Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/96008
標題: The effect of S-adenosylmethionine and cysteine on protein folding of human cystathionine β-synthase
S-腺苷甲硫胺酸以及半胱胺酸對人類胱硫醚乙型合成酶蛋白質折疊之影響
作者: Ci-Yin Huang
黃慈吟
關鍵字: S-腺苷甲硫胺酸
半胱胺酸
人類胱硫醚乙型合成酶
蛋白質折疊
S-adenosylmethionine
cysteine
human cystathionine β-synthase
protein folding
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摘要: 人類胱硫醚乙型合成酶(hCBS)為含硫胺基酸代謝中的關鍵酵素,透過轉硫作用的進行,可以代謝有毒的中間產物,高半胱胺酸,再進一步生合成人體所需的胺基酸,半胱胺酸(cysteine);除此之外,hCBS也參與麩胱甘肽以及H2S的生成。近期研究發現,S-腺苷甲硫胺酸(S-adenosylmethionine,SAM)與hCBS結合後,會引起hCBS構型的改變並且調節酵素本身的活性,而這一項研究也揭示了蛋白質構型特性與結合小分子之間微妙的關係。已知目前有許多小分子會與hCBS結合,不過,小分子對hCBS折疊性質的影響仍然未知,因此,我們將進一步探討小分子對hCBS構型折疊的影響。在本研究中,我們成功建構了兩種含有hCBS基因的重組質體並且優化了此蛋白質的純化流程以增加產量,由於產量及純度的考量,我們最後選擇hCBS-pET32a作為蛋白質的表現質體用於純化製程。我們利用Pulse proteolysis進行hCBS的熱力學穩定度實驗,實驗結果發現,hCBS可能是一個具有多重構型狀態的蛋白質,而SAM以及cysteine會針對特定構型產生影響;另外,我們也進行了hCBS的展開動力學實驗,結果顯示了,SAM以及cysteine對於hCBS的展開速度有著不同的影響。而這樣的結果提供我們有關於小分子參與代謝調控的參考資訊,也許可以進一步應用在疾病治療以及藥物的開發相關研究上。
Human cystathionine β-synthase(hCBS)is a key enzyme in sulfur amino acid metabolism. This enzyme can catabolize of homocysteine, which is a toxic metabolite, via transsulfuration. Moreover, hCBS also participates in the biosynthesis of cysteiene, glutathionine and H2S. Recently, hCBS was found to be allosterically activated by S-adenosylmethionine, which could trigger a conformational change of hCBS through binding. It reveals that the folding properties of this protein may be potentially affected by ligands binding. Interestingly, there are lots of ligands that interact with hCBS with unclear effect on the folding properties of this enzyme. In this study, we carefully investigated the ligand binding effect on the folding of hCBS. For obtaining hCBS, we have successfully constructed two recombinant plasmids and optimized the purification conditions for the protein purification. Because of the better purity and production, we chose hCBS-pET32a to express hCBS for subsequent experiments. We have meaured the thermodynamic stability and unfolding kinetics of hCBS by pulse proteolysis. Our results suggest that hCBS is a protein with multiple conformational state and ligands may affect specific conformation. Additionally, SAM and cysteine were found to have different influence on the unfolding rate of hCBS. This study provides valuable information for understanding the role of ligands in the metabolic regulation. This information would be possibly applied to disease treatments and drug development in the future.
URI: http://hdl.handle.net/11455/96008
文章公開時間: 2018-07-31
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