以單分子偵測技術探討ATP合成脢gamma亞基的構型變化

Abstract

隨著生化科技時代的來臨，研究生物體的結構以及運動方式，而解開許多生物遺傳及特性的奧秘，成為生物化學的研究主流。本篇論文利用單分子偵測技 術，探討如何解開生物體的運動模式。單分子偵測由於可以讓生物分子處於活性 狀態時，同步觀察其動態行為，近幾年來，成為探索許多生物機制的利器。

葉綠體 ATP 合成脢( Chloroplast ATP Synthase，CF0F1 )之γ 亞基( γ subunit )具有一個特殊的區域--- dithiol containing domain，藉由其中的 disulfide bond(Cys199，Cys205)會因環境不同而呈現氧化或還原作用來調控 ATPase 的狀態。根據模擬結果發現，γ亞基的構型會因為 ATPase 的狀態不同而改變；當 Cys199，Cys205 氧化成 disulfide bond 時，γ亞基變成一個較為緊密的構型 (close state)，造成γ亞基不易旋轉使得 ATPase 活性下降(latent)。當Cys199，Cys205為還原態，因為 dithiol domain 向上移動呈 open state，使γ亞基可以自由旋轉而提升活性( active )。γ亞基共有四個 cystienes(Cys89，Cys199，Cys205，Cys322)，所以我們必須從菠菜中萃取出氧化態 ATPase，保留Cys89和Cys322作為標記(labeling)染料的位置，利用 Hydroxyapatite Columns純化出 γ亞基，先由圓二色光譜儀 (CD)確認為自然態(native state)，再利用 ESI-MS 及 MALDI-TOF MS 確認染料標記的數目及標記的位置，最後利用單分子螢光偵測技術觀察γ亞基在native state以及 unfoling 之間的構型變化。本篇論文從如何由菠菜中萃取出 ATP 合成脢談起，再由其中純化出 γ 亞基，再談到標記染料並利用質譜儀確認標記染料的個數，最後再利用單分子螢光偵測技術觀察蛋白質構型變化。

The chloroplast F0F1-ATP synthase - ATPase is a tiny rotary motor responsible for coupling ATP synthesis and hydrolysis to the light-driven electrochemical proton gradient. Reversible oxidation/reduction of a dithiol, located within a special regulatory domain of the subunit of the chloroplast F1 enzyme, switches the enzyme between an inactive and an active state. This regulatory mechanism is unique to the ATP synthase of higher plants. Molecular dynamic studies predicted that the dithiol domain exists in a range of conformations between two extreme states；the fully open state, with the dithiol reduced ,and a fully closed state with dithiol oxidized to form a disulfide bridge. The open conformation is predicted to represent the activated state and the closed conformation is inactivated state. We isolated CF0F1 from spinach leaves. The γ subunit of spinach CF1 contains four cysteinyl sulfydryls, two of which (Cys199 and Cys205) are linked together to form the disulfide bridge. We isolated γ subunit from spinach ATPase by Hydroxyapatite Columns, and confirmed the native state of γ subunit by CD spectroscopy. We labeled TMR and Cy5 as donor and acceptor on γ subunit, confirm the numbers of dyes on γ subunit by ESI-MS, and confirmed the positions of dye on γ subunit by MALDI-TOF MS. Finally, we observed the change of conformations of γ subunit between folding and unfolding state by single molecular detection methods.