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標題: | 瓜胺酸側鏈長度在β-髮夾穩定性的影響及 標靶愛滋病毒相關核醣核酸的胜肽研究 Effect of Citrulline Side Chain Length on β-Hairpin Stability, and Towards Targeting HIV-Related RNA motifs |
作者: | Po-Yi Wu 吳柏宜 |
指導教授: | 陳平(Richard P. Cheng) |
關鍵字: | 瓜胺酸化,β-折板,β-髮夾,人類免疫缺乏病毒, Citrullination,β-Sheet,β-Hairpin,HIV, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 去亞胺化又稱瓜胺酸化是一種發生在生物體內的後轉譯修飾。將精胺酸轉換成瓜胺酸。此種修飾可以提升親油性和改變氫鍵的網絡,而改變蛋白質的結構、增進蛋白質的折疊及穩定性。除了瓜胺酸外,有三個與之相似的衍生物,差別與側鏈上亞甲基的數目。因此,我們利用二維的核磁共振光譜技術來探討瓜胺酸側鏈長度對於β-折板的穩定性。此研究結果可被利用來調節β-折板的穩定性進而發展β-髮夾的材料。 愛滋病仍然是21世紀人類為無法治療的疾病,造成愛滋病最常見的即是人類免疫缺乏(HIV-1)病毒。Rev responsive element (RRE) 位於在病毒的5’LTR基因組裡。Rev蛋白質會與RRE RNA結合來調控基因表現。一旦Rev胜肽和RRE RNA結合,Rev會從random coil結構變成α-螺旋。氫鍵替代物是一種用於穩定α-螺旋跨連結系統,藉由共價鍵C=C-C-N取代一般常見的(i,i+4) C=O…C-N氫鍵來穩定短的α-螺旋。預期鎖螺旋Rev胜肽親和性會高於自然的Rev,競爭RRE RNA以抑制調控基因表現。本篇成功合成出目標產物。藉由電泳偏移分析Rev胜肽對於RRE RNA的結合親和力,結果顯示鎖螺旋Rev的解離常數比自然的Rev胜肽的高相當多。此外,利用圓二色光譜鑑定其二級結構。 在人類免疫缺乏病毒Tat蛋白質中的一段胜肽序列:Tat(47-57)具有與TAR RNA結合能力來調控基因轉錄。在我們先前的研究當中,以Tat(47-57)當模組優化了一種胜肽TatC4,TatC4是同時具有與TAR RNA高的結合力及高抗胰蛋白酶(trypsin)水解能力。但在高濃度tRNA的競爭者下(65 μg/mL),與TAR RNA的結合力大幅下降了,顯示其專一性的不足。預測原因可能是胜肽過多的正電荷與核內其他帶負電物質產生吸引力,於是選擇性在各個位子上胍基上做去亞胺化,移除正電荷,探討移除哪個位子上的正電荷可提升TatC4的專一性。結果顯示不同位置的修飾所造成的親和力下降程度不同,對未來專一性的研究提供了有用的知識。 Citrullination is the term used for the post-translational modification of arginine to citrulline. Citrullination is also called deimination due to the conversion of a guanidium group to a urea group. This PTM increases hydrophobicity and alters the hydrogen bonding profile, leading to changes in protein structure, folding, and stability. Citrulline and three citrulline analogs with different side chain lengths: (S)-2-amino-6-ureido-hexanoic acid (Auh), (S)-2-amino-4-ureido-butyric acid (Aub), and (S)-2-amino-3-ureido-propionic acid (Aup), were studied. We investigated citrulline and its analogs in model peptides to study the effect of arginine deimination on β-sheet stability by 2D-NMR spectroscopy. The results showed that increasing the citrulline side chain length slightly affects the folded population except for Auh. The peptide with the longest side chain length (Auh, four methylenes) showed the highest folded population. This may be useful for modulating sheet stability and for developing β-hairpin based materials. Acquired immunodeficiency syndrome (AIDS) remains an incurable disease in the 21st century. Rev response element (RRE), located on the 5’LTR, is responsible for the function of the Rev protein in trans to modulate gene expression. The conformation of the Rev peptide converts from a random coil to an α-helix, upon forming the RRE RNA-Rev peptide complex. Hydrogen bond surrogate is a covalent system to stabilize the α-helix. In this system, the C=C-C-N covalent bond replaces the hydrogen bond between the ith carbonyl and the i+4th amine to form a stable short helix. This constrained helix may provide higher affinity compared to wild-type Rev to disrupt gene corresponding expression. In this study, the desired cyclized Rev peptide was synthesized. The dissociation constant (KD) for binding RRE RNA was determined by electrophoretic mobility shift assays (EMSA). The KD of the cyclized Rev peptide for bind RRE RNA was higher than wild-type Rev peptide. Moreover, the cyclized Rev peptide showed lower helicity compared to wild-type Rev by circular dichroism (CD) spectroscopy. There are 11 amino acids in the arginine-rich domain of Tat protein (human immunodeficiency virus transactivator of transcription protein, residues 47-57). The corresponding Tat peptide binds to the transactivator response element (TAR) RNA. In our previous study, TatC4 was designed based on Tat47-57. TatC4 exhibited both higher affinity and higher resistance to trypsin proteolysis. However, at high concentrations of tRNA (65 μg/mL), the affinity of TatC4 for TAR RNA decreased dramatically. This indicated low binding specificity of TatC4 for TAR RNA, suggesting undesired attractive interactions between the high density of positively charged side chains and non-target negatively charged compounds. To improve the binding specificity of TatC4, deiminated side chains were explored. The result showed varying degrees of decreased affinity with different positions compared to TatC4. Although a further optimized sequence was not revealed, this knowledge should contribute to further development of Tat peptides for specific TAR RNA binding. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53901 |
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