請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77533
標題: | 帶電荷胺基酸側鏈長短對於ABC異元三聚體膠原蛋白穩定度的影響 Effect of Charged Amino Acid Side Chain Length on ABC Heterotrimeric Collagen Triple Helix Stability |
作者: | Chen-Hsu Yu 余宸旭 |
指導教授: | 陳平 |
關鍵字: | 異元三聚體膠原蛋白,非自然界帶電荷胺基酸,變溫變性實驗,粒線體穿透胜?,細胞攝取量, heterotrimeric collagen triple helix,non-natural charged amino acid, thermal denaturation,mitochondria penetrating peptides,mitochondrial localization,cellular uptake, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 膠原蛋白是人體中最常見的一種蛋白質,由三條右旋的polyproline II螺旋所組成。Xaa-Yaa-Gly是一個在膠原蛋白中常出現的重複序列。脯胺酸(proline)、羥脯胺酸(hydroxyproline)及甘胺酸(glycine)是膠原蛋白中很常出現的胺基酸。除此之外,膠原蛋白也含有高於預期的帶電荷胺基酸,因此在此研究中將不同側鏈長短的帶電荷胺基酸引入異元三聚體膠原蛋白中來研究帶電荷胺基酸側鏈長短對於膠原蛋白穩定性的影響。引入的帶電荷胺基酸分別是天門冬胺酸(Asp)、谷氨酸(Glu)、Aad(側鏈較Glu多一個亞甲基)、Dap(側鏈較Lys少三個亞甲基)、Dab(側鏈較Lys少兩個亞甲基)、Orn(側鏈較Lys少一個亞甲基)及賴氨酸(Lys)。利用圓二色光譜儀來測量膠原蛋白在改變溫度下二級結構的變化以求得其變性的熱力學參數。在更改X位置為負電荷胺基酸的結果中,膠原蛋白穩定度的趨勢為Glu>Aad>Asp;在更改Y位置為負電荷胺基酸的結果中,膠原蛋白穩定度的趨勢為Aad>Glu>Asp。對於在Y位置更改為賴氨酸類似物膠原蛋白的穩定度趨勢為Dab>Dap>Lys>Orn。此結果對於未來設計引入離子對的膠原蛋白有幫助。
粒線體是細胞中不可或缺的胞器,有許多功能包括產生能量、細胞分化、細胞凋亡及細胞的訊息傳遞。當粒線體功能失調時,便會產生許多疾病。一般的藥物無法穿透到粒線體主要是因為粒線體難以通透的內膜。先前對於目標粒線體藥物的研究指出疏水性及正電荷是兩項影響粒線體穿膜的重要因素。在此研究中,將不同疏水性的非自然界胺基酸及不同側鏈長短的精氨酸類似物組合,期望合成出可表現粒線體穿膜的胜肽。將粒線體穿透胜肽接上螢光基團可研究其在細胞中的位置。利用螢光顯微鏡及流式細胞儀來檢測此胜肽在細胞中的位置及含量。研究結果顯示在粒線體穿透胜肽上接了螢光基團fluorescein會使其無法穿透進細胞,而接了螢光基團thiazole orange的粒線體穿透胜肽可進入細胞到達粒線體。未來需合成出更多接有螢光基團thiazole orange的粒線體穿透胜肽組合來研究精氨酸側鏈長短對於粒線體穿透胜肽的影響。 Collagen is the most abundant protein in the human body. Collagen forms a right-handed triple helix that consists of three polyproline II-like polypeptide chains. The Xaa-Yaa-glycine repeat are frequently observed in collagen triple helices. Besides, collagen contains more charged residues than expected. Therefore, investigating the effect of charged residues on collagen triple helix should shed light on the stability of collagen. This research focuses on the effect of charged amino acid side chain length on collagen triple helix stability. The collagen triple helices were designed based on an ABC-type heterotrimer collagen. All peptides were synthesized by Fmoc-based solid phase peptide synthesis. After purification, circular dichroism (CD) was used to monitor the thermal denaturation of collagen triple helices and further derive the thermodynamic parameters. The melting temperature, HTm and Gunfold were derived from the thermal denaturation data. The experimental results showed the stability of ABC heterotrimeric collagen triple helices upon incorporating the negatively charged residue at the Xaa position followed the trend Glu>Aad>Asp. The stability of ABC heterotrimeric collagen triple helices upon incorporating the negatively charged residue at the Yaa position followed the trend Aad>Glu>Asp. The stability of ABC heterotrimeric collagen triple helices upon incorporating the positively charged residue at the Yaa position followed the trend Dab>Dap>Lys>Orn. Negatively charged residues with longer side chains provided more collagen triple helix stability, perhaps due to formation of the intrastrand hydrogen bond between the side chain and the earlier glycine carbonyl. For collagen triple helices with a lysine analog incorporated, no correlation was observed between the side chain length and the collagen triple helix stability. Incorporating a charged residue at the Yaa position to replace hydroxyproline resulted in more significant changes in the collagen triple helix stability compared to the Xaa position. These results will be useful for developing collagen triple helices containing aspartic acid and lysine analogs. Mitochondrion is an important organelle in the cell. Mitochondria perform many functions including energy generation, cell proliferation, cell death programming, and signaling. Mitochondria dysfunction leads to many diseases such as mitochondrial disorder, diabetes, cardiomyopathy and endocrinopathy. Therefore, drug delivery targeting mitochondria is important to the remedy of mitochondria-related diseases. The major difficulty in mitochondrial delivery is the impervious mitochondrial inner membrane. Mitochondria penetrating peptides are drug delivery vehicles for targeting mitochondria. Lipophilicity and positive charge are the major factors affecting mitochondria localization. Besides, peptides with clustered positive charges result in increasing transportation across the plasma membrane, whereas peptides with separated positive charges lead to mitochondrial localization. Altering the side chain length also influences the cellular uptake of the peptide. The mitochondria penetrating peptides were designed upon altering the hydrophobic residues and the side chain length of arginine in mitochondria penetrating peptides. Fluorescence group was attached to the mitochondria penetrating peptides to be visualized by fluorescence microscopy. After purification, the localization and uptake of mitochondria penetrating peptides were analyzed by fluorescence microscopy and flow cytometry. Peptides with fluorescein led to low cellular uptake of the mitochondria penetrating peptides into Hela cells. Conversely, peptides with thiazole orange exhibited higher cellular uptake and mitochondrial localization. Conjugation with fluorescein results in the total charge of one positive charge, which hardly crossed the negatively charged plasma membrane. Therefore, thiazole orange conjugated mitochondria penetrating peptides is required to investigate the effect of arginine side chain length on mitochondria penetrating peptides. These results will lead to further designs of mitochondria penetrating peptides. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77533 |
DOI: | 10.6342/NTU201802802 |
全文授權: | 未授權 |
顯示於系所單位: | 化學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-107-R05223205-1.pdf 目前未授權公開取用 | 6.4 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。