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標題: | 側鏈長度對離子對作用力在β-Hairpin中及對離胺酸側鏈長度對Tat辨認核糖核酸的影響 Effect of Side Chain Length on Ion-Pairing Interaction in β-Hairpins and on RNA Recognition for Tat47-57 Derivatives |
作者: | Shing-Lung Liu 劉興龍 |
指導教授: | 陳平 |
關鍵字: | β-摺板,β-髮夾,離子對作用力,人類免疫缺乏病毒,核糖核酸辨認,電泳位移分析, β-sheet,β-hairpin,ion pairing interaction,HIV,RNA recognition,gel shift assay, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | β-摺板在蛋白質中是一個很重要的結構,而它的結構能夠被側向交叉股離子對作用力所穩定。離子對作用力是由兩個正負電荷相反的胺基酸交互作用而形成的。然而,在自然界帶電荷的胺基酸中,它們具有不同長度的側鏈。為了去探討這些不同長度的側鏈對β-摺板中側向交叉股離子對作用力的影響,我們決定用β-髮夾當作我們研究的對象,因為β-髮夾是β-摺板中最簡單的單元。β-髮夾是由兩條反平行的β-摺板,中間連接著一個β-回轉所組成的。我們利用β-髮夾的結構,去探討Asp-Agh、Glu-Agh、Aad-Agh間作用力對β-髮夾結構穩定度的影響。我們所設計的β-髮夾是有12個殘基的胜肽,它的β-回轉是由D-Pro-Gly所組成的。當D-Pro被它的鏡像異構物L-Pro替換時,整個胜肽的結構會被破壞,進而可以當作是完全沒有摺疊的狀態。當引用了兩個Cys在胜肽的兩端,進行氧化形成雙硫鍵,使整個胜肽被環化並且強制使它的結構進行摺疊,而可以視為是具有完全摺疊的狀態。我們利用固相胜肽合成,並用高效能液相層析儀來進行純化。完成純化後,我們利用核磁共振儀來分析胜肽的結構。胜肽中各個胺基酸α氫原子的化學位移被我們指派。β-髮夾結構的程度和自由能是由α氫原子的化學位移所計算得到。而β-髮夾的結構也由3JNHα 偶合常數、NOE訊號和Wüthrich圖表所判斷。離子對作用力的貢獻則是用雙突變循環所計算出來。研究的結果指出,當負電荷的側鏈長度最長時,對β-髮夾結構貢獻的穩定能量就越多。
人類免疫缺乏病毒中的鹼性區域Tat47-57對核糖核酸的辨認很重要。當Tat蛋白辨認核糖核酸的TAR,人類免疫缺乏病毒就會進行增生,並且造成人類免疫缺乏症候群。在精胺酸豐富的Tat47-57中,存在著兩個和精胺酸帶不同官能基的離胺酸。為了探討這兩個離胺酸的側鏈長度對核糖核酸辨認的影響,我們將離胺酸替換成長度較短的離胺酸類似物Dap、Dab和Orn。Tat47-57衍生物對核糖核酸辨認的專一性,是由加入了poly-dIdC或tRNA的條件下進行電泳位移分析實驗來得到。實驗的結果指出,對於核糖核酸的辨認,51號位置的離胺酸比50號位置的離胺酸還要重要。而不管有沒有加入poly-dIdC,Tat衍生物對核糖核酸的辨認都沒有很明顯的差異。但是當加入了tRNA,Tat衍生物對核糖核酸的辨認就下降了。在這些衍生物當中,50號位置改為Dap的Tat衍生物在poly-dIdC或tRNA的存在下,都有很好的核糖核酸辨認能力 β-Sheet is one of the important structures in proteins. The structure of β-sheets can be stabilized by lateral cross-strand interactions. In particular, ion pairing interactions between two oppositely charged amino acids can contribute to β-sheet stability. Interestingly, natural charged amino acids have different number of methylenes in the side chain. To investigate the effect of side chain length on lateral cross-strand ion pairing interactions in β-sheets, we decided to study β-hairpins, which are the simplest unit in β-sheets. β-Hairpins which are composed of two antiparallel β-strands connected by a β-turn were used to investigate the effects of Asp-Agh, Glu-Agh, and Aad-Agh ion pairing interactions on the β-hairpin structure stability. The β-hairpins we designed were 12-residue peptides using the DPro-Gly segment as the β-turn. Replacing DPro with LPro abolished the β-hairpin conformation and was regarded as the unfolded state. Disulfide bonds formed by cysteines were used for cyclization to enforce the β-hairpin formation. Peptides were synthesized by solid phase peptide synthesis, purified by HPLC, and were analyzed by NMR spectroscopy (TOCSY, ROESY, NOESY, DQF-COSY). The chemical shifts of the protons were assigned sequence specifically. The β-hairpin population and the free energy of folding were derived from the chemical shifts of the α-protons. The structure and the side chain interactions of the β-hairpin were characterized by the 3JNHα coupling constants, NOEs, and Wüthrich diagrams. The energetic contribution of the ion pairing interactions was derived from the double mutant cycle. The results showed that the ion pairing interaction of the longest negatively charged amino acid contributes the highest stabilizing free energy to the β-hairpin structure. The basic region of the human immunodeficiency virus (HIV) Tat protein (Tat47-57), which binds the trans-activation responsive region (TAR) RNA, is crucial for the proliferation of HIV-1 virus, which causes acquired immune deficiency syndrome. In the arginine-rich Tat47-57 peptide, there are two Lys residues which have different functionalities compared to Arg. To study the effect of side chain length of these Lys residues on binding TAR RNA, the Lys residues were replaced with Dap, Dab, and Orn, which are Lys analogs with different number of methylenes in the side chain. The RNA binding specificity for the Tat-derived peptides were studied by gel shift assays in the presence of poly-dIdC or tRNA. The results showed that the Lys at position 51 was more important for RNA recognition than position 50 in the basic region of Tat peptide. The presence of poly-dIdC had almost no effect on the binding affinity of the Tat-derived peptides, but tRNA did. The Dap mutant at position 50 had the highest affinity to TAR RNA in the presence of tRNA. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65395 |
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