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標題: | 甲基化賴胺酸對α螺旋與β折板結構穩定度及對核糖核酸辨識與細胞穿透之影響 Effect of Lysine Methylation on α-Helix Propensity, β-Sheet Propensity, RNA Recognition, and Cell Penetration |
作者: | Mu-Chun Liu 劉牧群 |
指導教授: | 陳平(Richard P. Cheng) |
關鍵字: | 甲基化,賴胺酸,α螺旋,β折板,核糖核酸辨識,細胞穿透, lysine,methylation,α-Helix,β-Sheet,RNA Recognition,Cell Penetration, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 轉譯後修飾在蛋白質行為上的影響非常重要,而賴胺酸的甲基化是其中一種非常常見的範例,甲基化可能會造成蛋白質結構上的變化以及功能上的活化或抑制。賴胺酸甲基化在生物體中有三種形式:單甲基、雙甲基和三甲基,不同程度的甲基化可能對蛋白質也有不同層級和效果的影響。在結構方面,三種甲基化賴胺酸被置入於兩種基本二級結構的胜肽模板中來研究其對二級結構穩定度的影響:α-螺旋和β-折板。圓二色光譜儀用來測量胜肽的螺旋程度,而折板的結構資訊則使用二維核磁共振光譜來分析。
於功能方面,本論文則是探討於人類免疫缺乏病毒的感染過程中非常重要的調控蛋白:Tat。此蛋白中存在著一段富含正電荷胺基酸的區域(RKKRRQRR)並且其已被研究出可以高選擇性地與特定RNA結構結合藉以大幅度提高病毒蛋白的表現量。這段非常重要的序列同時也賦予Tat蛋白穿透細胞膜的能力來入侵其他細胞。而賴胺酸的甲基化被證實與此蛋白的活化和抑制息息相關。因此,我們合成這段胜肽並在50和51號分別置入三種甲基化賴胺酸來研究其對辨認特定RNA以及穿透細胞能力的影響。與RNA之間的解離常數由膠體電泳計算,而穿透細胞的能力則使用流式細胞儀來進行測試。 Post-translational modification dominates many protein behaviors. Methylation of lysine impacts both protein function and structure. There are three variations of methylated lysines that were identified in proteins. It is logical to assume that different numbers of methyl groups attached onto the side chain amino group should have different degrees of effects on proteins. In this study, various types of methylated lysines are placed into two basic secondary structures: the α-helix and the simplest β-sheet model “β-hairpin”, to investigate the effect of lysine methylation on structural stability. The fraction helix of the helical peptides was determined by circular dichorism spectroscopy. The structural information of the hairpin peptides was analyzed by 2D NMR. Lysine methylation also plays an important part in many biological processes. The regulatory Tat protein contains a basic region (RKKRRQRRR, residue 49 to 57) which specifically binds to the trans-activating responsive (TAR) element to modulate HIV-1 RNA transcription. The binding between HIV-1 Tat protein and TAR RNA is essential for HIV-1 virus to efficiency produce full-length viral RNA. To study the effect of lysine methylation on RNA recognition and cellular uptake, two lysine residues Lys50 and Lys51 were replaced with monothylated, dimethylated, and trimethylated lysines. The dissociation constant for the Tat derived peptide-TAR RNA complexes was determined by gel shift assay. The cellular uptake efficiency of Tat derived peptide into Jurkat cell was assessed by flow cytometry. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5213 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 化學系 |
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