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標題: | 後轉譯修飾對胜肽結構與功能的影響 Effect of Post-Translational Modifications on Peptide Structure and Function |
作者: | Yun-Chiao Yao 姚云喬 |
指導教授: | 陳平(Richard P. Cheng) |
關鍵字: | 胜?,後轉譯修飾, peptide,post-translational modification,PTM, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 「後轉譯修飾」為發生在蛋白質轉譯之後的化學修飾,後轉譯修飾影響蛋白質的結構以及功能,增加了生物體的複雜度。在此篇論文中,我們探討後轉譯修飾對胜肽結構與功能的影響,包含磷酸化對α-螺旋和β-摺板結構之影響、醣化對β-摺板結構之影響、以及改變側鏈長度的瓜胺酸對Tat胜肽辨認核醣核酸與細胞穿透之影響。 磷酸化是最常見的後轉譯修飾,參與如細胞訊息傳遞等生物功能。磷酸化在絲胺酸及蘇胺酸的側鏈加上一個磷酸基團,隨酸鹼值變化可能攜帶零到二個負電荷。我們利用設計好的胜肽系統,研究磷酸化之絲胺酸和蘇胺酸對於α-螺旋和β-摺板結構穩定性的影響。研究顯示,磷酸化讓α-螺旋和β-髮夾的穩定度下降,只有當磷酸化發生在α-螺旋的N端時,才具有特別的穩定效果。 醣化蛋白質參與蛋白質的正確摺疊以及膜蛋白辨認等生物功能,可分為作用在天門冬醯胺的氮連接醣化和作用在絲胺酸和蘇胺酸的氧連接醣化。我們將接上單醣的天門冬醯胺、絲胺酸、和蘇胺酸置入設計好的β-髮夾胜肽系統中,研究醣化對穩定度的影響。根據結果,β-髮夾的穩定性不受氮連接醣化影響,而氧連接醣化則因與單醣連接的α/β位向不同而影響不同。 瓜胺酸化會將精胺酸的胍基置換為尿素基團,改變胺基酸的電荷及氫鍵網絡。我們將瓜胺酸及其改變側鏈長度的衍生物置入HIV-1 Tat(47-57)胜肽中,取代自然界Tat中52、53或55位置的精胺酸,研究其對Tat之TAR RNA辨認能力和細胞穿透能力的影響。研究結果顯示,比起自然界Tat胜肽,任一側鏈長度的瓜胺酸化都使Tat與TAR RNA的結合能力下降,也使細胞穿透能力下降。而Tat之瓜胺酸化對RNA辨認及細胞穿透的影響程度與位置相關,側鏈長短則沒有特別趨勢。 Post-translational modifications (PTMs) are chemical modifications of a protein after its translation. PTMs broaden the range of protein functionalities. Phosphorylation, glycosylation, and citrullination are PTMs that play key roles in various biological processes. Protein structure affects protein function. As such, the structural effects of these PTMs are important for understanding the corresponding biological processes at the molecular level. In these studies, we investigated the effect of phosphorylation on α-helix and β-sheet, the effect of glycosylation on β-sheet, and the effect of citrulline side chain length on RNA recognition and cellular uptake for Tat derived peptide. Phosphorylation is the most common PTM. The conversion of a neutral hydroxyl side chain to a pH dependent negatively charged phosphate group may be a useful conformational switch in protein domain restructuring. Many cell signal transductions and enzyme activations are based on protein phosphorylation. Focusing on structural effects, phosphorylation of Ser and Thr reduced both α-helix and β-sheet stability, except at the N-cap position of a helix. It has been estimated that half of all proteins are glycoproteins. One major function of glycosylation is to provide additional recognition epitopes for protein receptors. In addition, glycosylation is related to the mechanism of protein folding and transcriptional control. Glycosylation can be divided into N-glycosylation on the amide nitrogen of Asn, and O-glycosylation on the hydroxyl oxygen of Ser or Thr. In this research, the effect of glycosylation on β-sheet stability was investigated using amino acids attached with monosaccharide. The hairpin stability of the Asn-containing peptide remained the same upon glycosylation, suggesting that N-linked glycosylation has minimal effect. In the contrast, the effect of O-linked glycosylation on β-hairpin stability depended on glycosylation type (the stereochemistry at the anomeric carbon). Citrullination converts a guanidinium group into a urea group, removing the positive charge and changing the hydrogen bonding pattern. We used the HIV-1 Tat peptide as a model to examine the effect of citrulline side chain length on peptide function. There are two functions of the Tat peptide: TAR RNA recognition and cellular uptake. Upon introducing citrulline analogs in positions 52, 53, or 55 at Tat peptides, the results showed that the effect of citrullination was position dependent and that the effect of different side chain lengths was not significant. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54002 |
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