標題: 利用時間解析螢光顯影顯微技術研究二氫葉酸還原?自身動態與其催化功能之間的關係
Linking the conformational fluctuations of Dihydrofolate reductase (DHFR) to its catalytic function
作者: 高雅婷
KAO YA-TING
國立交通大學生物科技學系(所)
公開日期: 2013
摘要: 此研究計晝著重於探討蛋白質結構變動與其酵素催化功能之間的連結。酵素催化反 應本身是個包含結構變化的動態過程,然而對於蛋白質結構變動與酵素催化之關聯的深 入探討仍不足。我的實驗組提出利用單一蛋白質/酵素時間解析螢光顯影顯微技術研究探 討二羞窠鑀遗廣游自身動態與其催化功能之間的關係。二氫葉酸還原酶利用其輔酶分子 催化還原二氫葉酸為四氫葉酸,反應過程有一氫負離子轉移(hydride transfer),分子間相 互距離及蛋白質動態狀態均會影響此一轉移過程。我的實驗組將在實驗方法上獨特地利 用酵素反應中所必需的輔酶分子為光學偵測分子,而非一般所用的染料分子;並直接藉 由輔酶分子的螢光放光,將酵素反應過程以螢光明暗變化來顯示,提供一個有系統的研 究方法。最為重要:研究的結果能提供我們對酵素自身結構動態與酵素化學反應之間的 關聯有進一步深刻理解,進而我們能夠藉由外在變因,例如:基因變異、微空間黏滯度、 溫度控制與酸鹼度等,來影響酵素結構動態,最終改變並提高酵素反應的效率與能力。 並進一步可用以設計相關藥物來改變此酵素整體動態,進而抑制酵素作用。
The objective of the proposed research is to understand the linkage between protein conformational fluctuations and enzyme catalysis. Enzyme catalysis is an inherently dynamic process. Binding of a cofactor or substrate and release of a product are often accompanied by conformational changes. In addition, catalytic reaction involves conformational changes at atomic level occurring along the reaction coordinate. However, how protein conformational fluctuations, such as active-site loop movements, hinge-bending motions, and reorientation of protein domains or entire protein subunits, prompting catalytic processes remains unclear. To obtain insight of the interrelationship between protein conformational fluctuations and catalytic function, we will follow the single-molecule fluorescence trajectory along the evolution of the ligands binding/release and catalytic hydride transfer in dihydrofolate reductase (DHFR). DHFR catalyzes the reduction of dihydrofolate by hydride transfer from its cofactor, nicotinamide adenine dinucleotide phosphate (NADPH), to form tetrahydrofolate which is essential for the biosynthesis of purines, pyrimidines and amino acids. We will follow the fluorescence trajectories of the cofactor (NADPH) to determinate the real time catalytic turnover and temporal conformational fluctuations. With site-directed mutagenesis and under various thermal and pH conditions, the catalytic rate and its temporal fluctuations will be further investigated. These studies will advance the understanding of how protein fluctuations facilitate cofactor and substrate binding/release, how protein flexibility relates to the catalytic function, and which residues play essential roles in prompting catalysis. The specific aims of the project are as following: (I) Investigate protein conformational fluctuations at different liganded states and during the catalytic activity.
官方說明文件#: NSC101-2113-M009-024-MY2
URI: http://hdl.handle.net/11536/96447
https://www.grb.gov.tw/search/planDetail?id=2854895&docId=404814
顯示於類別:研究計畫