results. CD spectroscopy indicated that all ourinvestigated peptides remained somewhat helical. The positive charge on Arg243 is very important for binding DNA, mutating other ariginines can modulate and even increase the binding affinity. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64682 |
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