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Title: | 正電荷胺基酸側鏈長短在膠原蛋白X位置及Y位置對於穩定度的影響 Effect of Positively Charged Amino Acid Side Chain Length at the X Position and the Y Position on Collagen Triple Helix Stability |
Authors: | Yan-Chen Chen 陳彥臣 |
Advisor: | 陳平 |
Keyword: | 膠原蛋白,帶電荷胺基酸,圓二色性光譜儀,熱分解與熱重組,CD47,訊息控制蛋白α, collagen,charged amino acid,circular dichroism spectroscopy,thermal denaturation/ renaturation,CD47,SIRPα, |
Publication Year : | 2018 |
Degree: | 碩士 |
Abstract: | 膠原蛋白是人體內最重要的蛋白質,用來連接、支撐人體的物質。帶電荷胺基酸在膠原蛋白裡出現的頻率很高,且超出預期。在膠原蛋白鏈上的帶電荷胺基酸能用靜電作用力彼此吸引或排斥。本篇聚焦在精胺酸、賴胺酸及其衍生物在膠原蛋白的X位置及Y位置的影響。精胺酸(Arg)的衍生類似物有三個,分別是側鏈較多一個亞甲基的Agh、少一個亞甲基的Agb以及少兩個亞甲基的Agp;賴胺酸(Lys)的衍生類似物有三個,分別是側鏈少一個亞甲基的Orn、少兩個亞甲基的Dab以及少三個亞甲基的Dap。本篇以ABC異元三聚體來當作基礎設計膠原蛋白,在合成上,以Fmoc保護基的固相胜肽合成,將膠原蛋白純化後,利用圓二色性光譜儀(CD)來量測膠原蛋白平衡狀態的熱分解與熱重組,其數據可以推導出膠原蛋白非摺疊的熱力學參數。結果顯示出,在X位置引入賴胺酸及其衍生類似物的膠原蛋白穩定度的趨勢為Dab>Orn>Lys>Dap;在X位置引入精胺酸及其衍生類似物的膠原蛋白穩定度趨勢為Agh>Arg>Agb>Agp;在Y位置引入精胺酸及其衍生類似物的膠原蛋白穩定度趨勢為Agb>Arg>Agh>Agp。這些結果顯示出帶電荷胺基酸的側鏈長短對於膠原蛋白穩定度的影響,將可以在往後設計膠原蛋白上有提供些貢獻。
CD47膜蛋白在人類紅血球表面有表現,並且可以與訊息控制蛋白α (SIRPα)做結合產生一個雙向的訊息傳導,使得抑制脾臟或紅髓巨噬細胞的吞噬。CD47膜外區域上的FG loop會插入SIRPα膜外區域的凹槽來與SIRPα做結合,其中CD47的FG loop含有許多帶電荷胺基酸。利用修飾過後的CD47片段(已修飾的FG loop,Self,SelfL,SelfH,SelfHL與類似物)來探討不同帶電荷胺基酸的側鏈長短對於CD47與SIRPα的親和力的影響。FG loop的谷胺酸(Glu)與精胺酸(Arg)被置換成側鏈較長或較短的類似衍生物,在序列101位置的亮胺酸(Leu)被置換成D-脯胺酸(D-Pro)以限制住轉彎(turn)的形狀,在序列104位置的谷胺酸被置換成其衍生類似物Aad以提升β摺版的傾向。CD47與SIRPα的親和力將會用分裂螢光素酶法(Split-luciferase assay)來量測。CD47與SIRPα的結合作用將來可以應用在癌症或其他疾病的藥物上,作為阻擋吞噬作用的用途。 Collagen is the most abundant protein in the human body. Collagen is the substance that holds the whole body together. Charged amino acids appear more frequently than expected in collagen triple helices. Strands with charged amino acids form electrostatic interactions with other strands with charged amino acids. This thesis focuses on arginine (Arg) and Arg analogs with longer (Agh) and shorter side chains (Agb and Agp) at the Xaa and Yaa positions, lysine (Lys) and Lys analogs with shorter side chains (Orn, Dab, and Dap) at the Xaa position. The collagen triple helices were designed based on ABC heterotrimers. All peptides were synthesized by Fmoc-based solid phase peptide synthesis. After purification, thermal denaturation/ renaturation with full equilibrium was monitored by circular dichroism (CD) spectroscopy. The data was used to derive the unfolding thermodynamic parameters. The results showed the stability of collagen triple helices with Lys and analogs at the Xaa position followed the trend: Dab>Orn>Lys>Dap. The stability of collagen triple helices with Arg analogs at the Xaa position followed the trend: Agh>Arg>Agb>Agp. The stability of collagen triple helices with Arg analogs at the Yaa position followed the trend: Agb>Arg>Agh>Agp. These results demonstrate the importance of charged amino acid side chain length on collagen triple helix stability. The results are thus important for designing collagen triple helices. CD47 transmembrane protein is expressed on red blood cells to bind human signal regulatory protein α (SIRPα). The binding generates a bidirectional signal to inhibit splenic or red pulp macrophages. The extracellular domain of CD47 binds SIRPα using the FG loop, where the FG loop inserts into a wide groove on the surface of extracellular domain of SIRPα. The FG loop has many charged amino acids. Modified CD47 fragment peptides (Self, SelfL, SelfH, SelfHL and analogs) were used to understand the effect of different charged amino acid side chain length on the CD47 binding affinity to SIRPα. Charged residues (Glu or Arg) on the SelfHL were changed to longer or shorter side chain length. Leu101 on the FG loop was changed to DPro to confine the turn structure. Glu103 was changed to Aad to increase the β-sheet propensity. The modified CD47/SIRPα binding affinity will be determined by the split luciferase assay. The CD47/SIRPα interaction applied to nanoparticles has the potential in blocking phagocytosis as therapeutic drug targets in cancer or other diseases in the near future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77535 |
DOI: | 10.6342/NTU201802971 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 化學系 |
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