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  • 學位論文

合成含2-氧基組胺酸的胜肽並鑑定其交互作用蛋白質與結合模體

Synthesis of 2-oxohistidine-containing peptides and identification of their interacting proteins and binding motifs

指導教授 : 戴桓青

摘要


老化和神經退化等疾病皆與蛋白質的氧化極為相關。在生物體中,活性氧化物質的存在是導致蛋白質氧化的主因。在蛋白質上,與金屬離子螯合能力佳的組胺酸為常見的氧化傷害目標之一。近期研究中發現,組胺酸氧化所形成的產物為2-氧基組胺酸,雖然其物理性質和生化特性尚未被了解透徹,但有機會作為蛋白質氧化受損的生物標記物。 我們發展了能有效合成胜肽中含有2-氧基組胺酸殘基方法,並且將合成的三條含有2-氧基組胺酸殘基的胜肽接上螢光染料作為蛋白質微陣列晶片分析E. coli K12蛋白質體的探針。我們的目標是找出在細胞中能辨識2-氧基組胺酸殘基的潛在蛋白。利用三條胜肽探針、螢光偏振和解離常數的測量成功驗證十個交互作用的蛋白。這十種蛋白質中有九個似乎參與細胞的氧化還原相關功能。我們亦利用生物資訊分析得到可能的結合模體位置。並以生物資訊來預測出人體蛋白質體中也存在具有相似結合模體之蛋白為S100A1。後續也驗證了S100A1確實能與三條含有2-氧基組胺酸殘基胜肽之間產生交互作用。存在細菌及人類蛋白質中的可能結合模體恰好皆為裸露在蛋白質外部的一段α螺旋,這意味著該模體有機會與其它蛋白質相互作用。 為了後續證明蛋白質中的結合模體與2-氧基組胺酸殘基之間交互作用的真實性,我們合成了hemE和yqjG這兩個蛋白質的螺旋構型結合模體胜肽,並對於模體胜肽和2-氧基組胺酸分別接合上螢光共振能量轉移螢光對以進行分子之間的交互作用分析。結果顯示這兩種胜肽之間並沒有交互作用產生,而圓二色光譜顯示模體胜肽之二級結構無形成α螺旋,可能為交互作用失敗的主因。結合區域亦可能不僅是一小段α螺旋,因此我們尚未得知此交互作用所需要的結構條件。

並列摘要


Protein oxidation has been associated with aging and neurodegenerative disorders. Histidine is a major target for metal-catalyzed oxidation due to its metal chelating property. 2-Oxohistidine, the major products of histidine oxidation, respectively, have been recently identified as markers of oxidative damage in biological systems, but their biophysical and biochemical properties are understudied. We developed efficient methods to generate 2-oxohistidine side chains, from peptides. The 2-oxohistidine-containing peptides were conjugated with DyLight fluorophores to make probes for protein microarray analysis against E.coli proteomes. Our goal is to identify cellular proteins that potentially interact with oxidized peptides. Ten interacting proteins were successfully validated using a three peptide probes, fluorescence polarization assay as well as binding constant measurements. We discovered that 9 out of 10 identified proteins seemed to be involved in redox-related cellular functions, and we found a putative consensus binding motif by bioinformatic analysis. A similar binding motif was also identified on human protein S100A1 by bioinformatic prediction. And we validated that S100A1 could interact with all three 2-oxohistidine containing peptides that we synthesized. These putative binding motifs in both bacteria and human were located within α-helices and faced the outside of proteins, which meant that they had the chance to interact with the other proteins. To identify the interaction between consensus binding motifs and 2-oxohitidine peptide, helical binding motifs from hemE and yqjG were synthesized as individual peptide probes. Binding motif peptides and 2-oxohistidine peptides were labeled with different fluorophores to construct FRET pairs for molecular interaction assays. However, there were no detectable interactions by FRET assays, and CD spectra showed a lack of alpha helical content in the secondary structure of binding motif peptides, which may be the main reason for non-interaction. It may suggest that the functional binding domain was probably larger than just a single helix, and we still do not understand its structural requirements.

參考文獻


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